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Sample records for modeling site-specific wild

  1. Site-Specific Seismic Site Response Model for the Waste Treatment Plant, Hanford, Washington

    Energy Technology Data Exchange (ETDEWEB)

    Rohay, Alan C.; Reidel, Steve P.

    2005-02-24

    This interim report documents the collection of site-specific geologic and geophysical data characterizing the Waste Treatment Plant site and the modeling of the site-specific structure response to earthquake ground motions.

  2. Construction of Mouse Melanoma Cell Models of Wild-type Overexpression Connexin 43 and Site-specific Mutant Connexin 43%过表达及定点突变缝隙连接蛋白Cx43小鼠黑色素瘤细胞模型的构建

    Institute of Scientific and Technical Information of China (English)

    李玢; 张广献; 刘娟; 赵青; 谭宇蕙; 吴映雅; 易华; 杜标炎

    2014-01-01

    目的:构建小鼠黑色素瘤细胞(B16)过表达野生型及点突变型缝隙连接蛋白43(Connexin43, Cx43)细胞模型,为以缝隙连接(Gap Junction, GJ)为靶点的中药复方、中药单药及药物单体的研究提供可靠阳性对照和阴性对照。方法构建野生型Cx43、突变型Cx43G21R、突变型Cx43G138R重组荧光蛋白融合慢病毒表达质粒,用定点突变技术获得Cx43G21R和Cx43G138R突变体,对上述3种质粒进行双酶切和测序鉴定,并分别包装病毒感染B16细胞,使B16细胞过表达野生型Cx43、突变型Cx43G21R、突变型Cx43G138R。 Western blot检测Cx43蛋白表达水平变化,荧光示踪法观察缝隙连接通讯(Gap Junction Intercellular Communication, GJIC)功能变化。结果①酶切及测序证明,成功构建 pLVCx43-mCherry、 pLVCx43-mCherryG21R、 pLVCx43-mCherryG138R重组荧光蛋白融合慢病毒表达质粒。②成功感染B16细胞并筛选稳定过表达Cx43、 Cx43G21R、Cx43G138R细胞株, Western blot检测显示上述细胞株Cx43蛋白表达均高于对照组。③过表达野生型Cx43后B16细胞GJIC功能较对照组增强;过表达突变型Cx43后B16细胞GJIC功能较对照组减弱。结论过表达野生型Cx43可增强B16细胞GJIC功能,过表达突变型Cx43可抑制B16细胞GJIC功能。%Objective To construct mouse B16 melanoma cell models of wild-type overexpression Cx43 and site-specific mutant Cx43, thus to provide reliable positive and negative control for the study of gap junction targeted Chinese herbal formula, Chinese medicine ingredient, and drug monomer. Methods We constructed the recombined fluorescent protein(mCherry) infused with Lentivirus expression plasmid of wild-type Cx43(pLVCx43-mCherry), mutant R (pLVCx43G21R-mCherry) and mutant Cx43G138R(pLVCx43 G138R-mCherry). The Cx43G21R and Cx43G138R mutants were obtained by site-specific mutagenesis. The three kinds of plasmid were performed double restriction

  3. Development of a site specific environmental radiation assessment model for Youngkwang NPPs

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Yang Geun; Song, Myung Jae; Shon, Soon Hwan; Shin, Sang Woon; Lee, Gap Bock; Yang, Kyung Hwa [Korea Electric Power Research Institute, Taejon (Korea, Republic of); Kim, Seong Pyung; Kim, Jeong Kyu; Shin, Dae Yoon; Kim, Hyun Ku; Lee, Kyung Jin [Chosun University, Kwangju (Korea, Republic of)

    1996-12-31

    During the normal operation of nuclear power plants, a very small amount of radionuclide materials is released to the environment. The radionuclide releases to the environment may give rise to some additional radiation dose through a number of pathways. With a few exception, radionuclide transports are highly dependent on the environmental conditions such as topological features, atmospheric and oceanic conditions and socio-environmental characteristics, etc. The dose calculation in Korea are presently being performed using the models in US NRC Reg. Guide. However, these models which were originally released by the US NRC in 1977 are inadequate to deal with environmental movement of radionuclides in consideration of complex terrain, tidal condition of the Yellow Sea, and Youngkwang site specific conditions. KEPRI has developed the models to consider the site specific characteristics around Youngkwang NPPs, to give the realistic dose assessment, and to improve assurance and reliability in public dose calculation. (author). 41 figs., 70 refs.

  4. On the validity of evolutionary models with site-specific parameters.

    Directory of Open Access Journals (Sweden)

    Konrad Scheffler

    Full Text Available Evolutionary models that make use of site-specific parameters have recently been criticized on the grounds that parameter estimates obtained under such models can be unreliable and lack theoretical guarantees of convergence. We present a simulation study providing empirical evidence that a simple version of the models in question does exhibit sensible convergence behavior and that additional taxa, despite not being independent of each other, lead to improved parameter estimates. Although it would be desirable to have theoretical guarantees of this, we argue that such guarantees would not be sufficient to justify the use of these models in practice. Instead, we emphasize the importance of taking the variance of parameter estimates into account rather than blindly trusting point estimates - this is standardly done by using the models to construct statistical hypothesis tests, which are then validated empirically via simulation studies.

  5. Testing the atmospheric dispersion model of CSA N288.1 with site-specific data

    CERN Document Server

    Chouhan, S L

    2001-01-01

    The atmospheric dispersion component of CSA Standard N288. 1, which provides guidelines for calculating derived release limits, has been tested. Long-term average concentrations of tritium in air were predicted using site-specific release rates and meteorological data and compared with measured concentrations at 43 monitoring sites at all CANDU stations in Canada. The predictions correlate well with the observations but were found to be conservative, overestimating by about 50% on average. The model overpredicted 84% of the time, with the highest prediction lying a factor of 5.5 above the corresponding observation. The model underpredicted the remaining 16% of the time, with the lowest prediction about one-half of the corresponding measurement. Possible explanations for this bias are discussed but no single reason appears capable of accounting for the discrepancy. Rather, the tendency to overprediction seems to result from the cumulative effects of a number of small conservatisms in the model. The model predi...

  6. Biodiversity in models of cyclic dominance is preserved by heterogeneity in site-specific invasion rates

    CERN Document Server

    Szolnoki, Attila

    2016-01-01

    Global, population-wide oscillations in models of cyclic dominance may result in the collapse of biodiversity due to the accidental extinction of one species in the loop. Previous research has shown that such oscillations can emerge if the interaction network has small-world properties, and more generally, because of long-range interactions among individuals or because of mobility. But although these features are all common in nature, global oscillations are rarely observed in actual biological systems. This begets the question what is the missing ingredient that would prevent local oscillations to synchronize across the population to form global oscillations. Here we show that, although heterogeneous species-specific invasion rates fail to have a noticeable impact on species coexistence, randomness in site-specific invasion rates successfully hinders the emergence of global oscillations and thus preserves biodiversity. Our model takes into account that the environment is often not uniform but rather spatiall...

  7. Oncologic doses of zoledronic acid induce site specific suppression of bone modelling in rice rats.

    Science.gov (United States)

    Exposto, C R; Oz, U; Callard, J S; Allen, M J; Khurana, H; Atri, A D'; Mo, X; Fernandez, S A; Tatakis, D N; Edmonds, K; Westgate, P M; Huja, S S

    2017-06-01

    To examine the effect of zoledronic acid (ZOL) on cortical bone modelling and healing of extraction sockets in the jaw bones of a rodent model. We hypothesized ZOL suppresses both the bone formation in the modelling mode in the jaw bones and alters the extraction site healing. Rice rats were administered saline solution and two dose regimens of ZOL: 0.1 mg/kg, twice a week, for 4 weeks (n=17, saline=8 & ZOL=9) and a higher dose of 0.4 mg/kg, weekly, for 9 weeks (n=30, saline=15 & ZOL=15). Two pairs of fluorochrome bone labels were administered. Extraction of maxillary teeth was performed in maxilla. Mineral apposition rate, mineralizing surface and bone formation rate (BFR) were quantified on periodontal (PDL), alveolar and basal bone surfaces, and in the trabecular bone of proximal tibia. Bone volume (BV) was evaluated at extraction sockets. Multivariate Gaussian models were used to account for repeated measurements, and analyzes were conducted in SAS V9.3. ZOL suppressed bone modelling (BFR/BS) at the PDL surfaces in the mandible (Pmodelling mode in the jaws demonstrates the site specific effects of ZOL in rice rats. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Biodiversity in models of cyclic dominance is preserved by heterogeneity in site-specific invasion rates

    Science.gov (United States)

    Szolnoki, Attila; Perc, Matjaž

    2016-12-01

    Global, population-wide oscillations in models of cyclic dominance may result in the collapse of biodiversity due to the accidental extinction of one species in the loop. Previous research has shown that such oscillations can emerge if the interaction network has small-world properties, and more generally, because of long-range interactions among individuals or because of mobility. But although these features are all common in nature, global oscillations are rarely observed in actual biological systems. This begets the question what is the missing ingredient that would prevent local oscillations to synchronize across the population to form global oscillations. Here we show that, although heterogeneous species-specific invasion rates fail to have a noticeable impact on species coexistence, randomness in site-specific invasion rates successfully hinders the emergence of global oscillations and thus preserves biodiversity. Our model takes into account that the environment is often not uniform but rather spatially heterogeneous, which may influence the success of microscopic dynamics locally. This prevents the synchronization of locally emerging oscillations, and ultimately results in a phenomenon where one type of randomness is used to mitigate the adverse effects of other types of randomness in the system.

  9. Probing Site-Specific Structural Information of Peptides at Model Membrane Interface In Situ.

    Science.gov (United States)

    Ding, Bei; Panahi, Afra; Ho, Jia-Jung; Laaser, Jennifer E; Brooks, Charles L; Zanni, Martin T; Chen, Zhan

    2015-08-19

    Isotope labeling is a powerful technique to probe detailed structures of biological molecules with a variety of analytical methods such as NMR and vibrational spectroscopies. It is important to obtain molecular structural information on biological molecules at interfaces such as cell membranes, but it is challenging to use the isotope labeling method to study interfacial biomolecules. Here, by individually (13)C═(16)O labeling ten residues of a peptide, Ovispirin-1, we have demonstrated for the first time that a site-specific environment of membrane associated peptide can be probed by the submonolayer surface sensitive sum frequency generation (SFG) vibrational spectroscopy in situ. With the peptide associated with a single lipid bilayer, the sinusoidal trend of the SFG line width and peak-center frequency suggests that the peptide is located at the interface beneath the lipid headgroup region. The constructive interferences between the isotope labeled peaks and the main peptide amide I peak contributed by the unlabeled components were used to determine the membrane orientation of the peptide. From the SFG spectral peak-center frequency, line width, and polarization dependence of the isotope labeled units, we deduced structural information on individual units of the peptide associated with a model cell membrane. We also performed molecular dynamics (MD) simulations to understand peptide-membrane interactions. The physical pictures described by simulation agree well with the SFG experimental result. This research demonstrates the feasibility and power of using isotope labeling SFG to probe molecular structures of interfacial biological molecules in situ in real time.

  10. Comparing measured and modelled soil carbon: which site-specific variables are linked to high stability?

    Science.gov (United States)

    Robertson, Andy; Schipanski, Meagan; Ma, Liwang; Ahuja, Lajpat; McNamara, Niall; Smith, Pete; Davies, Christian

    2016-04-01

    Changes in soil carbon (C) stocks have been studied in depth over the last two decades, as net greenhouse gas (GHG) sinks are highlighted to be a partial solution to the causes of climate change. However, the stability of this soil C is often overlooked when measuring these changes. Ultimately a net sequestration in soils is far less beneficial if labile C is replacing more stable forms. To date there is no accepted framework for measuring soil C stability, and as a result there is considerable uncertainty associated with the simulated impacts of land management and land use change when using process-based systems models. However, a recent effort to equate measurable soil C fractions to model pools has generated data that help to assess the impacts of land management, and can ultimately help to reduce the uncertainty of model predictions. Our research compiles this existing fractionation data along with site metadata to create a simplistic statistical model able to quantify the relative importance of different site-specific conditions. Data was mined from 23 published studies and combined with original data to generate a dataset of 100+ land use change sites across Europe. For sites to be included they required soil C fractions isolated using the Zimmermann et al. (2007) method and specific site metadata (mean annual precipitation, MAP; mean annual temperature, MAT; soil pH; land use; altitude). Of the sites, 75% were used to develop a generalized linear mixed model (GLMM) to create coefficients where site parameters can be used to predict influence on the measured soil fraction C stocks. The remaining 25% of sites were used to evaluate uncertainty and validate this empirical model. Further, four of the aforementioned sites were used to simulate soil C dynamics using the RothC, DayCent and RZWQM2 models. A sensitivity analysis (4096 model runs for each variable applying Latin hypercube random sampling techniques) was then used to observe whether these models place

  11. A class frequency mixture model that adjusts for site-specific amino acid frequencies and improves inference of protein phylogeny

    Directory of Open Access Journals (Sweden)

    Li Karen

    2008-12-01

    Full Text Available Abstract Background Widely used substitution models for proteins, such as the Jones-Taylor-Thornton (JTT or Whelan and Goldman (WAG models, are based on empirical amino acid interchange matrices estimated from databases of protein alignments that incorporate the average amino acid frequencies of the data set under examination (e.g JTT + F. Variation in the evolutionary process between sites is typically modelled by a rates-across-sites distribution such as the gamma (Γ distribution. However, sites in proteins also vary in the kinds of amino acid interchanges that are favoured, a feature that is ignored by standard empirical substitution matrices. Here we examine the degree to which the pattern of evolution at sites differs from that expected based on empirical amino acid substitution models and evaluate the impact of these deviations on phylogenetic estimation. Results We analyzed 21 large protein alignments with two statistical tests designed to detect deviation of site-specific amino acid distributions from data simulated under the standard empirical substitution model: JTT+ F + Γ. We found that the number of states at a given site is, on average, smaller and the frequencies of these states are less uniform than expected based on a JTT + F + Γ substitution model. With a four-taxon example, we show that phylogenetic estimation under the JTT + F + Γ model is seriously biased by a long-branch attraction artefact if the data are simulated under a model utilizing the observed site-specific amino acid frequencies from an alignment. Principal components analyses indicate the existence of at least four major site-specific frequency classes in these 21 protein alignments. Using a mixture model with these four separate classes of site-specific state frequencies plus a fifth class of global frequencies (the JTT + cF + Γ model, significant improvements in model fit for real data sets can be achieved. This simple mixture model also reduces the long

  12. Bioavailability of particulate metal to zebra mussels: Biodynamic modelling shows that assimilation efficiencies are site-specific

    Energy Technology Data Exchange (ETDEWEB)

    Bourgeault, Adeline, E-mail: bourgeault@ensil.unilim.fr [Cemagref, Unite de Recherche Hydrosystemes et Bioprocedes, 1 rue Pierre-Gilles de Gennes, 92761 Antony (France); FIRE, FR-3020, 4 place Jussieu, 75005 Paris (France); Gourlay-France, Catherine, E-mail: catherine.gourlay@cemagref.fr [Cemagref, Unite de Recherche Hydrosystemes et Bioprocedes, 1 rue Pierre-Gilles de Gennes, 92761 Antony (France); FIRE, FR-3020, 4 place Jussieu, 75005 Paris (France); Priadi, Cindy, E-mail: cindy.priadi@eng.ui.ac.id [LSCE/IPSL CEA-CNRS-UVSQ, Avenue de la Terrasse, 91198 Gif-sur-Yvette (France); Ayrault, Sophie, E-mail: Sophie.Ayrault@lsce.ipsl.fr [LSCE/IPSL CEA-CNRS-UVSQ, Avenue de la Terrasse, 91198 Gif-sur-Yvette (France); Tusseau-Vuillemin, Marie-Helene, E-mail: Marie-helene.tusseau@ifremer.fr [IFREMER Technopolis 40, 155 rue Jean-Jacques Rousseau, 92138 Issy-Les-Moulineaux (France)

    2011-12-15

    This study investigates the ability of the biodynamic model to predict the trophic bioaccumulation of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni) and zinc (Zn) in a freshwater bivalve. Zebra mussels were transplanted to three sites along the Seine River (France) and collected monthly for 11 months. Measurements of the metal body burdens in mussels were compared with the predictions from the biodynamic model. The exchangeable fraction of metal particles did not account for the bioavailability of particulate metals, since it did not capture the differences between sites. The assimilation efficiency (AE) parameter is necessary to take into account biotic factors influencing particulate metal bioavailability. The biodynamic model, applied with AEs from the literature, overestimated the measured concentrations in zebra mussels, the extent of overestimation being site-specific. Therefore, an original methodology was proposed for in situ AE measurements for each site and metal. - Highlights: > Exchangeable fraction of metal particles did not account for the bioavailability of particulate metals. > Need for site-specific biodynamic parameters. > Field-determined AE provide a good fit between the biodynamic model predictions and bioaccumulation measurements. - The interpretation of metal bioaccumulation in transplanted zebra mussels with biodynamic modelling highlights the need for site-specific assimilation efficiencies of particulate metals.

  13. GIS Modelling for Site-Specific Nitrogen Fertilization towards Soil Sustainability

    Directory of Open Access Journals (Sweden)

    Antonis Papadopoulos

    2015-05-01

    Full Text Available Farmers need to make decisions that in most cases incorporate the concept of prediction and can hardly be revoked. One such decision is the application of fertilizing inputs. During past crop management and decision-making on fertilizing practices, many significant errors have been recorded, which have led and continue to lead to reduced production and environmental burden. The methodology followed in this paper involves the use of GIS, fuzzy logic and expert knowledge, in order to model physical processes associated with nitrogen balance in cultivated ecosystems and to evaluate the capabilities of or limitations on the use of certain fertilizers, based on spatial criteria. An original spatial decision support system was designed, developed and applied in a given study area. The system is composed of two modules (“fertilizing rate” and “fertilizing type”, making use of soil, climate and cultivation practices’ data, as recorded in the area of interest in quantitative or categorical form. The results of the application spatially classify the involved area according to its demand for nitrogen on the basis of the characteristics of each sub-region. The “fertilizing rate” module suggests reduced fertilizing doses of nitrogenous fertilizers compared to those already applied in the area. The system further divides the area into zones where specific types of fertilizers should be applied, giving a certain prescription for the method and time of application.

  14. Environmental transportation of tritium and estimation of site-specific model parameters for Kaiga site, India.

    Science.gov (United States)

    Reji, T K; Ravi, P M; Ajith, T L; Dileep, B N; Hegde, A G; Sarkar, P K

    2012-04-01

    Tritium content in air moisture, soil water, rain water and plant water samples collected around the Kaiga site, India was estimated and the scavenging ratio, wet deposition velocity and ratio of specific activities of tritium between soil water and air moisture were calculated and the results are interpreted. Scavenging ratio was found to vary from 0.06 to 1.04 with a mean of 0.46. The wet deposition velocity of tritium observed in the present study was in the range of 3.3E-03 to 1.1E-02 m s(-1) with a mean of 6.6E-03 m s(-1). The ratio of specific activity of tritium in soil moisture to that in air moisture ranged from 0.17 to 0.95 with a mean of 0.49. The specific activity of tritium in plant water in this study varied from 73 to 310 Bq l(-1). The present study is very useful for understanding the process and modelling of transfer of tritium through air/soil/plant system at the Kaiga site.

  15. Site Specific Weed Control Technologies

    DEFF Research Database (Denmark)

    Christensen, Svend; Søgaard, Henning Tangen; Kudsk, Per;

    2009-01-01

    describe the basic parts of site specific weed control technologies, comprising of weed sensing systems, weed management models and precision weed control implements. A review of state-of-the-art technologies shows that several weed sensing systems and precision implements have been developed over the last...... of knowledge about the economic and environmental potential for increasing the resolution of weed control. The integration of site-specific information on weed distribution, weed species composition and density, and the effect on crop yield, is decisive for successful site-specific weed management.   Keywords......Site-specific weed control technologies are defined as machinery or equipment embedded with technologies that detect weeds growing in a crop and, taking into account predefined factors such as economics, takes action to maximise the chances of successfully controlling them. In the article, we...

  16. Skeletal site-specific response to ovariectomy in a rat model: change in bone density and microarchitecture.

    Science.gov (United States)

    Liu, Xi Ling; Li, Chun Lei; Lu, Weijia William; Cai, Wei Xin; Zheng, Li Wu

    2015-04-01

    Ovariectomized (OVX) rat model has been widely used in osteoporosis-related studies. However, the discrepancies in age and skeletal sites being investigated make it difficult to compare the results from different studies. The purpose of this study was to provide information of systemic skeletal site-specific changes in a stable OVX rat model. Thirty-three 6-month Spraque-Dawley female rats were used. Fifteen rats underwent ovariectomy, and fifteen received sham surgery. Three animals without any surgery were sacrificed at week 0 to serve as baseline. Three animals in the OVX and sham group, respectively, were euthanized at week 2, 4, 12, 24 and 36 post-surgery. Ten bone sites, including parietal bone, interparietal bone, maxilla, mandible, humerus, ulna, femur, tibia, lumber vertebra, and ilium, were subjected to micro-CT. Overall, long bones, lumber vertebra, and ilium showed similar trend of bone loss post-OVX, with tibia and femur suffered the most bone loss and spine the least (decreased by 75.0%, 70.4% and 36.6% in bone mineral density BMD at week 36 from base line, respectively). Upon OVX, jaw bones and cranial bones only showed a minor reduction in BMD (decreased by 1~3% from baseline) at week 36. Significant deterioration of trabecular structure was detected in long bones, lumber vertebra, and ilium post-OVX, while jaw bones remained relatively stable. This study for the first time assessed the systemic site-specific bone loss and microarchitecture changes in OVX rat model. It provided valuable information for selecting bone site and observation time in osteoporosis-related study. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Site-Specific Innovation

    DEFF Research Database (Denmark)

    Reeh, Henrik; Hemmersam, Peter

    2015-01-01

    , but also places for emerging lifestyles and cultural processes. In this transformation process, harbours provide arenas for a new urban dynamics, involving multiple sectors and functions in society, as opposed to the monoculturalism prevailing in the ages of the industrial city. To us, the critical...... question is how innovation may contribute to urban life and site-specific qualities....

  18. Site-Specific Innovation

    DEFF Research Database (Denmark)

    Reeh, Henrik; Hemmersam, Peter

    2015-01-01

    Currently, cities across the Northern European region are actively redeveloping their former industrial harbours. Indeed, harbours areas are essential in the long-term transition from industrial to information and experience societies; harbours are becoming sites for new businesses and residences...... question is how innovation may contribute to urban life and site-specific qualities....

  19. Site-specific Microzonation Study in Delhi Metropolitan City by 2-D Modelling of SH and P-SV Waves

    Science.gov (United States)

    Parvez, Imtiyaz A.; Vaccari, F.; Panza, G. F.

    Delhi - the capital of India lies on a severe earthquake hazard threat not only from local earthquakes but also from Himalayan events just 200-250 km apart. The seismic ground motion in a part of Delhi City is computed with a hybrid technique based on the modal summation and the finite-difference scheme for site-specific strong ground motion modelling. Complete realistic SH and #E5/E5#-SV wave seismograms are computed along two geological cross sections, (1) north-south, from Inter State Bus Terminal (ISBT) to Sewanagar and (2) east-west, from Tilak Bridge to Punjabi Bagh. Two real earthquake sources of July 15, 1720 (MMI=IX, M=7.4) and August 27, 1960 (M=6.0) have been used in modelling. The response spectra ratio (RSR), i.e. the response spectra computed from the signals synthesized along the laterally varying section and normalized by the response spectra computed from the corresponding signals, synthesized for the bedrock reference regional model, have been determined. As expected, the sedimentary cover causes an increase of the signal amplitude, particularly in the radial and transverse components. To further check the site-effects, we reversed the source location to the other side of the cross section and recomputed the site amplifications. There are only a few sites where a large amplification is invariant with respect to the two source locations considered. The RSR ranges between 5 to 10 in the frequency range from 2.8 to 3.7 Hz for the radial and transverse components of motion along the NS cross section. Along the EW cross section RSR varies between 3.5 to 7.5 in the frequency range from 3.5 to 4.1 Hz. The amplification of the vertical component is considerable at high frequency (>4 Hz.) whereas it is negligible in lower frequency range.

  20. Measurement and Modeling of Site-specific Nitrogen and Oxygen Isotopic Composition of Atmospheric Nitrous Oxide at Mace Head, Ireland

    Science.gov (United States)

    McClellan, M. J.; Saikawa, E.; Prinn, R. G.; Ono, S.

    2015-12-01

    Global mixing ratios of atmospheric nitrous oxide (N2O), a potent greenhouse gas, have increased nearly linearly from the beginning of the modern industrial period to today, with the current global average in excess of 325 ppb. This increase can be largely attributed to anthropogenic activity above the level of N2O emissions from natural biotic sources. The effect of N2O on Earth's climate is twofold: in the troposphere, N2O is radiatively active and chemically inert, while it serves as a reactive source of ozone-destroying nitrogen oxides in the stratosphere. The marked altitudinal divide in its reactivity means that all stages in the N2O life cycle—emission, transport, and destruction—must be examined to understand the overall effect of N2O on Earth's climate. However, the understanding of the total impact of N2O is incomplete, as there remain significant uncertainties in the global budget of this gas. Due to unique isotopic substitutions (15N and 18O) made by different N2O sources and stratospheric chemical reactions, the measurement of N2O isotopic ratios in ambient air can help identify the distribution and magnitude of distinct source types. We present the first year of site-specific nitrogen and oxygen isotopic composition data from the MIT Stheno-tunable infrared direct absorption spectroscopy (TILDAS) instrument at Mace Head, Ireland. Aided by the Stheno preconcentration system, Stheno-TILDAS can achieve measurement precisions of 0.10‰ or greater for all isotopic ratios (δ15N and δ18O) in ambient N2O. We further compare these data to the results from Model for Ozone and Related Tracers version 4 (MOZART-4) simulations, including N2O isotopic fractionation processes and MERRA/GEOS-5 reanalysis meteorological fields. These results will form the basis of future Bayesian inverse modeling simulations that will constrain global N2O source, circulation, and sink dynamics better.

  1. Calculating soil gas fluxes from gas concentration profiles: can we use standard DS models or should we use site-specific DS models?

    Science.gov (United States)

    Paulus, Sinikka; Jochheim, Hubert; Wirth, Stephan; Maier, Martin

    2015-04-01

    The apparent gas diffusion coefficient in soil (DS) is an important parameter describing soil aeration. It also links the profiles of soil gas concentration and soil gas flux using Fick's law. Soil gas diffusivity depends mainly on the structure of the pore system and the soil moisture status. There are several standard DS-models available that can easily be used for calculating DS. Another, more laborious option is to calibrate site specific DS models on soil core samples from the respective profile. We tested 4 standard DS models and a site-specific model and compared the resulting soil gas fluxes in two forest soils. Differences between the models were substantial. Another very important effect, however, is that standard DS models are usually derived from a single soil moisture measurement (device), that can result in an substantial offset in soil moisture estimation. The mean soil moisture content at a depth can be addressed more accurately by taking several soil cores. As a consequence, using standard DS models in combination with a single soil moisture measurement is less reliable than using site-specific models based on several soil samples.

  2. Intercomparison of atmospheric reanalysis data in the Arctic region: To derive site-specific forcing data for terrestrial models

    Science.gov (United States)

    Mori, J.; Saito, K.; Machiya, H.; Yabuki, H.; Ikawa, H.; Ohta, T.; Iijima, Y.; Kotani, A.; Suzuki, R.; Miyazaki, S.; Sato, A.; Hajima, T.; Sueyoshi, T.

    2015-12-01

    An intercomparison project for the Arctic terrestrial (physical and ecosystem) models, GTMIP, is conducted, targeting at improvements in the existing terrestrial schemes, as an activity of the Terrestrial Ecosystem research group in the Arctic of Japan GRENE Arctic Climate Change Research Project (GRENE-TEA). For site simulations for four GRENE-TEA sites (i.e., Fairbanks/AK, Kevo/Finland, Tiksi and Yakutsk/Siberia), we needed to prepare continuous, site-fit forcing data ready to drive the models. Due to scarcity of site observations in the region, however, it was difficult to make such data directly from the observations. Therefore, we decided to create a backbone dataset (Level 0 or Lv0) first by utilizing the reanalysis data to derive the site-specific data (Level 1 or Lv1). For selection of the best dataset for our purpose, we compared four atmospheric reanalysis datasets, i.e., ERA Interim, JRA-55, NCEP/NCAR Reanalysis 1, and NCEP-DOE Reanalysis 2, in terms of the climatic reproducibility (w.r.t. temperature at 2 m and precipitation) in the region north of 60°N. CRU for temperature and GPCP for precipitation were also used for monthly-mean ground-level climate. As we will show ERA-Interim showed the smallest bias for both the parameters in terms of RMSE. Especially, air temperature in the cold period was reproduced better in ERA-Interim than is in JRA-55 or other reanalysis products. Therefore, we created Lv0 from ERA-Interim. Comparison between the site observations and Lv0 showed good agreement except for wind speed at all sites and air temperature at Tiksi, a coastal site in the eastern Siberia. Air temperature of ERA-Interim showed significantly continental characteristics while the site observation more coastal. The 34-year-long, hourly, site-fit continuous data (Lv1) for each of the GRENE-TEA sites was then created from the Lv0 values at the grid point closest to the site, by merging with the observations.

  3. Development of a regression model to predict copper toxicity to Daphnia magna and site-specific copper criteria across multiple surface-water drainages in an arid landscape.

    Science.gov (United States)

    Fulton, Barry A; Meyer, Joseph S

    2014-08-01

    The water effect ratio (WER) procedure developed by the US Environmental Protection Agency is commonly used to derive site-specific criteria for point-source metal discharges into perennial waters. However, experience is limited with this method in the ephemeral and intermittent systems typical of arid climates. The present study presents a regression model to develop WER-based site-specific criteria for a network of ephemeral and intermittent streams influenced by nonpoint sources of Cu in the southwestern United States. Acute (48-h) Cu toxicity tests were performed concurrently with Daphnia magna in site water samples and hardness-matched laboratory waters. Median effect concentrations (EC50s) for Cu in site water samples (n=17) varied by more than 12-fold, and the range of calculated WER values was similar. Statistically significant (α=0.05) univariate predictors of site-specific Cu toxicity included (in sequence of decreasing significance) dissolved organic carbon (DOC), hardness/alkalinity ratio, alkalinity, K, and total dissolved solids. A multiple-regression model developed from a combination of DOC and alkalinity explained 85% of the toxicity variability in site water samples, providing a strong predictive tool that can be used in the WER framework when site-specific criteria values are derived. The biotic ligand model (BLM) underpredicted toxicity in site waters by more than 2-fold. Adjustments to the default BLM parameters improved the model's performance but did not provide a better predictive tool compared with the regression model developed from DOC and alkalinity.

  4. Aespoe modelling task force - experiences of the site specific flow and transport modelling (in detailed and site scale)

    Energy Technology Data Exchange (ETDEWEB)

    Gustafson, Gunnar [Chalmers Univ. of Technology, Goeteborg (Sweden); Stroem, A.; Wikberg, P. [Swedish Nuclear Fuel and Waste Management Co. , Stockholm (Sweden)

    1998-09-01

    The Aespoe Task Force on modelling of groundwater flow and transport of solutes was initiated in 1992. The Task Force shall be a forum for the organisations supporting the Aespoe Hard Rock Laboratory Project to interact in the area of conceptual and numerical modelling of groundwater flow and solute transport in fractured rock. Much emphasis is put on building of confidence in the approaches and methods in use for modelling of groundwater flow and nuclide migration in order to demonstrate their use for performance and safety assessment. The modelling work within the Task Force is linked to the experiments performed at the Aespoe Laboratory. As the first Modelling Task, a large scale pumping and tracer experiment called LPT2 was chosen. This was the final part of the characterisation work for the Aespoe site before the construction of the laboratory in 1990. The construction of the Aespoe HRL access tunnel caused an even larger hydraulic disturbance on a much larger scale than that caused by the LPT2 pumping test. This was regarded as an interesting test case for the conceptual and numerical models of the Aespoe site developed during Task No 1, and was chosen as the third Modelling Task. The aim of Task 3 can be seen from two different perspectives. The Aespoe HRL project saw it as a test of their ability to define a conceptual and structural model of the site that can be utilised by independent modelling groups and be transformed to a predictive groundwater flow model. The modelling groups saw it as a means of understanding groundwater flow in a large fractured rock volume and of testing their computational tools. A general conclusion is that Task 3 has served these purposes well. Non-sorbing tracers tests, made as a part of the TRUE-experiments were chosen as the next predictive modelling task. A preliminary comparison between model predictions made by the Aespoe Task Force and the experimental results, shows that most modelling teams predicted breakthrough from

  5. WAG 2 remedial investigation and site investigation site-specific work plan/health and safety checklist for the sediment transport modeling task

    Energy Technology Data Exchange (ETDEWEB)

    Holt, V.L.; Baron, L.A.

    1994-05-01

    This site-specific Work Plan/Health and Safety Checklist (WP/HSC) is a supplement to the general health and safety plan (HASP) for Waste Area Grouping (WAG) 2 remedial investigation and site investigation (WAG 2 RI&SI) activities [Health and Safety Plan for the Remedial Investigation and Site Investigation of Waste Area Grouping 2 at the Oak Ridge National Laboratory, Oak Ridge, Tennessee (ORNL/ER-169)] and provides specific details and requirements for the WAG 2 RI&SI Sediment Transport Modeling Task. This WP/HSC identifies specific site operations, site hazards, and any recommendations by Oak Ridge National Laboratory (ORNL) health and safety organizations [i.e., Industrial Hygiene (IH), Health Physics (HP), and/or Industrial Safety] that would contribute to the safe completion of the WAG 2 RI&SI. Together, the general HASP for the WAG 2 RI&SI (ORNL/ER-169) and the completed site-specific WP/HSC meet the health and safety planning requirements specified by 29 CFR 1910.120 and the ORNL Hazardous Waste Operations and Emergency Response (HAZWOPER) Program Manual. In addition to the health and safety information provided in the general HASP for the WAG 2 RI&SI, details concerning the site-specific task are elaborated in this site-specific WP/HSC, and both documents, as well as all pertinent procedures referenced therein, will be reviewed by all field personnel prior to beginning operations.

  6. Establishment of a Site-Specific Tropospheric Model Based on Ground Meteorological Parameters over the China Region.

    Science.gov (United States)

    Zhou, Chongchong; Peng, Bibo; Li, Wei; Zhong, Shiming; Ou, Jikun; Chen, Runjing; Zhao, Xinglong

    2017-07-27

    China is a country of vast territory with complicated geographical environment and climate conditions. With the rapid progress of the Chinese BeiDou satellite navigation system (BDS); more accurate tropospheric models must be applied to improve the accuracy of navigation and positioning. Based on the formula of the Saastamoinen and Callahan models; this study develops two single-site tropospheric models (named SAAS_S and CH_S models) for the Chinese region using radiosonde data from 2005 to 2012. We assess the two single-site tropospheric models with radiosonde data for 2013 and zenith tropospheric delay (ZTD) data from four International GNSS Service (IGS) stations and compare them to the results of the Saastamoinen and Callahan models. The experimental results show that: the mean accuracy of the SAAS_S model (bias: 0.19 cm; RMS: 3.19 cm) at all radiosonde stations is superior to those of the Saastamoinen (bias: 0.62 cm; RMS: 3.62 cm) and CH_S (bias: -0.05 cm; RMS: 3.38 cm) models. In most Chinese regions; the RMS values of the SAAS_S and CH_S models are about 0.51~2.12 cm smaller than those of their corresponding source models. The SAAS_S model exhibits a clear improvement in the accuracy over the Saastamoinen model in low latitude regions. When the SAAS_S model is replaced by the SAAS model in the positioning of GNSS; the mean accuracy of vertical direction in the China region can be improved by 1.12~1.55 cm and the accuracy of vertical direction in low latitude areas can be improved by 1.33~7.63 cm. The residuals of the SAAS_S model are closer to a normal distribution compared to those of the Saastamoinen model. Single-site tropospheric models based on the short period of the most recent data (for example 2 years) can also achieve a satisfactory accuracy. The average performance of the SAAS_S model (bias: 0.83 cm; RMS: 3.24 cm) at four IGS stations is superior to that of the Saastamoinen (bias: -0.86 cm; RMS: 3.59 cm) and CH_S (bias: 0.45 cm; RMS: 3.38 cm

  7. Calculation of site specific characterisation factors for metal ecotoxicity using decoupled multi species fate and exposure modelling

    DEFF Research Database (Denmark)

    Birkved, Morten; Strandesen, Maria; Larsen, HF

    2004-01-01

    Calculation of characterisation factors (CF’s) for metal ecotoxicity typically involves fate and exposure modelling of metals in multi-media models developed for assessment of organic compounds. Metals do not follow the fate patterns of organic chemicals, and the results will therefore most likel...

  8. Soil erosion model predictions using parent material/soil texture-based parameters compared to using site-specific parameters

    Science.gov (United States)

    R. B. Foltz; W. J. Elliot; N. S. Wagenbrenner

    2011-01-01

    Forested areas disturbed by access roads produce large amounts of sediment. One method to predict erosion and, hence, manage forest roads is the use of physically based soil erosion models. A perceived advantage of a physically based model is that it can be parameterized at one location and applied at another location with similar soil texture or geological parent...

  9. Calculation of site specific characterisation factors for metal ecotoxicity using decoupled multi species fate and exposure modelling

    DEFF Research Database (Denmark)

    Birkved, Morten; Strandesen, Maria; Larsen, HF;

    2004-01-01

    Calculation of characterisation factors (CF’s) for metal ecotoxicity typically involves fate and exposure modelling of metals in multi-media models developed for assessment of organic compounds. Metals do not follow the fate patterns of organic chemicals, and the results will therefore most likel...

  10. Evaluation and application of site-specific data to revise the first-order decay model for estimating landfill gas generation and emissions at Danish landfills

    DEFF Research Database (Denmark)

    Mou, Zishen; Scheutz, Charlotte; Kjeldsen, Peter

    2015-01-01

    Methane (CH4) generated from low-organic waste degradation at four Danish landfills was estimated by three first-order decay (FOD) landfill gas (LFG) generation models (LandGEM, IPCC, and Afvalzorg). Actual waste data from Danish landfills were applied to fit model (IPCC and Afvalzorg) required...... categories. In general, the single-phase model, LandGEM, significantly overestimated CH4 generation, because it applied too high default values for key parameters to handle low-organic waste scenarios. The key parameters were biochemical CH4 potential (BMP) and CH4 generation rate constant (k.......Implications: Landfill operators use the first-order decay (FOD) models to estimate methane (CH4) generation. A single-phase model (LandGEM) and a traditional model (IPCC) could result in overestimation when handling a low-organic waste scenario. Site-specific data were important and capable of calibrating key parameter...

  11. Settings for Physical Activity – Developing a Site-specific Physical Activity Behavior Model based on Multi-level Intervention Studies

    DEFF Research Database (Denmark)

    Troelsen, Jens; Klinker, Charlotte Demant; Breum, Lars

    Settings for Physical Activity – Developing a Site-specific Physical Activity Behavior Model based on Multi-level Intervention Studies Introduction: Ecological models of health behavior have potential as theoretical framework to comprehend the multiple levels of factors influencing physical...... activity (PA). The potential is shown by the fact that there has been a dramatic increase in application of ecological models in research and practice. One proposed core principle is that an ecological model is most powerful if the model is behavior-specific. However, based on multi-level interventions...... of the facilities. Despite the conceptual and contextual differences PA behavior is also affected by cultural and social values related to the specific site which not alone can be explained by intrapersonal, interpersonal or organizational factors. Discussion: The Ecological Model of Four Domains of Active Living...

  12. Settings for Physical Activity – Developing a Site-specific Physical Activity Behavior Model based on Multi-level Intervention Studies

    DEFF Research Database (Denmark)

    Troelsen, Jens; Klinker, Charlotte Demant; Breum, Lars

    Settings for Physical Activity – Developing a Site-specific Physical Activity Behavior Model based on Multi-level Intervention Studies Introduction: Ecological models of health behavior have potential as theoretical framework to comprehend the multiple levels of factors influencing physical...... of new established outdoor facilities for PA among 12 – 85 years old citizens (N=10,434). The SPACE and the WCMC study used objective measurements of PA combined with e-surveys, the VDPA study is based on self-reported e-survey data. Results: Merging the data from the three intervention studies clarifies...... activity (PA). The potential is shown by the fact that there has been a dramatic increase in application of ecological models in research and practice. One proposed core principle is that an ecological model is most powerful if the model is behavior-specific. However, based on multi-level interventions...

  13. Angiotensin-converting enzyme-2 (ACE2): comparative modeling of the active site, specificity requirements, and chloride dependence.

    Science.gov (United States)

    Guy, Jodie L; Jackson, Richard M; Acharya, K Ravi; Sturrock, Edward D; Hooper, Nigel M; Turner, Anthony J

    2003-11-18

    Angiotensin-converting enzyme 2 (ACE2), a homologue of ACE, represents a new and potentially important target in cardio-renal disease. A model of the active site of ACE2, based on the crystal structure of testicular ACE, has been developed and indicates that the catalytic mechanism of ACE2 resembles that of ACE. Structural differences exist between the active site of ACE (dipeptidyl carboxypeptidase) and ACE2 (carboxypeptidase) that are responsible for the differences in specificity. The main differences occur in the ligand-binding pockets, particularly at the S2' subsite and in the binding of the peptide carboxy-terminus. The model explains why the classical ACE inhibitor lisinopril is unable to bind to ACE2. On the basis of the ability of ACE2 to cleave a variety of biologically active peptides, a consensus sequence of Pro-X-Pro-hydrophobic/basic for the protease specificity of ACE2 has been defined that is supported by the ACE2 model. The dipeptide, Pro-Phe, completely inhibits ACE2 activity at 180 microM with angiotensin II as the substrate. As with ACE, the chloride dependence of ACE2 is substrate-specific such that the hydrolysis of angiotensin I and the synthetic peptide substrate, Mca-APK(Dnp), are activated in the presence of chloride ions, whereas the cleavage of angiotensin II is inhibited. The ACE2 model is also suggestive of a possible mechanism for chloride activation. The structural insights provided by these analyses for the differences in inhibition pattern and substrate specificity among ACE and its homologue ACE2 and for the chloride dependence of ACE/ACE2 activity are valuable in understanding the function and regulation of ACE2.

  14. Modelling site-specific N2O emission factors from Austrian agricultural soils for targeted mitigation measures (NitroAustria)

    Science.gov (United States)

    Amon, Barbara; Zechmeister-Boltenstern, Sophie; Kasper, Martina; Foldal, Cecilie; Schiefer, Jasmin; Kitzler, Barbara; Schwarzl, Bettina; Zethner, Gerhard; Anderl, Michael; Sedy, Katrin; Gaugitsch, Helmut; Dersch, Georg; Baumgarten, Andreas; Haas, Edwin; Kiese, Ralf

    2016-04-01

    Results from a previous project "FarmClim" highlight that the IPCC default emission factor is not able to reflect region specific N2O emissions from Austrian arable soils. The methodology is limited in identifying hot spots and hot moments of N2O emissions. When estimations are based on default emission factors no recommendations can be given on optimisation measures that would lead to a reduction of soil N2O emissions. The better the knowledge is about Nitrogen and Carbon budgets in Austrian agricultural managed soils the better the situation can be reflected in the Austrian GHG emission inventory calculations. Therefore national and regionally modelled emission factors should improve the evidence for national deviation from the IPCC default emission factors and reduce the uncertainties. The overall aim of NitroAustria is to identify the drivers for N2O emissions on a regional basis taking different soil types, climate, and agricultural management into account. We use the LandscapeDNDC model to update the N2O emission factors for N fertilizer and animal manure applied to soils. Key regions in Austria were selected and region specific N2O emissions calculated. The model runs at sub-daily time steps and uses data such as maximum and minimum air temperature, precipitation, radiation, and wind speed as meteorological drivers. Further input data are used to reflect agricultural management practices, e.g., planting/harvesting, tillage, fertilizer application, irrigation and information on soil and vegetation properties for site characterization and model initialization. While at site scale, arable management data (crop cultivation, rotations, timings etc.) is obtained by experimental data from field trials or observations, at regional scale such data need to be generated using region specific proxy data such as land use and management statistics, crop cultivations and yields, crop rotations, fertilizer sales, manure resulting from livestock units etc. The farming

  15. Improvement of AEP Predictions Using Diurnal CFD Modelling with Site-Specific Stability Weightings Provided from Mesoscale Simulation

    Science.gov (United States)

    Hristov, Y.; Oxley, G.; Žagar, M.

    2014-06-01

    The Bolund measurement campaign, performed by Danish Technical University (DTU) Wind Energy Department (also known as RISØ), provided significant insight into wind flow modeling over complex terrain. In the blind comparison study several modelling solutions were submitted with the vast majority being steady-state Computational Fluid Dynamics (CFD) approaches with two equation k-epsilon turbulence closure. This approach yielded the most accurate results, and was identified as the state-of-the-art tool for wind turbine generator (WTG) micro-siting. Based on the findings from Bolund, further comparison between CFD and field measurement data has been deemed essential in order to improve simulation accuracy for turbine load and long-term Annual Energy Production (AEP) estimations. Vestas Wind Systems A/S is a major WTG original equipment manufacturer (OEM) with an installed base of over 60GW in over 70 countries accounting for 19% of the global installed base. The Vestas Performance and Diagnostic Centre (VPDC) provides online live data to more than 47GW of these turbines allowing a comprehensive comparison between modelled and real-world energy production data. In previous studies, multiple sites have been simulated with a steady neutral CFD formulation for the atmospheric surface layer (ASL), and wind resource (RSF) files have been generated as a base for long-term AEP predictions showing significant improvement over predictions performed with the industry standard linear WAsP tool. In this study, further improvements to the wind resource file generation with CFD are examined using an unsteady diurnal cycle approach with a full atmospheric boundary layer (ABL) formulation, with the unique stratifications throughout the cycle weighted according to mesoscale simulated sectorwise stability frequencies.

  16. PROCOV: maximum likelihood estimation of protein phylogeny under covarion models and site-specific covarion pattern analysis

    Directory of Open Access Journals (Sweden)

    Wang Huai-Chun

    2009-09-01

    Full Text Available Abstract Background The covarion hypothesis of molecular evolution holds that selective pressures on a given amino acid or nucleotide site are dependent on the identity of other sites in the molecule that change throughout time, resulting in changes of evolutionary rates of sites along the branches of a phylogenetic tree. At the sequence level, covarion-like evolution at a site manifests as conservation of nucleotide or amino acid states among some homologs where the states are not conserved in other homologs (or groups of homologs. Covarion-like evolution has been shown to relate to changes in functions at sites in different clades, and, if ignored, can adversely affect the accuracy of phylogenetic inference. Results PROCOV (protein covarion analysis is a software tool that implements a number of previously proposed covarion models of protein evolution for phylogenetic inference in a maximum likelihood framework. Several algorithmic and implementation improvements in this tool over previous versions make computationally expensive tree searches with covarion models more efficient and analyses of large phylogenomic data sets tractable. PROCOV can be used to identify covarion sites by comparing the site likelihoods under the covarion process to the corresponding site likelihoods under a rates-across-sites (RAS process. Those sites with the greatest log-likelihood difference between a 'covarion' and an RAS process were found to be of functional or structural significance in a dataset of bacterial and eukaryotic elongation factors. Conclusion Covarion models implemented in PROCOV may be especially useful for phylogenetic estimation when ancient divergences between sequences have occurred and rates of evolution at sites are likely to have changed over the tree. It can also be used to study lineage-specific functional shifts in protein families that result in changes in the patterns of site variability among subtrees.

  17. Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

    KAUST Repository

    Reymer, A.

    2009-07-08

    To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleo-protein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.

  18. Site-specific analysis of gene expression in early osteoarthritis using the Pond-Nuki model in dogs

    Directory of Open Access Journals (Sweden)

    Kuroki Keiichi

    2006-10-01

    Full Text Available Abstract Background Osteoarthritis (OA is a progressive and debilitating disease that often develops from a focal lesion and may take years to clinically manifest to a complete loss of joint structure and function. Currently, there is not a cure for OA, but early diagnosis and initiation of treatment may dramatically improve the prognosis and quality of life for affected individuals. This study was designed to determine the feasibility of analyzing changes in gene expression of articular cartilage using the Pond-Nuki model two weeks after ACL-transection in dogs, and to characterize the changes observed at this time point. Methods The ACL of four dogs was completely transected arthroscopically, and the contralateral limb was used as the non-operated control. After two weeks the dogs were euthanatized and tissues harvested from the tibial plateau and femoral condyles of both limbs. Two dogs were used for histologic analysis and Mankin scoring. From the other two dogs the surface of the femoral condyle and tibial plateau were divided into four regions each, and tissues were harvested from each region for biochemical (GAG and HP and gene expression analysis. Significant changes in gene expression were determined using REST-XL, and Mann-Whitney rank sum test was used to analyze biochemical data. Significance was set at (p Results Significant differences were not observed between ACL-X and control limbs for Mankin scores or GAG and HP tissue content. Further, damage to the tissue was not observed grossly by India ink staining. However, significant changes in gene expression were observed between ACL-X and control tissues from each region analyzed, and indicate that a unique regional gene expression profile for impending ACL-X induced joint pathology may be identified in future studies. Conclusion The data obtained from this study lend credence to the research approach and model for the characterization of OA, and the identification and validation of

  19. Statistical and Economic Techniques for Site-specific Nematode Management.

    Science.gov (United States)

    Liu, Zheng; Griffin, Terry; Kirkpatrick, Terrence L

    2014-03-01

    Recent advances in precision agriculture technologies and spatial statistics allow realistic, site-specific estimation of nematode damage to field crops and provide a platform for the site-specific delivery of nematicides within individual fields. This paper reviews the spatial statistical techniques that model correlations among neighboring observations and develop a spatial economic analysis to determine the potential of site-specific nematicide application. The spatial econometric methodology applied in the context of site-specific crop yield response contributes to closing the gap between data analysis and realistic site-specific nematicide recommendations and helps to provide a practical method of site-specifically controlling nematodes.

  20. Validation of individual-based Markov-like stochastic process model of insect behaviour and a ‘virtual farm’ concept for enhancement of site-specific IPM

    Directory of Open Access Journals (Sweden)

    Slawomir Antoni Lux

    2016-08-01

    Full Text Available The paper reports application of a Markov-like stochastic process agent-based model and a ‘virtual farm’ concept for enhancement of site-specific Integrated Pest Management. Conceptually, the model represents a ‘bottom-up ethological’ approach and emulates behaviour of the ‘primary IPM actors’ - large cohorts of individual insects - within seasonally changing mosaics of spatiotemporally complex faming landscape, under the challenge of the local IPM actions. Algorithms of the proprietary PESTonFARM model were adjusted to reflect behaviour and ecology of R. cerasi. Model parametrization was based on compiled published information about R. cerasi and the results of auxiliary on-farm experiments. The experiments were conducted on sweet cherry farms located in Austria, Germany and Belgium. For each farm, a customised model-module was prepared, reflecting its spatiotemporal features. Historical data about pest monitoring, IPM treatments and fruit infestation were used to specify the model assumptions and calibrate it further. Finally, for each of the farms, virtual IPM experiments were simulated and the model-generated results were compared with the results of the real experiments conducted on the same farms. Implications of the findings for broader applicability of the model and the ‘virtual farm’ approach - were discussed.

  1. Validation of Individual-Based Markov-Like Stochastic Process Model of Insect Behavior and a "Virtual Farm" Concept for Enhancement of Site-Specific IPM.

    Science.gov (United States)

    Lux, Slawomir A; Wnuk, Andrzej; Vogt, Heidrun; Belien, Tim; Spornberger, Andreas; Studnicki, Marcin

    2016-01-01

    The paper reports application of a Markov-like stochastic process agent-based model and a "virtual farm" concept for enhancement of site-specific Integrated Pest Management. Conceptually, the model represents a "bottom-up ethological" approach and emulates behavior of the "primary IPM actors"-large cohorts of individual insects-within seasonally changing mosaics of spatiotemporally complex faming landscape, under the challenge of the local IPM actions. Algorithms of the proprietary PESTonFARM model were adjusted to reflect behavior and ecology of R. cerasi. Model parametrization was based on compiled published information about R. cerasi and the results of auxiliary on-farm experiments. The experiments were conducted on sweet cherry farms located in Austria, Germany, and Belgium. For each farm, a customized model-module was prepared, reflecting its spatiotemporal features. Historical data about pest monitoring, IPM treatments and fruit infestation were used to specify the model assumptions and calibrate it further. Finally, for each of the farms, virtual IPM experiments were simulated and the model-generated results were compared with the results of the real experiments conducted on the same farms. Implications of the findings for broader applicability of the model and the "virtual farm" approach-were discussed.

  2. Quantification of alterations in cortical bone geometry using Site Specificity Software in mouse models of aging and the responses to ovariectomy and altered loading

    Directory of Open Access Journals (Sweden)

    Gabriel L Galea

    2015-04-01

    Full Text Available Investigations into the effect of (remodelling stimuli on cortical bone in rodents normally rely on analysis of changes in bone mass and architecture at a narrow cross-sectional site. However, it is well established that the effects of axial loading produce site-specific changes throughout bones’ structure. Non-mechanical influences (e.g. hormones can be additional to or oppose locally-controlled adaptive responses and may have more generalized effects. Tools currently available to study site-specific cortical bone adaptation are limited. Here we applied novel Site-Specificity software to measure bone mass and architecture at each 1% site along the length of the mouse tibia from standard micro-computed tomography (μCT images. Resulting measures are directly comparable to those obtained through μCT analysis (R2 > 0.96. Site-Specificity Analysis was used to compare a number of parameters in tibiae from young adult (19-week-old versus aged (19-month-old mice; ovariectomized and entire mice; limbs subjected to short periods of axial loading or disuse induced by sciatic neurectomy. Age was associated with uniformly reduced cortical thickness and site-specific decreases in cortical area most apparent in the proximal tibia. Mechanical loading site-specifically increased cortical area and thickness in the proximal tibia. Disuse uniformly decreased cortical thickness and decreased cortical area in the proximal tibia. Ovariectomy uniformly reduced cortical area without altering cortical thickness. Differences in polar moment of inertia between experimental groups were only observed in the proximal tibia. Ageing and ovariectomy also altered eccentricity in the distal tibia. In summary, Site-Specificity Analysis provides a valuable tool for measuring changes in cortical bone mass and architecture along the entire length of a bone. Changes in the (remodelling response determined at a single site may not reflect the response at different locations within

  3. Evaluation of the Biotic Ligand Model relative to other site-specific criteria derivation methods for copper in surface waters with elevated hardness.

    Science.gov (United States)

    Van Genderen, Eric; Gensemer, Robert; Smith, Carrie; Santore, Robert; Ryan, Adam

    2007-08-30

    The goal of this study was to evaluate the reliability of the Biotic Ligand Model to predict Cu toxicity in very hard surface water (>200 mg/L as CaCO(3)), relative to current copper criteria methodologies (hardness-based equation and the water-effect ratio; WER). To test these methods, we conducted acute Cu toxicity tests with three aquatic test species (Ceriodaphnia dubia, Daphnia pulex and Pimephales promelas) in seven surface waters. The sites were representative of effluent-dependent or effluent-dominated streams common to the arid western United States of America (arid West) and a wide range of water quality variables were tested. In addition, concurrent Cu toxicity tests were conducted in laboratory waters that were matched to hardness and alkalinity of the sites to facilitate calculation of WER values. Results were used to characterize empirical relationships between water quality characteristics and Cu toxicity, and to compare measured Cu toxicity with Biotic Ligand Model (BLM) predictions. Acute toxicity tests were also conducted with C. dubia and P. promelas in a range of Ca or Mg-dominated hardness concentrations to determine the independent effects of Ca or Mg on Cu toxicity at high hardness levels. Conclusions from this study suggest that the BLM generates appropriate criteria for the waters tested in this study when compared to the hardness-based equation or WER approach. Although the historical site-specific methods are useful for surface waters with hardness alkalinity, Ca, Mg and Na). Therefore, the BLM offers an improved alternative to the hardness-based and WER approaches, particularly for situations where the current methods would be under-protective of sensitive aquatic life.

  4. Development of a Site-Specific Kinetic Model for Chlorine Decay and the Formation of Chlorination By-Products in Seawater

    Directory of Open Access Journals (Sweden)

    Suhur Saeed

    2015-07-01

    Full Text Available Chlorine is used commonly to prevent biofouling in cooling water systems. The addition of chlorine poses environmental risks in natural systems due to its tendency to form chlorination by-products (CBPs when exposed to naturally-occurring organic matter (NOM. Some of these CBPs can pose toxic risks to aquatic and benthic species in the receiving waters. It is, therefore, important to study the fate of residual chlorine and CBPs to fully understand the potential impacts of chlorination to the environment. The goal of this study was to develop improved predictions of how chlorine and CBP concentrations in seawater vary with time, chlorine dose and temperature. In the present study, chlorination of once-through cooling water at Ras Laffan Industrial City (RLIC, Qatar, was studied by collecting unchlorinated seawater from the RLIC cooling water system intake, treating it with chlorine and measuring time series of chlorine and CBP concentrations. Multiple-rate exponential curves were used to represent fast and slow chlorine decay and CBP formation, and site-specific chlorine kinetic relationships were developed. Through extensive analysis of laboratory measurements, it was found that only some of the control parameters identified in the literature were important for predicting residual chlorine and CBP concentrations for this specific location. The new kinetic relationships were able to significantly improve the predictability and validity of Generalized Environmental Modeling System for Surfacewaters (GEMSS-chlorine kinetics module (CKM, a three-dimensional hydrodynamic and chlorine kinetics and transport model when applied for RLIC outfall studies using actual field measurements.

  5. SU-E-T-760: Tolerance Design for Site-Specific Range in Proton Patient QA Process Using the Six Sigma Model

    Energy Technology Data Exchange (ETDEWEB)

    Lah, J [Myongji Hospital, Goyang, Gyeonggi-do (Korea, Republic of); Shin, D [National Cancer Center, Goyang-si, Gyeonggi-do (Korea, Republic of); Kim, G [University of California, San Diego, La Jolla, CA (United States)

    2015-06-15

    Purpose: To show how tolerance design and tolerancing approaches can be used to predict and improve the site-specific range in patient QA process in implementing the Six Sigma. Methods: In this study, patient QA plans were selected according to 6 site-treatment groups: head &neck (94 cases), spine (76 cases), lung (89 cases), liver (53 cases), pancreas (55 cases), and prostate (121 cases), treated between 2007 and 2013. We evaluated a model of the Six Sigma that determines allowable deviations in design parameters and process variables in patient-specific QA, where possible, tolerance may be loosened, then customized if it necessary to meet the functional requirements. A Six Sigma problem-solving methodology is known as DMAIC phases, which are used stand for: Define a problem or improvement opportunity, Measure process performance, Analyze the process to determine the root causes of poor performance, Improve the process by fixing root causes, Control the improved process to hold the gains. Results: The process capability for patient-specific range QA is 0.65 with only ±1 mm of tolerance criteria. Our results suggested the tolerance level of ±2–3 mm for prostate and liver cases and ±5 mm for lung cases. We found that customized tolerance between calculated and measured range reduce that patient QA plan failure and almost all sites had failure rates less than 1%. The average QA time also improved from 2 hr to less than 1 hr for all including planning and converting process, depth-dose measurement and evaluation. Conclusion: The objective of tolerance design is to achieve optimization beyond that obtained through QA process improvement and statistical analysis function detailing to implement a Six Sigma capable design.

  6. Site-specific functionalization for chemical speciation of Cr(III) and Cr(VI) using polyaniline impregnated nanocellulose composite: equilibrium, kinetic, and thermodynamic modeling

    Science.gov (United States)

    Jain, Priyanka; Varshney, Shilpa; Srivastava, Shalini

    2017-07-01

    Site-specific functionalizations are the emergent attention for the enhancement of sorption latent of heavy metals. Limited chemistry has been applied for the fabrication of diafunctionalized materials having potential to tether both environmentally stable oxidation states of chromium (Cr(III) and Cr(VI). Polyaniline impregnated nanocellulose composite (PANI-NCC) has been fabricated using click chemistry and explored for the removal of Cr(III) and Cr(VI) from hydrological environment. The structure, stability, morphology, particle size, surface area, hydrophilicity, and porosity of fabricated PANI-NCC were characterized comprehensively using analytical techniques and mathematical tools. The maximum sorption performance of PANI-NCC was procured for (Cr(III): 47.06 mg g-1; 94.12 %) and (Cr(VI): 48.92 mg g-1; 97.84 %) by equilibrating 0.5 g sorbent dose with 1000 mL of 25 mg L-1 chromium conc. at pH 6.5 and 2.5 for Cr(III) and Cr(VI), respectively. The sorption data showed a best fit to the Langmuir isotherm and pseudo-second-order kinetic model. The negative value of ∆ G° (-8.59 and -11.16 kJ mol-1) and ∆ H° (66.46 × 10-1 and 17.84 × 10-1 kJ mol-1), and positive value of ∆ S° (26.66 and 31.46 J mol-1K-1) for Cr(III) and Cr(VI), respectively, reflect the spontaneous, feasibility, and exothermic nature of the sorption process. The application of fabricated PANI-NCC for removing both the forms of chromium in the presence of other heavy metals was also tested at laboratory and industrial waste water regime. These findings open up new avenues in the row of high performance, scalable, and economic nanobiomaterial for the remediation of both forms of chromium from water streams.

  7. Site-specific weed control technologies

    DEFF Research Database (Denmark)

    Christensen, S; Søgaard, H T; Kudsk, P

    2009-01-01

    Site-specific weed control technologies are defined as machinery or equipment embedded with technologies that detect weeds growing in a crop and, taking into account predefined factors such as economics, takes action to maximise the chances of successfully controlling them. In the article, we...... describe the basic parts of site specific weed control technologies, comprising of weed sensing systems, weed management models and precision weed control implements. A review of state-of-the-art technologies shows that several weed sensing systems and precision implements have been developed over the last...... two decades, though barriers prevent their breakthrough. Most important among these is the lack of a truly robust weed recognition method, owing to mutual shading among plants and limitations in the capacity of highly accurate spraying and weeding apparatuses.   Another barrier is the lack...

  8. Nanoparticles for Site Specific Genome Editing

    Science.gov (United States)

    McNeer, Nicole Ali

    translate gene therapies for diseases of the blood and immune system to clinical practice. In addition, we have expanded the use of this technology to an additional nonhematopoietic model system: correction of the human cystic fibrosis transmembrane receptor gene in human bronchial epithelial cells. The work presented here represents (1) the first use of biodegradable nanoparticles for PNA delivery, (2) the first direct in vivo site-specific genome modification in human cells, and (3) the first use of triplex-PNA technology for site-specific genome editing in cystic fibrosis.

  9. A Mouse Model That Reproduces the Developmental Pathways and Site Specificity of the Cancers Associated With the Human BRCA1 Mutation Carrier State.

    Science.gov (United States)

    Liu, Ying; Yen, Hai-Yun; Austria, Theresa; Pettersson, Jonas; Peti-Peterdi, Janos; Maxson, Robert; Widschwendter, Martin; Dubeau, Louis

    2015-10-01

    Predisposition to breast and extrauterine Müllerian carcinomas in BRCA1 mutation carriers is due to a combination of cell-autonomous consequences of BRCA1 inactivation on cell cycle homeostasis superimposed on cell-nonautonomous hormonal factors magnified by the effects of BRCA1 mutations on hormonal changes associated with the menstrual cycle. We used the Müllerian inhibiting substance type 2 receptor (Mis2r) promoter and a truncated form of the Follicle stimulating hormone receptor (Fshr) promoter to introduce conditional knockouts of Brca1 and p53 not only in mouse mammary and Müllerian epithelia, but also in organs that control the estrous cycle. Sixty percent of the double mutant mice developed invasive Müllerian and mammary carcinomas. Mice carrying heterozygous mutations in Brca1 and p53 also developed invasive tumors, albeit at a lesser (30%) rate, in which the wild type alleles were no longer present due to loss of heterozygosity. While mice carrying heterozygous mutations in both genes developed mammary tumors, none of the mice carrying only a heterozygous p53 mutation developed such tumors (P < 0.0001), attesting to a role for Brca1 mutations in tumor development. This mouse model is attractive to investigate cell-nonautonomous mechanisms associated with cancer predisposition in BRCA1 mutation carriers and to investigate the merit of chemo-preventive drugs targeting such mechanisms.

  10. A Mouse Model That Reproduces the Developmental Pathways and Site Specificity of the Cancers Associated With the Human BRCA1 Mutation Carrier State

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2015-10-01

    Full Text Available Predisposition to breast and extrauterine Müllerian carcinomas in BRCA1 mutation carriers is due to a combination of cell-autonomous consequences of BRCA1 inactivation on cell cycle homeostasis superimposed on cell-nonautonomous hormonal factors magnified by the effects of BRCA1 mutations on hormonal changes associated with the menstrual cycle. We used the Müllerian inhibiting substance type 2 receptor (Mis2r promoter and a truncated form of the Follicle stimulating hormone receptor (Fshr promoter to introduce conditional knockouts of Brca1 and p53 not only in mouse mammary and Müllerian epithelia, but also in organs that control the estrous cycle. Sixty percent of the double mutant mice developed invasive Müllerian and mammary carcinomas. Mice carrying heterozygous mutations in Brca1 and p53 also developed invasive tumors, albeit at a lesser (30% rate, in which the wild type alleles were no longer present due to loss of heterozygosity. While mice carrying heterozygous mutations in both genes developed mammary tumors, none of the mice carrying only a heterozygous p53 mutation developed such tumors (P < 0.0001, attesting to a role for Brca1 mutations in tumor development. This mouse model is attractive to investigate cell-nonautonomous mechanisms associated with cancer predisposition in BRCA1 mutation carriers and to investigate the merit of chemo-preventive drugs targeting such mechanisms.

  11. Habitat Suitability Index Models: Eastern wild turkey

    Science.gov (United States)

    Schroeder, Richard L.

    1985-01-01

    A review and synthesis of existing information were used to develop a Habitat Suitability Index (HSI) model for the eastern wild turkey (Meleagris gallopavo sylvestris). The model consolidates habitat use information into a framework appropriate for field application, and is scaled to produce an index between 0.0 (unsuitable habitat) to 1.0 (optimum habitat). HSI models are designed to be used with Habitat Evaluation Procedures previously developed by the U.S. Fish and Wildlife Service.

  12. An Evaluation of Site-specific and Generalized Spatial Models of Aboveground Forest Biomass Based on Landsat Time-series and LiDAR Strip Samples in the Eastern U.S.A.

    Science.gov (United States)

    Deo, R. K.; Domke, G. M.; Russell, M.; Andersen, H. E.; Cohen, W. B.

    2016-12-01

    Aboveground forest biomass (AGB) contributes a large part of the terrestrial carbon and varies with forest types. While forest disturbance and land use changes significantly influence greenhouse gas (GHG) emissions, international conventions on climate change mitigation have identified forest conservation and management as an efficient mechanism for enhancing carbon storage in live plant biomass to offset the emissions. Regional or national scale assessment of AGB on forest lands facilitates strategic plans such as the national GHG inventory per the requirement of the United Nations Framework Convention on Climate Change (UNFCCC). Large area inventory of AGB can be efficiently carried out by using remotely sensed data and a generalized statistical model in contrast to using only field measurements and site-specific models. In this study, we have integrated specialized forest inventory plot data with spatial predictors derived from time-series Landsat imagery and LiDAR strip samples in four sites across the eastern U.S.A.- Minnesota (MN), Maine (ME), Pennsylvania-New Jersey (PANJ) and South Carolina (SC) - to formulate statistical models. Pixel-level polynomial curve fit was applied to the time-series Landsat variables to obtain projected metrics in the target year 2014. Two forms of models based on ordinary least-squares regression (OLR) and the random forest (RF) algorithm were developed for each site and with the pooled (generalized) dataset. The site-specific models were tested with the national forest inventory (NFI) data as well as specialized plot data of the other sites while the generalized models were tested with the NFI data only. We observed similar level of accuracies with the OLR and RF models at the same site. Although the RF models yielded larger values for the amount of explained variance (i.e., pseudo R2) in the site-specific models, the generalized model was also promising. The amount of variance explained with the site-site specific models were

  13. Stability of transmembrane amyloid β-peptide and membrane integrity tested by molecular modeling of site-specific Aβ42 mutations.

    Directory of Open Access Journals (Sweden)

    Chetan Poojari

    Full Text Available Interactions of the amyloid β-protein (Aβ with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer's disease. Natural mutations in Aβ42, such as the Arctic mutation (E22G have been shown to increase Aβ42 aggregation and neurotoxicity, leading to the early-onset of Alzheimer's disease. A correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ42 peptide it was further revealed that the aggregation of different Aβ42 mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ42 mutation and peptide-membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ42 variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ42 stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ42 peptide and a decreased stability for D23G compared to wild-type Aβ42, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.

  14. Response to Nuclear Regulatory Commission`s ten questions pertaining to site-specific models for use in the license termination rule: Multimedia Environmental Pollutant Assessment System (MEPAS)

    Energy Technology Data Exchange (ETDEWEB)

    Buck, J.W.; Whelan, G.; Strenge, D.L.; Hoopes, B.L.; McDonald, J.P.; Castleton, K.J.; Pelton, M.A.; Gelston, G.M.; Taira, R.Y. [Pacific Northwest National Lab., Richland, WA (United States)

    1998-05-01

    This paper is in response to the US Nuclear Regulatory Commission (NRC) ten questions posed at the Modeling Workshop held November 13 and 14, 1997. The ten questions were developed in advance of the workshop to allow model developers to prepare a presentation at the Workshop. This paper is an expanded version of the Multimedia Environmental Pollutant Assessment System (MEPAS) presentation given at the Modeling Workshop by Pacific Northwest National Laboratory (PNNL) staff. This paper is organized by the ten questions asked by the NRC, each section devoted to a single question. The current version of methodology is MEPAS 3.2 (NRC 1997) and the discussion in this paper will pertain to that version. In some cases, MEPAS 4.0, which is currently being developed under the Framework for Risk Analysis in Multimedia Environmental Systems (FRAMES) (Whelan et al. 1997), will be referenced to inform the reader of potential capabilities in the near future. A separate paper is included in the document that discusses the FRAMES concept.

  15. Site-Specific, Climate-Friendly Farming

    Science.gov (United States)

    Brown, D. J.; Brooks, E. S.; Eitel, J.; Huggins, D. R.; Painter, K.; Rupp, R.; Smith, J. L.; Stockle, C.; Vierling, L. A.

    2011-12-01

    Of the four most important atmospheric greenhouse gasses (GHG) enriched through human activities, only nitrous oxide (N2O) emissions are due primarily to agriculture. However, reductions in the application of synthetic N fertilizers could have significant negative consequences for a growing world population given the crucial role that these fertilizers have played in cereal yield increases since WWII. Increasing N use efficiency (NUE) through precision management of agricultural N in space and time will therefore play a central role in the reduction of agricultural N2O emissions. Precision N management requires a greater understanding of the spatio-temporal variability of factors supporting N management decisions such as crop yield, water and N availability, utilization and losses. We present an overview of a large, collaborative, multi-disciplinary project designed to improve our basic understanding of nitrogen (N), carbon (C) and water (H2O) spatio-temporal dynamics for wheat-based cropping systems on complex landscapes, and develop management tools to optimize water- and nitrogen-use efficiency for these systems and landscapes. Major components of this project include: (a) cropping systems experiments addressing nitrogen application rate and seeding density for different landscape positions; (b) GHG flux experiments and monitoring; (c) soil microbial genetics and stable isotope analyses to elucidate biochemical pathways for N2O production; (d) proximal soil sensing for construction of detailed soil maps; (e) LiDAR and optical remote sensing for crop growth monitoring; (f) hydrologic experiments, monitoring, and modeling; (g) refining the CropSyst simulation model to estimate biophysical processes and GHG emissions under a variety of management and climatic scenarios; and (h) linking farm-scale enterprise budgets to simulation modeling in order to provide growers with economically viable site-specific climate-friendly farming guidance.

  16. Neural network-based crop growth model to predict processing tomato yield on a site-specific basis using remotely sensed data

    Science.gov (United States)

    Koller, Michal

    Remote sensing is one of the major data acquisition tools available to rapidly acquire soil and plant related information over a wide area for use in precision agriculture. Green canopy has very specific reflectance characteristics distinguishing it from other materials such as soil and dry vegetative matter. Reflectance values in red (R) and near infra-red (NIR) spectral bands have been widely used for calculating normalized difference vegetation index (NDVI). Many researchers have related NDVI values to plant vigor, water stress, leaf area index (LAI) and/or yield. However, vegetative indices such as NDVI are usually sensitive to background reflectance characteristics. Often soil adjusted vegetation indices (SAVI) are used to minimize the background effect. In this study we have developed a relationship between the processing tomato yield and SAVI based on the R and NIR reflectance. Eight three band (R, NIR and green) aerial images were obtained at approximately two-week intervals during the 2000 processing tomato growing season. These images were analyzed to obtain SAVI values which were in turn related to LAI using regression techniques. A tuned neural network was developed to predict daily LAI values based on the biweekly experimental LAI values derived from aerial images. The coefficients of multiple determination between the actual LAI and neural network predicted LAI values were greater than 0.96 for all 56 grid points. The LAI values were numerically integrated over the whole growing season to obtain cumulative leaf area index days (CLAID). The CLAID values predicted from the neural network correlated very well with experimentally derived CLAID values (coefficient of determination, r2 = 0.83) indicating that the neural network model simulated processing tomato growth well. A crop growth model that was capable of predicting crop yield based on neural network predicted LAI values and CIMIS weather data was developed. Although predicted yield tended to be low

  17. Site-specificity in small architectural interventions

    DEFF Research Database (Denmark)

    Marie Bruun Jespersen, Line

    2016-01-01

    This paper is investigating the concepts of site-specificity, place and experience as they have been interpreted in a number of projects with at strong emphasis on the characteristics of the site, in the form of small architectural or artistic interventions. The idea of site-specificity is re......-emerging in contemporary architectural discourse in a series of projects, in e.g. the Norwegian project ”National Tourist Roads”. The small architectural interventions consequently work with enhancing or framing the qualities of site and aim at being a mediating element between man and nature. In this paper two main...... specificity in the small architectural interventions. The paper concludes that the small architectural interventions operate with a narrow concept of site, and that the large variety of artistic strategies and types of site-specificity found in contemporary art could serve as inspiration to open up...

  18. Preparation of metallochelating microbubbles and study on their site-specific interaction with rGFP-HisTag as a model protein.

    Science.gov (United States)

    Lukáč, Róbert; Kauerová, Zuzana; Mašek, Josef; Bartheldyová, Eliška; Kulich, Pavel; Koudelka, Štěpán; Korvasová, Zina; Plocková, Jana; Papoušek, František; Kolář, František; Schmidt, Roland; Turánek, Jaroslav

    2011-04-19

    The histidine-metallochelating lipid complex is one of the smallest high affinity binding units used as tools for rapid noncovalent binding of histidine tagged molecules, especially recombinant proteins. The advantage of metallochelating complex over protein-ligand complexes (e.g., streptavidine-biotin, glutathiontransferase-glutathion) consists in its very low immunogenicity, if any. This concept for the construction of surface-modified metallochelating microbubbles was proved with recombinant green fluorescent protein (rGFP) containing 6His-tag. This protein is easy to be detected by various fluorescence techniques as flow cytometry and confocal microscopy. Microbubbles (MB) composed of DPPC with various contents of metallochelating lipid DOGS-NTA-Ni were prepared by intensive shaking of the liposome suspension under the atmosphere of sulfur hexafluoride. For this purpose, the instrument 3M ESPE CapMix was used. Various techniques (static light scattering, flow cytometry, and optical microscopy) were compared and used for the measurements of the size distribution of MB. All three methods demonstrated that the prepared MB were homogeneous in their size, and the mean diameter of the MB in various batches was within the range of 2.1-2.8 μm (the size range of 1-10 μm). The presence of large MB (8-10 μm) was marginal. Counting of MB revealed that the average amount of MB prepared of 10 mg of phospholipid equaled approximately 10(9) MB/mL. Lyophilized MB were prepared with saccharose as a cryoprotectant. These MB were shown to be stable both in vitro (the estimated half-live of the MB in bovine serum at 37 °C was 3-7 min) and in vivo (mouse). The stability of the MB was affected by molar content of DOGS-NTA-Ni. DPPC-based metallochelating MB provided a clear and very contrast image of the ventricular cavity soon after the injection. Site selective and stable binding of rGFP-HisTag (as a model of His-tagged protein) onto the surface of metallochelating MB was

  19. Prospects for site specific weed management

    DEFF Research Database (Denmark)

    Christensen, Svend; Rasmussen, Jesper; Pedersen, Søren Marcus;

    2014-01-01

    Research on Site Specific Weed Management (SSWM) started in the late 80's. Since that moment, considerable research has been conducted on different aspects of SSWM, from fundamental studies on the spatial ecology of weeds to the applied development and testing of new technologies for weed detection...

  20. Site-Specific PEGylation of Therapeutic Proteins

    Directory of Open Access Journals (Sweden)

    Jonathan K. Dozier

    2015-10-01

    Full Text Available The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.

  1. Site-specific dissociation dynamics of H{sub 2}/D{sub 2} on Ag(111) and Co(0001) and the validity of the site-averaging model

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Xixi [Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131 (United States); Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China); Jiang, Bin [Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131 (United States); Department of Chemical Physics, University of Science and Technology of China, Hefei 230026 (China); Xie, Daiqian, E-mail: dqxie@nju.edu.cn, E-mail: hguo@unm.edu [Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China); Synergetic Innovation Center of Quantum Information and Quantum Physics, University of Science and Technology of China, Hefei, Anhui 230026 (China); Guo, Hua, E-mail: dqxie@nju.edu.cn, E-mail: hguo@unm.edu [Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131 (United States)

    2015-09-21

    Dissociative chemisorption of polyatomic molecules on metal surfaces involves high-dimensional dynamics, of which quantum mechanical treatments are computationally challenging. A promising reduced-dimensional approach approximates the full-dimensional dynamics by a weighted average of fixed-site results. To examine the performance of this site-averaging model, we investigate two distinct reactions, namely, hydrogen dissociation on Co(0001) and Ag(111), using accurate first principles potential energy surfaces (PESs). The former has a very low barrier of ∼0.05 eV while the latter is highly activated with a barrier of ∼1.15 eV. These two systems allow the investigation of not only site-specific dynamical behaviors but also the validity of the site-averaging model. It is found that the reactivity is not only controlled by the barrier height but also by the topography of the PES. Moreover, the agreement between the site-averaged and full-dimensional results is much better on Ag(111), though quantitative in neither system. Further quasi-classical trajectory calculations showed that the deviations can be attributed to dynamical steering effects, which are present in both reactions at all energies.

  2. RESRAD. Site-Specific Residual Radioactivity

    Energy Technology Data Exchange (ETDEWEB)

    Yu, C. [Argonne National Laboratory, IL (United States)

    1989-06-01

    RESRAD is designed to derive site-specific guidelines for allowable residual concentrations of radionuclides in soil. A guideline is defined as a radionuclide concentration or a level of radiation or radioactivity that is acceptable if a site is to be used without radiological restrictions. Guidelines are expressed as (1) concentrations of residual radionuclides in soil, (2) concentrations of airborne radon decay products, (3) levels of external gamma radiation, (4) levels of radioactivity from surface contamination, and (5) concentrations of residual radionuclides in air and water. Soil is defined as unconsolidated earth material, including rubble and debris that may be present. The controlling principles of all guidelines are (1) the annual radiation dose received by a member of the critical population group from the residual radioactive material - predicted by a realistic but reasonably conservative analysis and averaged over a 50 year period - should not exceed 100 mrem/yr, and (2) doses should be kept as low as reasonably achievable. All significant exposure pathways for the critical population group are considered in deriving soil guidelines. These pathways include direct exposure to external radiation from the contaminated soil material; internal radiation from inhalation of airborne radionuclides; and internal radiation from ingestion of plant foods grown in the contaminated soil, meat and milk from livestock fed with contaminated fodder and water, drinking water from a contaminated well, and fish from a contaminated pond.

  3. Optimization under Uncertainty of Site-Specific Turbine Configurations: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Quick, Julian; Dykes, Katherine; Graf, Peter; Zahle, Frederik

    2016-11-01

    Uncertainty affects many aspects of wind energy plant performance and cost. In this study, we explore opportunities for site-specific turbine configuration optimization that accounts for uncertainty in the wind resource. As a demonstration, a simple empirical model for wind plant cost of energy is used in an optimization under uncertainty to examine how different risk appetites affect the optimal selection of a turbine configuration for sites of different wind resource profiles. If there is unusually high uncertainty in the site wind resource, the optimal turbine configuration diverges from the deterministic case and a generally more conservative design is obtained with increasing risk aversion on the part of the designer.

  4. Experimental investigations and geochemical modelling of site-specific fluid-fluid and fluid-rock interactions in underground storage of CO2/H2/CH4 mixtures: the H2STORE project

    Science.gov (United States)

    De Lucia, Marco; Pilz, Peter

    2015-04-01

    work packages hosted at the German Research Centre for Geosciences (GFZ) focus on the fluid-fluid and fluid-rock interactions triggered by CO2, H2 and their mixtures. Laboratory experiments expose core samples to hydrogen and CO2/hydrogen mixtures under site-specific conditions (temperatures up to 200 °C and pressure up to 300 bar). The resulting qualitative and, whereas possible, quantitative data are expected to ameliorate the precision of predictive geochemical and reactive transport modelling, which is also performed within the project. The combination of experiments, chemical and mineralogical analyses and models is needed to improve the knowledge about: (1) solubility model and mixing rule for multicomponent gas mixtures in high saline formation fluids: no data are namely available in literature for H2-charged gas mixtures in the conditions expected in the potential sites; (2) chemical reactivity of different mineral assemblages and formation fluids in a broad spectrum of P-T conditions and composition of the stored gas mixtures; (3) thermodynamics and kinetics of relevant reactions involving mineral dissolution or precipitation. The resulting amelioration of site characterization and the overall enhancement in understanding the potential processes will benefit the operational reliability, the ecological tolerance, and the economic efficiency of future energy storing plants, crucial aspects for public acceptance and for industrial investors.

  5. Modelo de respuesta sitio-específica del maíz al nitrógeno y agua edáfica en un haplustol Site-specific corn nitrogen and soil water response model in haplustolls

    Directory of Open Access Journals (Sweden)

    María Celeste Gregoret

    2006-12-01

    potential were identified: rainfed high and low potential and high potential with irrigation. The data are from on-farm trials for the 2004-2005 crop season, in Manfredi (Córdoba with a uniform N rate along strips (0, 32, 64, 96, 129 and 161 kg N ha-¹ in the dry land and 0, 64, 137, 212, 279 and 351 kg N ha-¹ in the irrigated area and a randomized complete block design to estimate site-specific crop response functions. Spatial autocorrelation is taken into account in regression estimation of N response functions by landscape position, in the form a spatial autoregressive error structure. Results suggest that N response differs significantly by landscape position and by soil water availability. Yields ranged from 4.914 to 11,346 kg ha-¹ in the zone of low productivity, between 4,956 and 12,204 kg ha-¹ in the one of high productivity and between 7,830 and 14,387 kg ha-¹ in irrigated plots. The agronomical (DOA and economically (DOE optimal N rates also differed among management zones, being DOA= 285, 184 and 162 kg ha-1 for the zones from greater to smaller productivity, meanwhile DOE values were 171, 111 and 90 kg ha-1, respectively. Initial soil nitrogen and available water inclusion allowed us to build a single N response model for the rainfed management zones. Besides, irrigated experiment maize performance was well predicted by the same model type but excluding available water (non-limiting factor, as input. The model performed well using either available N at 60 or 200 cm soil depth, and available water al 200 cm depth. This model allowed us to recommend fertilization rates using variable rate application, maximizing net return to N, and minimizing environment contamination risks, by over dose input (i.e. N fertilization application. Our conclusions are preliminary because they come from a single year experiment, which did not include the whole range of expectated initial conditions. More scenarios are needed in order to validate the proposed model.

  6. Temporally-controlled site-specific recombination in zebrafish.

    Directory of Open Access Journals (Sweden)

    Stefan Hans

    Full Text Available Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER(T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM or its active metabolite, 4-hydroxy-tamoxifen (4-OHT. Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

  7. Temporally-controlled site-specific recombination in zebrafish.

    Science.gov (United States)

    Hans, Stefan; Kaslin, Jan; Freudenreich, Dorian; Brand, Michael

    2009-01-01

    Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER(T2). Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

  8. Appreciating Site-Specific Qualities in Urban Harbours

    DEFF Research Database (Denmark)

    Reeh, Henrik

    2015-01-01

    When “site-specificity” becomes a central value in city and harbor transformation, it soon proves necessary to address the ways in which scholars and professionals actually determine site-specific qualities in urban fabrics and social life. This paper delves into the above questions by means...... of site-specificity, even in the traditional harbor settings. Considered with conceptual care, such situations may teach us what it means to “appreciate site-specific qualities”....

  9. Site-Specific Genome Engineering in Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Sylvia Merkert

    2016-06-01

    Full Text Available The possibility to generate patient-specific induced pluripotent stem cells (iPSCs offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

  10. Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

    Science.gov (United States)

    Merkert, Sylvia; Martin, Ulrich

    2016-06-24

    The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

  11. Site-specific crop management using geophysical proximal sensors

    Science.gov (United States)

    Key components of site-specific crop management are (i) identifying the site-specific factors that influence within-field crop yield variation and (ii) spatially characterizing those factors. Geo-referenced measurements of apparent soil electrical conductivity (ECa) provide a potential means of cha...

  12. Innovation and Diffusion of Site-specific Crop Management

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus; Pedersen, Jørgen Lindgaard

    2006-01-01

    Site-specific crop management or precision farming is a highly complex managementsystem for site-specific input application of lime, fertilizers and pesticides in arable farming. The Global Positioning System (GPS)is the backbone of the system. To conduct precision farming several technical systems...

  13. The Application of TAPM for Site Specific Wind Energy Forecasting

    Directory of Open Access Journals (Sweden)

    Merlinde Kay

    2016-02-01

    Full Text Available The energy industry uses weather forecasts for determining future electricity demand variations due to the impact of weather, e.g., temperature and precipitation. However, as a greater component of electricity generation comes from intermittent renewable sources such as wind and solar, weather forecasting techniques need to now also focus on predicting renewable energy supply, which means adapting our prediction models to these site specific resources. This work assesses the performance of The Air Pollution Model (TAPM, and demonstrates that significant improvements can be made to only wind speed forecasts from a mesoscale Numerical Weather Prediction (NWP model. For this study, a wind farm site situated in North-west Tasmania, Australia was investigated. I present an analysis of the accuracy of hourly NWP and bias corrected wind speed forecasts over 12 months spanning 2005. This extensive time frame allows an in-depth analysis of various wind speed regimes of importance for wind-farm operation, as well as extreme weather risk scenarios. A further correction is made to the basic bias correction to improve the forecast accuracy further, that makes use of real-time wind-turbine data and a smoothing function to correct for timing-related issues. With full correction applied, a reduction in the error in the magnitude of the wind speed by as much as 50% for “hour ahead” forecasts specific to the wind-farm site has been obtained.

  14. Site-Specific Chemical Labeling of Long RNA Molecules

    DEFF Research Database (Denmark)

    Jahn, Kasper; Olsen, Eva Maria; Nielsen, Morten Muhlig

    2011-01-01

    Site-specific labeling of RNA molecules is a valuable tool for studying their structure and function. Here, we describe a new site-specific RNA labeling method, which utilizes a DNA-templated chemical reaction to attach a label at a specific internal nucleotide in an RNA molecule. The method...... is nonenzymatic and based on the formation of a four-way junction, where a donor strand is chemically coupled to an acceptor strand at a specific position via an activated chemical group. A disulfide bond in the linker is subsequently cleaved under mild conditions leaving a thiol group attached to the acceptor-RNA...... strand. The site-specific thiol-modified target RNA can then be chemically labeled with an optional group, here demonstrated by coupling of a maleimide-functionalized fluorophore. The method is rapid and allows site specific labeling of both in vitro and in vivo synthesized RNA with a broad range...

  15. Changing communities: documentary photography and site-specific installation

    OpenAIRE

    Voller, James

    2017-01-01

    This exegesis documents my Master of Fine practice-led research project exploring photography's intersection with site specificity. Through generating a dialogue about architectural changes in Melbourne's north and western suburbs, the project engages with ideas surrounding community engagement and engagement with photographic, site specific and archival practices. Beginning with the research question - How can documentary photography exist in local communities as site­ specific installation ...

  16. Simple mathematical models for interacting wild and transgenic mosquito populations.

    Science.gov (United States)

    Li, Jia

    2004-05-01

    Two discrete-time models for interacting populations of wild and genetically altered mosquito are presented, where the genetically altered mosquitoes are grouped into a single population without distinguishing their zygosity. The birth and death rates for both populations are density-dependent, and the mating rates between the mosquitoes are assumed to be either constant or proportional to the total populations for the two models, respectively. The existence and stability of the boundary and positive equilibria are investigated. In particular, it is shown that bifurcations from both boundary and positive equilibria can appear for the model with proportional mating rates. Stable equilibria, periodic-doubling bifurcations, aperiodic oscillations, and chaotic behavior are all illustrated by numerical simulations.

  17. Differential equations models for interacting wild and transgenic mosquito populations.

    Science.gov (United States)

    Li, Jia

    2008-07-01

    We formulate and study continuous-time models, based on systems of ordinary differential equations, for interacting wild and transgenic mosquito populations. We assume that the mosquito mating rate is either constant, proportional to total mosquito population size, or has a Holling-II-type functional form. The focus is on the model with the Holling-II-type functional mating rate that incorporates Allee effects, in order to account for mating difficulty when the size of the total mosquito populations is small. We investigate the existence and stability of both boundary and positive equilibria. We show that the Holling-II-type model is the more realistic and, by means of numerical simulations, that it exhibits richer dynamics.

  18. Site-specific criteria for the completion of landfill aftercare.

    Science.gov (United States)

    Laner, David; Fellner, Johann; Brunner, Paul H

    2012-09-01

    Municipal solid waste (MSW) landfills need to be managed after closure to assure long-term environmental compatibility. Aftercare can be completed when the authorities consider the landfill not likely to pose a threat to humans and the environment. In this work, a methodology for deriving site-specific aftercare completion criteria is presented and its application is illustrated via a case study. The evaluation method combines models addressing waste emission behavior, long-term barrier performance, and pollutant migration to assess the potential impact of landfill emissions on the environment. Based on the definition of acceptable impact levels at certain points of compliance, scenario- and pollutant-specific aftercare completion criteria are derived. The methodology was applied to a closed MSW landfill in Austria and potential aftercare durations were determined. While landfill gas emissions may become environmentally tolerable within decades at the site, leachate-related aftercare measures were expected to be necessary for centuries (primarily as a result of ammonium). Although the evaluation comes with large uncertainties, it allows for linking aftercare intensity and duration with respect to an environmentally compatible state of the landfill in the absence of aftercare. However, further case studies including regulatory review and acceptance are needed to use the methodology in a decision support tool on aftercare completion.

  19. Multispectral videography for site-specific farm management

    Science.gov (United States)

    Anderson, Gerald L.; Yang, C.

    1996-11-01

    Researchers are expending considerable effort to develop the technology and methodology needed to identify and map within-field management zones for site-specific farming. Much of the research has focused on the use of either a high-density geographically referenced grid of soil samples or mechanical yield sensor measurements that record geographic positions and production levels. In either case, complex spatial models are generally used to extrapolate the various soil variables and production level information across the entire field. Both procedures produce a wealth of information, however, the analysis of soil samples tend to be quite expensive and the accuracy of mechanical yield measurements does vary. This study represents an ongoing effort designed to evaluate remote sensing as a tool for determining within field management zones. Color-infrared aerial photography and multispectral videography were used in concert to map and stratify two grain sorghum fields into regions or zones of homogeneous spectral response. A limited number of soil and plant samples were acquired to characterize the biotic and edaphic conditions within each zone. Results obtained during the first year of the study indicated that multispectral video can be used to develop within field management zones. Simple univariate analysis indicated that soil pH, Ca, and Fe were important variables affecting yield. Analysis of the yield data indicated that the economic returns from 17% of the first field and 20% of the second field were insufficient to recoup planting costs. Multispectral video also proved instrumental in modeling the spatial variability of yield. A significant negative correlation (r2 greater than 0.90) was obtained between the red spectral band and crop yields for both fields. Stratification, in this case using image data, reduces the number of samples required to characterize a field by reducing the variance associated within each stratum. Image data also provided a

  20. Modelling the spread of innovation in wild birds.

    Science.gov (United States)

    Shultz, Thomas R; Montrey, Marcel; Aplin, Lucy M

    2017-06-01

    We apply three plausible algorithms in agent-based computer simulations to recent experiments on social learning in wild birds. Although some of the phenomena are simulated by all three learning algorithms, several manifestations of social conformity bias are simulated by only the approximate majority (AM) algorithm, which has roots in chemistry, molecular biology and theoretical computer science. The simulations generate testable predictions and provide several explanatory insights into the diffusion of innovation through a population. The AM algorithm's success raises the possibility of its usefulness in studying group dynamics more generally, in several different scientific domains. Our differential-equation model matches simulation results and provides mathematical insights into the dynamics of these algorithms. © 2017 The Author(s).

  1. Site specific protein labeling by enzymatic posttranslational modification.

    Science.gov (United States)

    Sunbul, Murat; Yin, Jun

    2009-09-07

    Site specific protein labeling plays a key role in elucidating the function of the proteins at the molecular level by revealing their locations in the cell, their interaction networks with other cellular components and the dynamic mechanisms of their bio-generation, trafficking and degradation in response to regulatory signals in a biological system. Site specific protein labeling is, in essence, artificial modification of proteins with new chemical entities at the posttranslational stage. Based on the analogy between protein labeling and protein posttranslational modification, enzymatic tools have been developed for site specific and efficient labeling of target proteins with chemical probes of diverse structures and functionalities. This perspective surveys a number of protein labeling methods based on the application of protein posttranslational modification enzymes.

  2. Innovation and Diffusion of Site-specific Crop Management

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus; Pedersen, Jørgen Lindgaard

    2006-01-01

    Site-specific crop management or precision farming is a highly complex managementsystem for site-specific input application of lime, fertilizers and pesticides in arable farming. The Global Positioning System (GPS)is the backbone of the system. To conduct precision farming several technical systems...... and data interpretations are needed. Precision farming is aiming to be used by large farms because of scale advantages. In reality adoption and benefits harvested are limited. We have analysed information from 28 experts and stakeholders, focus group meetings with 35 participants and a multinational farm...

  3. Innovation and diffusion of site-specific crop management

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus; Pedersen, Jørgen Lindgaard

    2004-01-01

    Site-specific crop management or precision farming (PF) is a highly complex management system for site-specific input application of lime, fertilizers and pesticides in arable farming. The Global Positioning System (GPS) is the backbone of the system. To conduct PF several technical systems...... and data interpretations are needed. FP is aiming to be used by large farms because of scale advantages. In reality adoption and benefits harvested are limited. We have analysed information from 28 experts and stakeholders and focus group meetings with 35 participants and a multinational farm survey...

  4. Oxygen as a site specific structural probe in neutron diffraction

    Energy Technology Data Exchange (ETDEWEB)

    Neuefeind, Joerg C [ORNL; Simonson, J Michael {Mike} [ORNL; Salmon, Phil [University of Bath; Zeidler, Anita [University of Bath; Fischer, Henry E [Institut Laue-Langevin (ILL); Rauch, Helmut [E141 Atominstitut der & #xD6; sterreichischen Universit& #xE4; ten,; Markland, Thomas [Columbia University; Lemmel, Hartmut [Technical University Vienna

    2011-01-01

    Oxygen is a ubiquitous element, playing an essential role in most scientific and technological disciplines, and is often incorporated within a structurally disordered material where examples include molten silicates in planetary science, glasses used for lasers and optical communication, and water in biological processes. Establishing the structure of a liquid or glassy oxide and thereby its relation to the functional properties of a material is not, however, a trivial task owing to the complexity associated with atomic disorder. Here we approach this challenge by measuring the bound coherent neutron scattering lengths of the oxygen isotopes with the sensitive technique of neutron interferometry. We find that there is a small but finite contrast of 0.204(6) fm between the scattering lengths of the isotope 18O and oxygen of natural isotopic abundance natO, contrary to tables of recommended values. This has enabled us to investigate the structure of both light and heavy water by exploiting, for the first time, the method of oxygen isotope substitution in neutron diffraction, thus circumventing many of the significant problems associated with more traditional methods in which hydrogen is substituted by deuterium. We find a difference of ~0.5% between the O-H and O-D intra-molecular bond distances which is much smaller than recent estimates based on diffraction data and is found to be in excellent agreement with path integral molecular dynamics simulations made with a flexible polarisable water model. Our results demonstrate the potential for using oxygen isotope substitution as a powerful and effective site specific probe in a plethora of materials, of pertinence as instrumentation at next generation neutron sources comes online

  5. Determination of site-specific glycan heterogeneity on glycoproteins

    DEFF Research Database (Denmark)

    Kolarich, Daniel; Jensen, Pia Hønnerup; Altmann, Friedrich

    2012-01-01

    and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide...

  6. Site-Specific Dance: Promoting Social Awareness in Choreography

    Science.gov (United States)

    MacBean, Arianne

    2004-01-01

    Site-specific dance, which is often defined as dance that occurs outside of the conventional theater space, challenges choreographers to look at, listen to, feel, and think about the space in which the dance is performed. It also asks audiences to be active participants in the performance experience. The dances have to be informed by the space and…

  7. Using site-specific soil samples as a substitution for improved hydrological and nonpoint source predictions.

    Science.gov (United States)

    Chen, Lei; Wang, Guobo; Zhong, Yucen; Zhao, Xin; Shen, Zhenyao

    2016-08-01

    Soil databases are one of the most important inputs for watershed models, and the quality of soil properties affects how well a model performs. The objectives of this study were to (1) quantify the sensitivity of model outputs to soil properties and to (2) use site-specific soil properties as a substitution for more accurate hydrological and nonpoint source (H/NPS) predictions. Soil samples were collected from a typical mountainous watershed in China, and the impacts of soil sample parameters on H/NPS predictions were quantified using the Soil and Water Assessment Tool (SWAT). The most sensitive parameters related to predicting flow, sediment, and total phosphorus (TP) mainly were the soil hydrological, the channel erosion processes, and the initial soil chemical environment, respectively. When the site-specific soil properties were used, the uncertainties (coefficient of variation) related to predicting the hydrology, sediment and TP decreased by 75∼80 %, 75∼84 %, and 46∼61 %, respectively. Based on changes in the Nash-Sutcliff coefficient, the model performance improved by 4.9 and 19.45 % for the hydrological and sediment model, accordingly. However, site-specific soil properties did not contribute to better TP predictions because of the high spatial variability of the soil P concentrations across the large watershed. Thus, although site-specific soil samples can be used to obtain more accurate H/NPS predictions, more sampling sites are required to apply this method in large watersheds.

  8. Development of site-specific locking plates for acetabular fractures.

    Science.gov (United States)

    Xu, Meng; Zhang, Li-Hai; Zhang, Ying-Ze; He, Chun-Qing; Zhang, Li-Cheng; Wang, Yan; Tang, Pei-Fu

    2013-05-01

    Site-specific locking plates have gained popularity for the treatment of fractures. However, the clinical use of a site-specific locking plate for acetabular fractures remains untested due to production limits. To design a universal site-specific locking plate for acetabular fractures, the 3-dimensional (3D) photographic records of 171 pelvises were retrospectively studied to generate a universal posterior innominate bone surface. Using 3D photographical processing software, the 3D coordinate system was reset according to bony landmarks and was scaled based on the acetabular diameter to allow a direct comparison between surfaces. The measured surface was separated into measurement units. At each measurement unit, the authors calculated the average z-axis values in all samples and obtained the 3D coordinate values of the point cloud that could be reconstructed into the universal surface. A plate was subsequently designed in 3D photographical processing software, and the orientation and distribution of locking screws was included. To manufacture a plate, the data were entered into Unigraphics NX version 6.0 software (Siemens PLM Software, Co, Ltd, Plano, Texas) and a CNC digital milling machine (FANUC Co, Ltd, Yamanashi, Japan). The resulting locking plate fit excellently with the reduced bone surface intraoperatively. Plate contouring was avoided intraoperatively. Universal 3.5-mm locking screws locked successfully into the plate, and their orientations were consistent with the design. No screw yielded to acetabular penetration. This method of designing a site-specific acetabular locking plate is practical, and the plates are suitable for clinical use. These site-specific locking plates may be an option for the treatment of acetabular fractures, particularly in elderly patients.

  9. Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

    Science.gov (United States)

    Taylor, John-Stephen

    2015-12-01

    How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

  10. Site-specific design optimization of wind turbines

    DEFF Research Database (Denmark)

    Fuglsang, P.; Bak, C.; Schepers, J.G.

    2002-01-01

    This article reports results from a European project, where site characteristics were incorporated into the design process of wind turbines, to enable site-specific design. Two wind turbines of different concept were investigated at six different sites comprising normal flat terrain, offshore...... of a new wind turbine. Both annual energy yield and design-determining loads depended on site characteristics, and this represented a potential for site-specific design. The maximum variation in annual energy yield was 37% and the maximum variation in blade root fatigue loads was 62%. Optimized site...... advantage of high-wind-speed sites. It was not possible to design a single wind turbine for all wind climates investigated, since the differences in the design loads were too large. Multiple-site wind turbines should be designed for generic wind conditions, which cover wind parameters encountered at flat...

  11. SITE-94. Site specific base data for the performance assessment

    Energy Technology Data Exchange (ETDEWEB)

    Geier, J. [ed.] [Clearwater Hardrock Consulting, Monmouth, OR (United States); Tiren, S. [Geosigma AB, Uppsala (Sweden); Dverstorp, B. [Swedish Nuclear Power Inspectorate, Stockholm (Sweden); Glynn, P. [U.S. Geological Survey, Reston, VA (United States)

    1996-06-01

    This report documents the site specific base data that were available, and the utilization of these data within SITE-94. A brief summary is given of SKB`s preliminary site investigations for the Aespoe Hard Rock Laboratory (HRL), which were the main source of site-specific data for SITE-94, and an overview is given of the field methods and instrumentation for the preliminary investigations. A compilation is given of comments concerning the availability and quality of the data for Aespoe, and specific recommendations are given for future site investigations. It was found that the HRL pre-investigations produced a large quantity of data which were, for the most part, of sufficient quality to be valuable for a performance assessment. However, some problems were encountered regarding documentation, procedural consistency, positional information, and storage of the data from the measurements. 77 refs, 4 tabs.

  12. Site-Specific Biomolecule Labeling with Gold Clusters

    OpenAIRE

    Ackerson, Christopher J.; Powell, Richard D.; Hainfeld, James F.

    2010-01-01

    Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (...

  13. Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

    Science.gov (United States)

    Cook, Brendon E; Adumeau, Pierre; Membreno, Rosemery; Carnazza, Kathryn E; Brand, Christian; Reiner, Thomas; Agnew, Brian J; Lewis, Jason S; Zeglis, Brian M

    2016-08-17

    In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate.

  14. Site-Specific ecological risk assessment. Case-study 2

    DEFF Research Database (Denmark)

    Jensen, John

    “Development of a decision support system for sustainable management of contaminated land by linking bioavailability, ecological risk and ground water pollution of organic pollutants”or in short “LIBERATION”. The presentation includes examples on how to scale and integrate the results from various scientific......The decision supporting and integrating assessment tool, TRIAD, is used site-specific on PAH- and heavy metal contaminated sites in Denmark. The various aspects of the TRIAD approach are used on a set of chemistry-, ecotoxicology- and ecology related data collected among others in the EU project...

  15. Site Specific Probable Maximum Precipitation Estimates and Professional Judgement

    Science.gov (United States)

    Hayes, B. D.; Kao, S. C.; Kanney, J. F.; Quinlan, K. R.; DeNeale, S. T.

    2015-12-01

    State and federal regulatory authorities currently rely upon the US National Weather Service Hydrometeorological Reports (HMRs) to determine probable maximum precipitation (PMP) estimates (i.e., rainfall depths and durations) for estimating flooding hazards for relatively broad regions in the US. PMP estimates for the contributing watersheds upstream of vulnerable facilities are used to estimate riverine flooding hazards while site-specific estimates for small water sheds are appropriate for individual facilities such as nuclear power plants. The HMRs are often criticized due to their limitations on basin size, questionable applicability in regions affected by orographic effects, their lack of consist methods, and generally by their age. HMR-51 for generalized PMP estimates for the United States east of the 105th meridian, was published in 1978 and is sometimes perceived as overly conservative. The US Nuclear Regulatory Commission (NRC), is currently reviewing several flood hazard evaluation reports that rely on site specific PMP estimates that have been commercially developed. As such, NRC has recently investigated key areas of expert judgement via a generic audit and one in-depth site specific review as they relate to identifying and quantifying actual and potential storm moisture sources, determining storm transposition limits, and adjusting available moisture during storm transposition. Though much of the approach reviewed was considered a logical extension of HMRs, two key points of expert judgement stood out for further in-depth review. The first relates primarily to small storms and the use of a heuristic for storm representative dew point adjustment developed for the Electric Power Research Institute by North American Weather Consultants in 1993 in order to harmonize historic storms for which only 12 hour dew point data was available with more recent storms in a single database. The second issue relates to the use of climatological averages for spatially

  16. Multiplexed site-specific electrode functionalization for multitarget biosensors.

    Science.gov (United States)

    Levrie, Karen; Jans, Karolien; Vos, Rita; Ardakanian, Niyousha; Verellen, Niels; Van Hoof, Chris; Lagae, Liesbet; Stakenborg, Tim

    2016-12-01

    Multitarget biosensors hold great promise to improve point-of-care diagnostics as they enable simultaneous detection of different biomolecular markers. Multiplexed detection of different markers, like genes, proteins, or a combination of both, propels advancement in numerous fields such as genomics, medical diagnosis and therapy monitoring. The functionalization of these biosensors, however, necessitates patterned immobilization of different bioreceptors, which remains challenging and time-consuming. We demonstrate a simple method for the patterned multiplexing of bioreceptors on a multi-electrode chip. By using the lithographically defined electrodes for surface functionalization, additional patterning steps become obsolete. Using the electrodes for self-aligned immobilization provides a spatial resolution that is limited by the electrode patterning process and that cannot be easily obtained by alternative dispensing or coating techniques. Via electrochemical reduction of diazonium salts combined with click chemistry, we achieved site-specific immobilization of two different ssDNA probes side by side on a single chip. This method was experimentally verified by cyclic voltammetry (CV), Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS), and specific target recognition was visualized by fluorescence microscopy. The combination of the electroaddressability of electrografting with the chemoselectivity of click chemistry, offers a versatile platform for highly efficient site-specific functionalization of multitarget biosensors.

  17. Site specific modification of the human plasma proteome by methylglyoxal.

    Science.gov (United States)

    Kimzey, Michael J; Kinsky, Owen R; Yassine, Hussein N; Tsaprailis, George; Stump, Craig S; Monks, Terrence J; Lau, Serrine S

    2015-12-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC-MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan-HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients.

  18. Towards soft robotic devices for site-specific drug delivery.

    Science.gov (United States)

    Alici, Gursel

    2015-01-01

    Considerable research efforts have recently been dedicated to the establishment of various drug delivery systems (DDS) that are mechanical/physical, chemical and biological/molecular DDS. In this paper, we report on the recent advances in site-specific drug delivery (site-specific, controlled, targeted or smart drug delivery are terms used interchangeably in the literature, to mean to transport a drug or a therapeutic agent to a desired location within the body and release it as desired with negligibly small toxicity and side effect compared to classical drug administration means such as peroral, parenteral, transmucosal, topical and inhalation) based on mechanical/physical systems consisting of implantable and robotic drug delivery systems. While we specifically focus on the robotic or autonomous DDS, which can be reprogrammable and provide multiple doses of a drug at a required time and rate, we briefly cover the implanted DDS, which are well-developed relative to the robotic DDS, to highlight the design and performance requirements, and investigate issues associated with the robotic DDS. Critical research issues associated with both DDSs are presented to describe the research challenges ahead of us in order to establish soft robotic devices for clinical and biomedical applications.

  19. Editing livestock genomes with site-specific nucleases.

    Science.gov (United States)

    Carlson, Daniel F; Tan, Wenfang; Hackett, Perry B; Fahrenkrug, Scott C

    2013-01-01

    Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10(-9). Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene's encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%-50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

  20. Site Specific Modification of the Human Plasma Proteome by Methylglyoxal

    Science.gov (United States)

    Kimzey, Michael J.; Kinsky, Owen R.; Yassine, Hussein N.; Tsaprailis, George; Stump, Craig; Monks, Terrence J.; Lau, Serrine S.

    2015-01-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC-MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan-HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients. PMID:26435215

  1. Site-specific tumor-targeted fluorescent contrast agents

    Science.gov (United States)

    Achilefu, Samuel I.; Bugaj, Joseph E.; Dorshow, Richard B.; Jimenez, Hermo N.; Rajagopalan, Raghavan; Wilhelm, R. Randy; Webb, Elizabeth G.; Erion, Jack L.

    2001-01-01

    Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effect of drugs, and enhances the contrast between normal and diseased tissues. In optical medicine, biocompatible dyes can be used as photo therapeutics or as contrast agents. Previous studies have shown that the use of covalent or non-covalent dye conjugates of carries such as antibodies, liposomes, and polysaccharides improves the delivery of such molecules to tumors. However, large biomolecules can elicit adverse immunogenic reactions and also result in prolonged blood circulation times, delaying visualization of target tissues. A viable alternative to this strategy is to use small bioactive molecule-dye conjugates. These molecules have several advantages over large biomolecules, including ease of synthesis of a variety of high purity compounds for combinatorial screening of new targets, enhanced diffusivity to solid tumors, and the ability to affect the pharmocokinetics of the conjugates by minor structural changes. Thus, we conjugated a near IR light absorbing dye to bioactive peptides that specifically target over expressed tumor receptors in established rat tumor lines. High tumor uptake of the conjugates was obtained without loss of either the peptide receptor affinity or the dye fluorescence. These findings demonstrate the efficacy of a small peptide-dye conjugate strategy for in vivo tumor imaging. Site-specific delivery of photodynamic therapy agents may also benefit form this approach.

  2. Proposed Site-Specific Response Spectra for Surabaya-Madura Bridge

    Directory of Open Access Journals (Sweden)

    Dyah Kusumastuti

    2008-01-01

    Full Text Available This paper presents a site-specific seismic hazard study to determine the recommended seismic design criteria for Suramadu Bridge. The study is performed using probabilistic seismic hazard approach to determine maximum acceleration and response spectra at bedrock and followed by local site effect analysis to determine maximum acceleration and response spectra at ground surface. The probabilistic seismic hazard analysis (PSHA is carried out using 3-dimension (3-D seismic source models (fault source model. Two hazard levels are analysed to represent 150 and 3,300 years return period of ground motion around site location. The local site effect analysis is performed using 1-dimension (1-D shear wave propagation theory to obtain peak ground acceleration and response spectra at ground surface. Finally, the site-specific surface response spectra with 5 percent damping are developed based on the mean plus one standard deviation concept from the result of local site effect analysis.

  3. Evolution of I-SceI Homing Endonucleases with Increased DNA Recognition Site Specificity

    Energy Technology Data Exchange (ETDEWEB)

    Joshi, Rakesh; Ho, Kwok Ki; Tenney, Kristen; Chen, Jui-Hui; Golden, Barbara L.; Gimble, Frederick S. (UIUC); (Purdue)

    2013-09-18

    Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G{sub +4} base pair for the wild-type A:T{sub +4} base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T{sub +4} were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T{sub +4} or the C:G{sub +4} base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G{sub +4} recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T{sub +4} target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G{sub +4} target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed -36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G{sub +4} substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.

  4. Impact of smoking and excess body weight on overall and site-specific cancer mortality risk

    OpenAIRE

    Meyer, Julia; Rohrmann, Sabine; Bopp, Matthias; Faeh, David

    2015-01-01

    Background: Smoking and excess body weight are major preventable risk factors for premature death. This study aimed at analyzing their single and combined association with site-specific cancer mortality. Methods: Our study population comprised 35,784 men and women of ages 14 to 99 years, who participated in population-based health surveys conducted 1977–1993 in Switzerland and were followed up for mortality until 2008. Multivariable Cox proportional hazards models were calculated for diffe...

  5. Site-specific gene expression patterns in oral cancer.

    Science.gov (United States)

    Frohwitter, Gesche; Buerger, Horst; Korsching, Eberhard; van Diest, Paul J; Kleinheinz, Johannes; Fillies, Thomas

    2017-05-10

    Squamous cell carcinomas (SCCs) are the most prevalent malignant tumours within the head and neck. Evidence exists that distinct genes are differentially regulated in SCCs of the oral cavity compared to other head and neck regions. Given this background, the aim of this study was to investigate whether such tumour site-specific gene expression can also be observed in different localizations within the oral cavity. Using tissue microarrays (TMAs), we investigated 76 SCCs of the floor of the mouth, 49 SCCs of the tongue and 68 SCCs of other anatomic regions within the oral cavity. The expression of 17 genes involved in cell cycle and growth control (p16, p21, p27, p53, cyclin D1, EGFR, c-kit, bcl-6), cell adhesion (alpha-, beta-, and gamma-catenin), and apoptosis/stress response genes (Hif-1-alpha, Glut 1, CA IX, caspase, hsp70, XIAP) were investigated by means of immunohistochemistry. The data were subjected to chi(2), interdependency and Kaplan-Meier analysis. Our study suggests a remote difference in the site-specific gene expression patterns of oral cancer. X-linked inhibitor of apoptosis (XIAP) showed a significantly higher expression (p oral cavity. The increased XIAP expression was further associated with significantly decreased overall survival in all cases of SCCs of the oral cavity (p Expression levels of p53, CA IX, beta-catenin, Hif-1-alpha, and c-kit were also observed to be inversely related between SCCs of the floor of the mouth and those of the tongue respectively, although these differences did not reach statistical significance. Overall and event-free survival did not differ in patients with T1/T2/N0 SCCs according to tumour localization. In summary, the protein expression patterns of SCCs of the oral cavity suggest the existence of a molecular and morphological spectrum of SCCs in the oral cavity. In particular the expression pattern of XIAP indicates distinct gene expression patterns between carcinomas of the floor of the mouth and oral tongue

  6. Site-specific growth of polymers on silica rods.

    Science.gov (United States)

    Peng, Bo; Soligno, Giuseppe; Kamp, Marlous; de Nijs, Bart; de Graaf, Joost; Dijkstra, Marjolein; van Roij, René; van Blaaderen, Alfons; Imhof, Arnout

    2014-12-28

    Colloids specifically developed for self-assembly (SA) into advanced functional materials have rapidly become more complex, as this complexity allows for more ways to optimize both the SA process and the properties of the resulting materials. For instance, by creating 'patchy' particles more open structures can be achieved through directional interactions. However, the number of ways in which site-specific chemistry can be achieved on particle surfaces is still limited. Here, we show how polymer patches can be specifically grown onto only the flat end of bullet-shaped silica rods by utilizing a subtle anisotropy in surface tension and shape caused by the growth mechanism of the rods. Conversely, if the bullet-shaped silica rods are used as 'Pickering-emulsion' stabilizers the same surface tension effects exclusively direct the orientation of the rods into a 'hedgehog-morphology'. Finally, we demonstrate how an external electric field can direct the particles in a 'vectorial' way.

  7. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish

    Directory of Open Access Journals (Sweden)

    Atsuo Kawahara

    2016-05-01

    Full Text Available The zebrafish (Danio rerio is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs and the clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR associated protein 9 (Cas9 system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.

  8. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish.

    Science.gov (United States)

    Kawahara, Atsuo; Hisano, Yu; Ota, Satoshi; Taimatsu, Kiyohito

    2016-05-13

    The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.

  9. Site-specific estimates of water yield applied in regional acid sensitivity surveys across western Canada

    Directory of Open Access Journals (Sweden)

    Patrick D. SHAW

    2010-08-01

    Full Text Available Runoff or water yield is an important input to the Steady-State Water Chemistry (SSWC model for estimating critical loads of acidity. Herein, we present site-specific water yield estimates for a large number of lakes (779 across three provinces of western Canada (Manitoba, Saskatchewan, and British Columbia using an isotope mass balance (IMB approach. We explore the impact of applying site-specific hydrology as compared to use of regional runoff estimates derived from gridded datasets in assessing critical loads of acidity to these lakes. In general, the average water yield derived from IMB is similar to the long-term average runoff; however, IMB results suggest a much larger range in hydrological settings of the lakes, attributed to spatial heterogeneity in watershed characteristics and landcover. The comparison of critical loads estimates from the two methods suggests that use of average regional runoff data in the SSWC model may overestimate critical loads for the majority of lakes due to systematic skewness in the actual runoff distributions. Implications for use of site-specific hydrology in regional critical loads assessments across western Canada are discussed.

  10. GIS-based procedure for site-specific risk assessment of pesticides for aquatic ecosystems.

    Science.gov (United States)

    Sala, Serenella; Vighi, Marco

    2008-01-01

    The EU Water Framework Directive states that the management of surface water must be based on a site-specific assessment of water quality, that is dependent on land use. As a result, to develop a robust chemical management policy for aquatic ecosystems, the ecotoxicological risk must be strictly related to the local conditions and characteristics of the system. This paper presents a methodology developed to assess the ecotoxicological risk of pesticides to site-specific aquatic ecosystems. Spatial and relational databases, provisional models and risk indices were integrated into Geographical Information Systems (GIS) to produce maps of exposure, effect and risk at watershed scale. Each active ingredient is characterised by a data set that includes input data as well as results represented by a risk assessment cartography. The aim of this procedure is to perform a site-specific risk assessment by integrating geographical distribution of predicted environmental concentrations (PECs), ecotoxicological effects and the potential/actual quality of the exposed ecosystem. Examples of pesticide risk maps for surface waters in Lombardia Region (Northern Italy) are shown.

  11. Modeling the status, trends, and impacts of wild bee abundance in the United States.

    Science.gov (United States)

    Koh, Insu; Lonsdorf, Eric V; Williams, Neal M; Brittain, Claire; Isaacs, Rufus; Gibbs, Jason; Ricketts, Taylor H

    2016-01-05

    Wild bees are highly valuable pollinators. Along with managed honey bees, they provide a critical ecosystem service by ensuring stable pollination to agriculture and wild plant communities. Increasing concern about the welfare of both wild and managed pollinators, however, has prompted recent calls for national evaluation and action. Here, for the first time to our knowledge, we assess the status and trends of wild bees and their potential impacts on pollination services across the coterminous United States. We use a spatial habitat model, national land-cover data, and carefully quantified expert knowledge to estimate wild bee abundance and associated uncertainty. Between 2008 and 2013, modeled bee abundance declined across 23% of US land area. This decline was generally associated with conversion of natural habitats to row crops. We identify 139 counties where low bee abundances correspond to large areas of pollinator-dependent crops. These areas of mismatch between supply (wild bee abundance) and demand (cultivated area) for pollination comprise 39% of the pollinator-dependent crop area in the United States. Further, we find that the crops most highly dependent on pollinators tend to experience more severe mismatches between declining supply and increasing demand. These trends, should they continue, may increase costs for US farmers and may even destabilize crop production over time. National assessments such as this can help focus both scientific and political efforts to understand and sustain wild bees. As new information becomes available, repeated assessments can update findings, revise priorities, and track progress toward sustainable management of our nation's pollinators.

  12. DNA origami metallized site specifically to form electrically conductive nanowires.

    Science.gov (United States)

    Pearson, Anthony C; Liu, Jianfei; Pound, Elisabeth; Uprety, Bibek; Woolley, Adam T; Davis, Robert C; Harb, John N

    2012-09-06

    DNA origami is a promising tool for use as a template in the design and fabrication of nanoscale structures. The ability to engineer selected staple strands on a DNA origami structure provides a high density of addressable locations across the structure. Here we report a method using site-specific attachment of gold nanoparticles to modified staple strands and subsequent metallization to fabricate conductive wires from DNA origami templates. We have modified DNA origami structures by lengthening each staple strand in select regions with a 10-base nucleotide sequence and have attached DNA-modified gold nanoparticles to the lengthened staple strands via complementary base-pairing. The high density of extended staple strands allowed the gold nanoparticles to pack tightly in the modified regions of the DNA origami, where the measured median gap size between neighboring particles was 4.1 nm. Gold metallization processes were optimized so that the attached gold nanoparticles grew until gaps between particles were filled and uniform continuous nanowires were formed. Finally, electron beam lithography was used to pattern electrodes in order to measure the electrical conductivity of metallized DNA origami, which showed an average resistance of 2.4 kΩ per metallized structure.

  13. Multispectral remote sensing for site-specific nitrogen fertilizer management

    Directory of Open Access Journals (Sweden)

    Nikrooz Bagheri

    2013-10-01

    Full Text Available The objective of this work was to evaluate the use of multispectral remote sensing for site-specific nitrogen fertilizer management. Satellite imagery from the advanced spaceborne thermal emission and reflection radiometer (Aster was acquired in a 23 ha corn-planted area in Iran. For the collection of field samples, a total of 53 pixels were selected by systematic randomized sampling. The total nitrogen content in corn leaf tissues in these pixels was evaluated. To predict corn canopy nitrogen content, different vegetation indices, such as normalized difference vegetation index (NDVI, soil-adjusted vegetation index (Savi, optimized soil-adjusted vegetation index (Osavi, modified chlorophyll absorption ratio index 2 (MCARI2, and modified triangle vegetation index 2 (MTVI2, were investigated. The supervised classification technique using the spectral angle mapper classifier (SAM was performed to generate a nitrogen fertilization map. The MTVI2 presented the highest correlation (R²=0.87 and is a good predictor of corn canopy nitrogen content in the V13 stage, at 60 days after cultivating. Aster imagery can be used to predict nitrogen status in corn canopy. Classification results indicate three levels of required nitrogen per pixel: low (0-2.5 kg, medium (2.5-3 kg, and high (3-3.3 kg.

  14. Cre-inducible site-specific recombination in zebrafish oligodendrocytes.

    Science.gov (United States)

    Pinzon-Olejua, Alejandro; Welte, Cornelia; Chekuru, Avinash; Bosak, Viktoria; Brand, Michael; Hans, Stefan; Stuermer, Claudia A O

    2017-01-01

    The conditional Cre/lox system has recently emerged as a valuable tool for studies on both embryonic and adult Zebrafish. Temporal control and site-specific recombination are achieved by using the ligand-inducible CreER(T2) and administration of the drug tamoxifen (TAM) or its active metabolite, 4-Hydroxytamoxifen (4-OHT). Here we report the generation of a transgenic Zebrafish line, which expresses an mCherry-tagged variant of CreER(T2) under the control of the myelin basic protein a (mbpa) promoter. Our analysis shows that larval and adult expression of the transgene recapitulates the endogenous mbpa expression pattern in oligodendrocytes. Furthermore, combination with a Cre-dependent EGFP reporter results in EGFP-expressing oligodendrocytes in the spinal cord, brain, and optic nerve in TAM- or 4-OHT-treated larvae and 4-month-old fish, but not in untreated controls. The transgenic Zebrafish line Tg(mbpa:mCherry-T2A-CreER(T2) ) elicits CreER(T2) expression specifically in myelinating glia cells. Cre-inducible targeted recombination of genes in oligodendrocytes will be useful to elucidate cellular and molecular mechanisms of myelination in vivo during development (myelination) and regeneration (remyelination) after injury to the central nervous system (CNS). It will also allow targeted expression and overexpression of genes of interest (transgenes) in oligodendrocytes at defined developmental and adult stages. Developmental Dynamics 246:41-49, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Genetic fate mapping using site-specific recombinases.

    Science.gov (United States)

    Legué, Emilie; Joyner, Alexandra L

    2010-01-01

    Understanding how cells are assembled in three dimensions to generate an organ, or a whole organism, is a pivotal question in developmental biology. Similarly, it is critical to understand how adult stem cells integrate into an existing organ during regeneration or in response to injury. Key to discovering the answers to these questions is being able to study the various behaviors of distinct cell types during development or regeneration. Fate mapping techniques are fundamental to studying cell behaviors such as proliferation, movement, and lineage segregation, as the techniques allow precursor cells to be marked and their descendants followed and characterized over time. The generation of transgenic mice, combined with the use of site-specific recombinases (SSR) in the mouse genome, has provided a means to develop powerful genetic fate mapping approaches. A key advantage of genetic fate mapping is that it allows cells to be genetically marked, and therefore the mark is transmitted to all the descendants of the initially marked cells. By making modifications to the SSRs that render their enzymatic activity inducible, and the development of an assortment of reporter alleles for marking cells, increasingly sophisticated genetic fate mapping studies can be performed. In this chapter, we review the four main genetic fate mapping methods that utilize intrachromosomal recombination to mark cells (cumulative, inducible, clonal, and intersectional) and one interchromosomal method, the tools required to carry out each approach, and the practical considerations that have to be taken into account before embarking on each type of genetic fate mapping study.

  16. The logistic model-generated carrying capacities for wild herbivores ...

    African Journals Online (AJOL)

    Jesse

    Modelled as discrete-time logistic equations with fixed carrying capacities, it captures the wildlife herbivore population dynamics. Time series data, covering a period ..... Building Models for Conservation and. Wildlife Management. (New York ...

  17. Active orientation models for face alignment in-the-wild

    NARCIS (Netherlands)

    Tzimiropoulos, Georgios; Alabort-i-Medina, Joan; Zafeiriou, Stefanos; Pantic, Maja

    2014-01-01

    We present Active Orientation Models (AOMs), generative models of facial shape and appearance, which extend the well-known paradigm of Active Appearance Models (AAMs) for the case of generic face alignment under unconstrained conditions. Robustness stems from the fact that the proposed AOMs employ a

  18. Site-specific hydrogen diffusion rates during clinopyroxene dehydration

    Science.gov (United States)

    Ferriss, Elizabeth; Plank, Terry; Walker, David

    2016-06-01

    The rate of hydrogen diffusion in clinopyroxene is relevant to interpreting hydrogen ("water") concentrations in xenoliths, phenocrysts, and clinopyroxene-hosted melt inclusions to provide insight into the deep-earth water cycle and volcanic explosivity. Here, we determine bulk and site-specific hydrogen diffusivities in two diopsides and an augite by heating initially homogeneous water-bearing samples in a 1-atm CO/CO2 gas-mixing furnace at 800-1000 °C and oxygen fugacity at the quartz-fayalite-magnetite buffer and observing H-loss profiles. The O-H stretching range between wavenumbers 3000 and 4000 cm-1 in FTIR spectra is resolved into 4-6 peaks, each of which is assumed to represent a distinct defect site for the hydrogen, to determine peak-specific diffusivities using our previously published whole-block method. For the diopside from the Kunlun Mts. in China, Arrhenius relations are reported for peaks at 3645, 3617, 3540, 3443, and 3355 cm-1 based on measurements at 816, 904, and 1000 °C. Bulk and site-specific diffusivities are determined for the same set of peaks at 904 °C for the second diopside (Jaipur). The augite (PMR-53) was a triangular thin slab, and hydrogen diffusivities were determined for bulk hydrogen and peaks at 3620, 3550, 3460, and 3355 cm-1 in the thickness direction at 800 °C. Bulk hydrogen diffusivity in the Jaipur diopside is consistent with previous work, and hydrogen diffusivity in augite PMR-53 is slightly lower than the fast direction diffusivities measured || [100] and [001]* in Jaipur diopside. Both diopsides show 1-2 orders of magnitude differences in the peaks-specific diffusivities, with the fastest diffusivities at 3450 cm-1 and the slowest at 3645 cm-1. However, the hydrogen diffusivities in Jaipur diopside are 2-4 orders of magnitude higher than those in Kunlun diopside for bulk hydrogen and all peaks. Thus, peak-specific differences cannot by themselves adequately explain the 5 orders of magnitude range in hydrogen

  19. Xer Site Specific Recombination: Double and Single Recombinase Systems

    Science.gov (United States)

    Castillo, Fabio; Benmohamed, Amal; Szatmari, George

    2017-01-01

    The separation and segregation of newly replicated bacterial chromosomes can be constrained by the formation of circular chromosome dimers caused by crossing over during homologous recombination events. In Escherichia coli and most bacteria, dimers are resolved to monomers by site-specific recombination, a process performed by two Chromosomally Encoded tyrosine Recombinases (XerC and XerD). XerCD recombinases act at a 28 bp recombination site dif, which is located at the replication terminus region of the chromosome. The septal protein FtsK controls the initiation of the dimer resolution reaction, so that recombination occurs at the right time (immediately prior to cell division) and at the right place (cell division septum). XerCD and FtsK have been detected in nearly all sequenced eubacterial genomes including Proteobacteria, Archaea, and Firmicutes. However, in Streptococci and Lactococci, an alternative system has been found, composed of a single recombinase (XerS) genetically linked to an atypical 31 bp recombination site (difSL). A similar recombination system has also been found in 𝜀-proteobacteria such as Campylobacter and Helicobacter, where a single recombinase (XerH) acts at a resolution site called difH. Most Archaea contain a recombinase called XerA that acts on a highly conserved 28 bp sequence dif, which appears to act independently of FtsK. Additionally, several mobile elements have been found to exploit the dif/Xer system to integrate their genomes into the host chromosome in Vibrio cholerae, Neisseria gonorrhoeae, and Enterobacter cloacae. This review highlights the versatility of dif/Xer recombinase systems in prokaryotes and summarizes our current understanding of homologs of dif/Xer machineries. PMID:28373867

  20. Lifetime growth in wild meerkats: incorporating life history and environmental factors into a standard growth model.

    Science.gov (United States)

    English, Sinéad; Bateman, Andrew W; Clutton-Brock, Tim H

    2012-05-01

    Lifetime records of changes in individual size or mass in wild animals are scarce and, as such, few studies have attempted to model variation in these traits across the lifespan or to assess the factors that affect them. However, quantifying lifetime growth is essential for understanding trade-offs between growth and other life history parameters, such as reproductive performance or survival. Here, we used model selection based on information theory to measure changes in body mass over the lifespan of wild meerkats, and compared the relative fits of several standard growth models (monomolecular, von Bertalanffy, Gompertz, logistic and Richards). We found that meerkats exhibit monomolecular growth, with the best model incorporating separate growth rates before and after nutritional independence, as well as effects of season and total rainfall in the previous nine months. Our study demonstrates how simple growth curves may be improved by considering life history and environmental factors, which may be particularly relevant when quantifying growth patterns in wild populations.

  1. Site-Specific Analyses for Demonstrating Compliance with 10 CFR 61 Performance Objectives - 12179

    Energy Technology Data Exchange (ETDEWEB)

    Grossman, C.J.; Esh, D.W.; Yadav, P.; Carrera, A.G. [U.S. Nuclear Regulatory Commission, 11545 Rockville Pike, Rockville, MD 20852 (United States)

    2012-07-01

    The U.S. Nuclear Regulatory Commission (NRC) is proposing to amend its regulations at 10 CFR Part 61 to require low-level radioactive waste disposal facilities to conduct site-specific analyses to demonstrate compliance with the performance objectives in Subpart C. The amendments would require licensees to conduct site-specific analyses for protection of the public and inadvertent intruders as well as analyses for long-lived waste. The amendments would ensure protection of public health and safety, while providing flexibility to demonstrate compliance with the performance objectives, for current and potential future waste streams. NRC staff intends to submit proposed rule language and associated regulatory basis to the Commission for its approval in early 2012. The NRC staff also intends to develop associated guidance to accompany any proposed amendments. The guidance is intended to supplement existing low-level radioactive waste guidance on issues pertinent to conducting site-specific analyses to demonstrate compliance with the performance objectives. The guidance will facilitate implementation of the proposed amendments by licensees and assist competent regulatory authorities in reviewing the site-specific analyses. Specifically, the guidance provides staff recommendations on general considerations for the site-specific analyses, modeling issues for assessments to demonstrate compliance with the performance objectives including the performance assessment, intruder assessment, stability assessment, and analyses for long-lived waste. This paper describes the technical basis for changes to the rule language and the proposed guidance associated with implementation of the rule language. The NRC staff, per Commission direction, intends to propose amendments to 10 CFR Part 61 to require licensees to conduct site-specific analyses to demonstrate compliance with performance objectives for the protection of public health and the environment. The amendments would require a

  2. Analysis of site-specific N-glycan remodeling in the endoplasmic reticulum and the Golgi

    Science.gov (United States)

    Hang, Ivan; Lin, Chia-wei; Grant, Oliver C; Fleurkens, Susanna; Villiger, Thomas K; Soos, Miroslav; Morbidelli, Massimo; Woods, Robert J; Gauss, Robert; Aebi, Markus

    2015-01-01

    The hallmark of N-linked protein glycosylation is the generation of diverse glycan structures in the secretory pathway. Dynamic, non-template-driven processes of N-glycan remodeling in the endoplasmic reticulum and the Golgi provide the cellular setting for structural diversity. We applied newly developed mass spectrometry-based analytics to quantify site-specific N-glycan remodeling of the model protein Pdi1p expressed in insect cells. Molecular dynamics simulation, mutational analysis, kinetic studies of in vitro processing events and glycan flux analysis supported the defining role of the protein in N-glycan processing. PMID:26240167

  3. Highly efficient site-specific transgenesis in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Michael Iacovos P

    2012-12-01

    Full Text Available Abstract Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate

  4. Modeling and mapping the probability of occurrence of invasive wild pigs across the contiguous United States.

    Directory of Open Access Journals (Sweden)

    Meredith L McClure

    Full Text Available Wild pigs (Sus scrofa, also known as wild swine, feral pigs, or feral hogs, are one of the most widespread and successful invasive species around the world. Wild pigs have been linked to extensive and costly agricultural damage and present a serious threat to plant and animal communities due to their rooting behavior and omnivorous diet. We modeled the current distribution of wild pigs in the United States to better understand the physiological and ecological factors that may determine their invasive potential and to guide future study and eradication efforts. Using national-scale wild pig occurrence data reported between 1982 and 2012 by wildlife management professionals, we estimated the probability of wild pig occurrence across the United States using a logistic discrimination function and environmental covariates hypothesized to influence the distribution of the species. Our results suggest the distribution of wild pigs in the U.S. was most strongly limited by cold temperatures and availability of water, and that they were most likely to occur where potential home ranges had higher habitat heterogeneity, providing access to multiple key resources including water, forage, and cover. High probability of occurrence was also associated with frequent high temperatures, up to a high threshold. However, this pattern is driven by pigs' historic distribution in warm climates of the southern U.S. Further study of pigs' ability to persist in cold northern climates is needed to better understand whether low temperatures actually limit their distribution. Our model highlights areas at risk of invasion as those with habitat conditions similar to those found in pigs' current range that are also near current populations. This study provides a macro-scale approach to generalist species distribution modeling that is applicable to other generalist and invasive species.

  5. Modeling parasite dynamics on farmed salmon for precautionary conservation management of wild salmon.

    Directory of Open Access Journals (Sweden)

    Luke A Rogers

    Full Text Available Conservation management of wild fish may include fish health management in sympatric populations of domesticated fish in aquaculture. We developed a mathematical model for the population dynamics of parasitic sea lice (Lepeophtheirus salmonis on domesticated populations of Atlantic salmon (Salmo salar in the Broughton Archipelago region of British Columbia. The model was fit to a seven-year dataset of monthly sea louse counts on farms in the area to estimate population growth rates in relation to abiotic factors (temperature and salinity, local host density (measured as cohort surface area, and the use of a parasiticide, emamectin benzoate, on farms. We then used the model to evaluate management scenarios in relation to policy guidelines that seek to keep motile louse abundance below an average three per farmed salmon during the March-June juvenile wild Pacific salmon (Oncorhynchus spp. migration. Abiotic factors mediated the duration of effectiveness of parasiticide treatments, and results suggest treatment of farmed salmon conducted in January or early February minimized average louse abundance per farmed salmon during the juvenile wild salmon migration. Adapting the management of parasites on farmed salmon according to migrations of wild salmon may therefore provide a precautionary approach to conserving wild salmon populations in salmon farming regions.

  6. Wild Fire Emissions for the NOAA Operational HYSPLIT Smoke Model

    Science.gov (United States)

    Huang, H. C.; ONeill, S. M.; Ruminski, M.; Shafran, P.; McQueen, J.; DiMego, G.; Kondragunta, S.; Gorline, J.; Huang, J. P.; Stunder, B.; Stein, A. F.; Stajner, I.; Upadhayay, S.; Larkin, N. K.

    2015-12-01

    Particulate Matter (PM) generated from forest fires often lead to degraded visibility and unhealthy air quality in nearby and downstream areas. To provide near-real time PM information to the state and local agencies, the NOAA/National Weather Service (NWS) operational HYSPLIT (Hybrid Single Particle Lagrangian Integrated Trajectory Model) smoke modeling system (NWS/HYSPLIT smoke) provides the forecast of smoke concentration resulting from fire emissions driven by the NWS North American Model 12 km weather predictions. The NWS/HYSPLIT smoke incorporates the U.S. Forest Service BlueSky Smoke Modeling Framework (BlueSky) to provide smoke fire emissions along with the input fire locations from the NOAA National Environmental Satellite, Data, and Information Service (NESDIS)'s Hazard Mapping System fire and smoke detection system. Experienced analysts inspect satellite imagery from multiple sensors onboard geostationary and orbital satellites to identify the location, size and duration of smoke emissions for the model. NWS/HYSPLIT smoke is being updated to use a newer version of USFS BlueSky. The updated BlueSky incorporates the Fuel Characteristic Classification System version 2 (FCCS2) over the continental U.S. and Alaska. FCCS2 includes a more detailed description of fuel loadings with additional plant type categories. The updated BlueSky also utilizes an improved fuel consumption model and fire emission production system. For the period of August 2014 and June 2015, NWS/HYSPLIT smoke simulations show that fire smoke emissions with updated BlueSky are stronger than the current operational BlueSky in the Northwest U.S. For the same comparisons, weaker fire smoke emissions from the updated BlueSky were observed over the middle and eastern part of the U.S. A statistical evaluation of NWS/HYSPLIT smoke predicted total column concentration compared to NOAA NESDIS GOES EAST Aerosol Smoke Product retrievals is underway. Preliminary results show that using the newer version

  7. Synthesis of Site-Specific Radiolabeled Antibodies for Radioimmunotherapy via Genetic Code Expansion.

    Science.gov (United States)

    Wu, Yiming; Zhu, Hua; Zhang, Bo; Liu, Fei; Chen, Jingxian; Wang, Yufei; Wang, Yan; Zhang, Ziwei; Wu, Ling; Si, Longlong; Xu, Huan; Yao, Tianzhuo; Xiao, Sulong; Xia, Qing; Zhang, Lihe; Yang, Zhi; Zhou, Demin

    2016-10-19

    Radioimmunotherapy (RIT) delivers radioisotopes to antigen-expressing cells via monoantibodies for the imaging of lesions or medical therapy. The chelates are typically conjugated to the antibody through cysteine or lysine residues, resulting in heterogeneous chelate-to-antibody ratios and various conjugation sites. To overcome this heterogeneity, we have developed an approach for site-specific radiolabeling of antibodies by combination of genetic code expansion and click chemistry. As a proof-of-concept study, model systems including anti-CD20 antibody rituximab, positron-emitting isotope (64)Cu, and a newly synthesized bifunctional linker (4-dibenzocyclooctynol-1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid, DIBO-DOTA) were used. The approach consists of three steps: (1) site-specific incorporation of an azido group-bearing amino acid (NEAK) via the genetic code expansion technique at the defined sites of the antibody as a "chemical handle"; (2) site-specific and quantitative conjugation of bifunctional linkers with the antibodies under a mild condition; and (3) radiolabeling of the chelate-modified antibodies with the appropriate isotope. We used heavy-chain A122NEAK rituximab as proof-of-concept and obtained a homogeneous radioconjugate with precisely two chelates per antibody, incorporated only at the chosen sites. The conjugation did not alter the binding and pharmacokinetics of the rituximab, as indicated by in vitro assays and in vivo PET imaging. We believe our research is a good supplement to the genetic code expansion technique for the development of novel radioimmunoconjugates.

  8. Craniofacial Statistical Deformation Models of Wild-type mice and Crouzon mice

    DEFF Research Database (Denmark)

    Ólafsdóttir, Hildur; Darvann, Tron Andre; Ersbøll, Bjarne Kjær

    2007-01-01

    of Micro CT scannings of the heads of wild-type (normal) mice and Crouzon mice were investigated. Statistical deformation models were built to assess the anatomical differences between the groups, as well as the within-group anatomical variation. Following the approach by Rueckert et al. we built an atlas...

  9. A maximum entropy model for predicting wild boar distribution in Spain

    Directory of Open Access Journals (Sweden)

    Jaime Bosch

    2014-09-01

    Full Text Available Wild boar (Sus scrofa populations in many areas of the Palearctic including the Iberian Peninsula have grown continuously over the last century. This increase has led to numerous different types of conflicts due to the damage these mammals can cause to agriculture, the problems they create in the conservation of natural areas, and the threat they pose to animal health. In the context of both wildlife management and the design of health programs for disease control, it is essential to know how wild boar are distributed on a large spatial scale. Given that the quantifying of the distribution of wild species using census techniques is virtually impossible in the case of large-scale studies, modeling techniques have thus to be used instead to estimate animals’ distributions, densities, and abundances. In this study, the potential distribution of wild boar in Spain was predicted by integrating data of presence and environmental variables into a MaxEnt approach. We built and tested models using 100 bootstrapped replicates. For each replicate or simulation, presence data was divided into two subsets that were used for model fitting (60% of the data and cross-validation (40% of the data. The final model was found to be accurate with an area under the receiver operating characteristic curve (AUC value of 0.79. Six explanatory variables for predicting wild boar distribution were identified on the basis of the percentage of their contribution to the model. The model exhibited a high degree of predictive accuracy, which has been confirmed by its agreement with satellite images and field surveys.

  10. Structural basis for the site-specific incorporation of lysine derivatives into proteins.

    Directory of Open Access Journals (Sweden)

    Veronika Flügel

    Full Text Available Posttranslational modifications (PTMs of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS:tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.

  11. Modeling the competition between antenna size mutant and wild type microalgae in outdoor mass culture.

    Science.gov (United States)

    de Mooij, Tim; Schediwy, Kira; Wijffels, René H; Janssen, Marcel

    2016-12-20

    Under high light conditions, microalgae are oversaturated with light which significantly reduces the light use efficiency. Microalgae with a reduced pigment content, antenna size mutants, have been proposed as a potential solution to increase the light use efficiency. The goal of this study was to investigate the competition between antenna size mutants and wild type microalgae in mass cultures. Using a kinetic model and literature-derived experimental data from wild type Chlorella sorokiniana, the productivity and competition of wild type cells and antenna size mutants were simulated. Cultivation was simulated in an outdoor microalgal raceway pond production system which was assumed to be limited by light only. Light conditions were based on a Mediterranean location (Tunisia) and a more temperate location (the Netherlands). Several wild type contamination levels were simulated in each mutant culture separately to predict the effect on the productivity over the cultivation time of a hypothetical summer season of 100days. The simulations demonstrate a good potential of antenna size reduction to increase the biomass productivity of microalgal cultures. However, it was also found that after a contamination with wild type cells the mutant cultures will be rapidly overgrown resulting in productivity loss.

  12. A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing.

    Science.gov (United States)

    Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B; Goth, Christoffer K; Petersen, Thomas Nordahl; Brunak, Søren; Bennett, Eric P; Levery, Steven B; Clausen, Henrik

    2011-11-18

    Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.

  13. Site-specific time heterogeneity of the substitution process and its impact on phylogenetic inference

    Directory of Open Access Journals (Sweden)

    Philippe Hervé

    2011-01-01

    Full Text Available Abstract Background Model violations constitute the major limitation in inferring accurate phylogenies. Characterizing properties of the data that are not being correctly handled by current models is therefore of prime importance. One of the properties of protein evolution is the variation of the relative rate of substitutions across sites and over time, the latter is the phenomenon called heterotachy. Its effect on phylogenetic inference has recently obtained considerable attention, which led to the development of new models of sequence evolution. However, thus far focus has been on the quantitative heterogeneity of the evolutionary process, thereby overlooking more qualitative variations. Results We studied the importance of variation of the site-specific amino-acid substitution process over time and its possible impact on phylogenetic inference. We used the CAT model to define an infinite mixture of substitution processes characterized by equilibrium frequencies over the twenty amino acids, a useful proxy for qualitatively estimating the evolutionary process. Using two large datasets, we show that qualitative changes in site-specific substitution properties over time occurred significantly. To test whether this unaccounted qualitative variation can lead to an erroneous phylogenetic tree, we analyzed a concatenation of mitochondrial proteins in which Cnidaria and Porifera were erroneously grouped. The progressive removal of the sites with the most heterogeneous CAT profiles across clades led to the recovery of the monophyly of Eumetazoa (Cnidaria+Bilateria, suggesting that this heterogeneity can negatively influence phylogenetic inference. Conclusion The time-heterogeneity of the amino-acid replacement process is therefore an important evolutionary aspect that should be incorporated in future models of sequence change.

  14. Modelling seasonal habitat suitability for wide-ranging species: Invasive wild pigs in northern Australia.

    Science.gov (United States)

    Froese, Jens G; Smith, Carl S; Durr, Peter A; McAlpine, Clive A; van Klinken, Rieks D

    2017-01-01

    Invasive wildlife often causes serious damage to the economy and agriculture as well as environmental, human and animal health. Habitat models can fill knowledge gaps about species distributions and assist planning to mitigate impacts. Yet, model accuracy and utility may be compromised by small study areas and limited integration of species ecology or temporal variability. Here we modelled seasonal habitat suitability for wild pigs, a widespread and harmful invader, in northern Australia. We developed a resource-based, spatially-explicit and regional-scale approach using Bayesian networks and spatial pattern suitability analysis. We integrated important ecological factors such as variability in environmental conditions, breeding requirements and home range movements. The habitat model was parameterized during a structured, iterative expert elicitation process and applied to a wet season and a dry season scenario. Model performance and uncertainty was evaluated against independent distributional data sets. Validation results showed that an expert-averaged model accurately predicted empirical wild pig presences in northern Australia for both seasonal scenarios. Model uncertainty was largely associated with different expert assumptions about wild pigs' resource-seeking home range movements. Habitat suitability varied considerably between seasons, retracting to resource-abundant rainforest, wetland and agricultural refuge areas during the dry season and expanding widely into surrounding grassland floodplains, savanna woodlands and coastal shrubs during the wet season. Overall, our model suggested that suitable wild pig habitat is less widely available in northern Australia than previously thought. Mapped results may be used to quantify impacts, assess risks, justify management investments and target control activities. Our methods are applicable to other wide-ranging species, especially in data-poor situations.

  15. Genome editing using artificial site-specific nucleases in zebrafish.

    Science.gov (United States)

    Hisano, Yu; Ota, Satoshi; Kawahara, Atsuo

    2014-01-01

    Zebrafish is a model vertebrate suitable for genetic analysis. Forward genetic analysis via chemical mutagenesis screening has established a variety of zebrafish mutants that are defective in various types of organogenesis, and the genes responsible for the individual mutants have been identified from genome mapping. On the other hand, reverse genetic analysis via targeted gene disruption using embryonic stem (ES) cells (e.g., knockout mouse) can uncover gene functions by investigating the phenotypic effects. However, this approach is mostly limited to mice among the vertebrate models because of the difficulty in establishing ES cells. Recently, new gene targeting technologies, such as the transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have been developed: that can directly introduce genome modifications at the targeted genomic locus. Here, we summarize these new and powerful genome editing techniques for the study of zebrafish.

  16. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    Science.gov (United States)

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  17. Model to Track Wild Birds for Avian Influenza by Means of Population Dynamics and Surveillance Information

    Science.gov (United States)

    Alba, Anna; Bicout, Dominique J.; Vidal, Francesc; Curcó, Antoni; Allepuz, Alberto; Napp, Sebastián; García-Bocanegra, Ignacio; Costa, Taiana; Casal, Jordi

    2012-01-01

    Design, sampling and data interpretation constitute an important challenge for wildlife surveillance of avian influenza viruses (AIV). The aim of this study was to construct a model to improve and enhance identification in both different periods and locations of avian species likely at high risk of contact with AIV in a specific wetland. This study presents an individual-based stochastic model for the Ebre Delta as an example of this appliance. Based on the Monte-Carlo method, the model simulates the dynamics of the spread of AIV among wild birds in a natural park following introduction of an infected bird. Data on wild bird species population, apparent AIV prevalence recorded in wild birds during the period of study, and ecological information on factors such as behaviour, contact rates or patterns of movements of waterfowl were incorporated as inputs of the model. From these inputs, the model predicted those species that would introduce most of AIV in different periods and those species and areas that would be at high risk as a consequence of the spread of these AIV incursions. This method can serve as a complementary tool to previous studies to optimize the allocation of the limited AI surveillance resources in a local complex ecosystem. However, this study indicates that in order to predict the evolution of the spread of AIV at the local scale, there is a need for further research on the identification of host factors involved in the interspecies transmission of AIV. PMID:22952962

  18. Model to track wild birds for avian influenza by means of population dynamics and surveillance information.

    Directory of Open Access Journals (Sweden)

    Anna Alba

    Full Text Available Design, sampling and data interpretation constitute an important challenge for wildlife surveillance of avian influenza viruses (AIV. The aim of this study was to construct a model to improve and enhance identification in both different periods and locations of avian species likely at high risk of contact with AIV in a specific wetland. This study presents an individual-based stochastic model for the Ebre Delta as an example of this appliance. Based on the Monte-Carlo method, the model simulates the dynamics of the spread of AIV among wild birds in a natural park following introduction of an infected bird. Data on wild bird species population, apparent AIV prevalence recorded in wild birds during the period of study, and ecological information on factors such as behaviour, contact rates or patterns of movements of waterfowl were incorporated as inputs of the model. From these inputs, the model predicted those species that would introduce most of AIV in different periods and those species and areas that would be at high risk as a consequence of the spread of these AIV incursions. This method can serve as a complementary tool to previous studies to optimize the allocation of the limited AI surveillance resources in a local complex ecosystem. However, this study indicates that in order to predict the evolution of the spread of AIV at the local scale, there is a need for further research on the identification of host factors involved in the interspecies transmission of AIV.

  19. Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

    Science.gov (United States)

    Basu, Sanjay; Aryan, Azadeh; Overcash, Justin M; Samuel, Glady Hazitha; Anderson, Michelle A E; Dahlem, Timothy J; Myles, Kevin M; Adelman, Zach N

    2015-03-31

    Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

  20. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

    Science.gov (United States)

    Liu, Jia; Shui, Sai-Lan

    2016-12-28

    The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing.

  1. Site-specific Quantification of Bone Quality using Highly Nonlinear Solitary Waves

    CERN Document Server

    Yang, Jinkyu; Borkowski, Sean; Silvestro, Claudio; De Nardo, Luigi; Daraio, Chiara; Ebramzadeh, Edward

    2010-01-01

    Osteoporosis is a well recognized problem affecting millions of individuals worldwide. Consequently, the need to effectively, efficiently, and affordably diagnose and identify those at risk is essential; moreover, site-specific assessment of bone quality is necessary, not only in the process of risk assessment, but may also be desirable for other applications. The present study evaluated a new one-dimensional granular crystal sensor, composed of a tightly packed chain of beads under Hertzian contact interaction, representing the most suitable fundamental component for solitary wave generation and propagation. First, the sensitivity of the novel sensor was tested using densities of rigid polyurethane foam, representing clinical bone quality ranging from healthy, to severely osteoporotic. Once the relationship between the signal response and known densities was established, the sensor was used to measure several sites located in the proximal femur of ten human cadaveric specimens. The accuracy of the model was ...

  2. Assessment of Wind Turbine for Site-Specific Conditions using Probabilistic Methods

    DEFF Research Database (Denmark)

    Heras, Enrique Gómez de las; Gutiérrez, Roberto; Azagra, Elena

    2013-01-01

    be very dependent on the site. The uncertainties on the wind properties depend on issues like the available wind data, the quality of the measurement sensors, the type of terrain or the accuracy of the engineering models for horizontal and vertical spatial extrapolation. An example is included showing two......This paper describes a new approach to assess the structural integrity of wind turbines for sitespecific conditions using probabilistic methods, taking into account the particular uncertainties associated to each site. This new approach intends to improve the site suitability analysis of wind...... turbines, helping to the decision making during the site assessment phase of wind farm designs. First, the design equation for the failure mode of interest is defined, where the loads associated to the site-specific wind conditions are compared with the design limits of the structural component. A limit...

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

    Science.gov (United States)

    Heinz, Andrea; Jung, Michael C; Duca, Laurent; Sippl, Wolfgang; Taddese, Samuel; Ihling, Christian; Rusciani, Anthony; Jahreis, Günther; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    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 specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P(1)'. MMP-7 shows a strong preference for Leu at P(1)', which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P(1)'. All three MMPs showed a clear preference for Pro at P(3) that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing.

  4. Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.

    Science.gov (United States)

    Qiu, Huawei; Boudanova, Ekaterina; Park, Anna; Bird, Julie J; Honey, Denise M; Zarazinski, Christine; Greene, Ben; Kingsbury, Jonathan S; Boucher, Susan; Pollock, Julie; McPherson, John M; Pan, Clark Q

    2013-03-20

    Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products. Eleven cysteine mutants were designed based on a structure model of the TSH-TSH receptor (TSHR) complex to create unique conjugation sites on both α and β subunits for site-specific conjugation. Sequential screening of mutant expression level, oligomerization tendency, and conjugation efficiency resulted in the identification of the αG22C rhTSH mutant for stable expression and scale-up PEGylation. The introduced cysteine in the αG22C rhTSH mutant was partially blocked when isolated from conditioned media and could only be effectively PEGylated after mild reduction with cysteine. This produced a higher reaction yield, ~85%, for the monoPEGylated product. Although the mutation had no effect on receptor binding, PEGylation of αG22C rhTSH led to a PEG size-dependent decrease in receptor binding. Nevertheless, the 40 kDa PEG αG22C rhTSH showed a prolonged duration of action compared to rhTSH in a rat pharmacodynamics model. Reverse-phase HPLC and N-terminal sequencing experiments confirmed site-specific modification at the engineered Cys 22 position on the α-subunit. This work is another demonstration of successful PEGylation of a cysteine-knot protein by an engineered cysteine mutation.

  5. Experiences from site-specific landslide early warning systems

    Directory of Open Access Journals (Sweden)

    C. Michoud

    2013-10-01

    Full Text Available Landslide early warning systems (EWSs have to be implemented in areas with large risk for populations or infrastructures when classical structural remediation measures cannot be set up. This paper aims to gather experiences of existing landslide EWSs, with a special focus on practical requirements (e.g., alarm threshold values have to take into account the smallest detectable signal levels of deployed sensors before being established and specific issues when dealing with system implementations. Within the framework of the SafeLand European project, a questionnaire was sent to about one-hundred institutions in charge of landslide management. Finally, we interpreted answers from experts belonging to 14 operational units related to 23 monitored landslides. Although no standard requirements exist for designing and operating EWSs, this review highlights some key elements, such as the importance of pre-investigation work, the redundancy and robustness of monitoring systems, the establishment of different scenarios adapted to gradual increasing of alert levels, and the necessity of confidence and trust between local populations and scientists. Moreover, it also confirms the need to improve our capabilities for failure forecasting, monitoring techniques and integration of water processes into landslide conceptual models.

  6. Disease spread models in wild and feral animal populations: application of artificial life models.

    Science.gov (United States)

    Ward, M P; Laffan, S W; Highfield, L D

    2011-08-01

    The role that wild and feral animal populations might play in the incursion and spread of important transboundary animal diseases, such as foot and mouth disease (FMD), has received less attention than is warranted by the potential impacts. An artificial life model (Sirca) has been used to investigate this issue in studies based on spatially referenced data sets from southern Texas. An incursion of FMD in which either feral pig or deer populations were infected could result in between 698 and 1557 infected cattle and affect an area of between 166 km2 and 455 km2 after a 100-day period. Although outbreak size in deer populations can be predicted bythe size of the local deer population initially infected, the resulting outbreaks in feral pig populations are less predictable. Also, in the case of deer, the size of potential outbreaks might depend on the season when the incursion occurs. The impact of various mitigation strategies on disease spread has also been investigated. The approach used in the studies reviewed here explicitly incorporates the spatial distribution and relationships between animal populations, providing a new framework to explore potential impacts, costs, and control strategies.

  7. Comparison of simulated crop yield patterns for site-specific management.

    NARCIS (Netherlands)

    Uffelen, van C.G.R.; Verhagen, J.; Bouma, J.

    1997-01-01

    Site-specific management (SSM) aims to maximise crop production and make efficient use of agrochemicals. This is achieved by varying farm management practices with consideration of local variability. Maps displaying relevant soil variability are needed to guide these site-specific practices. As it t

  8. FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

    Science.gov (United States)

    A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they h...

  9. 75 FR 2859 - Environmental Management Site-Specific Advisory Board, Hanford

    Science.gov (United States)

    2010-01-19

    ... Environmental Management Site-Specific Advisory Board, Hanford AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific... 4, 2010--9 a.m.-5 p.m. Friday, February 5, 2010--8:30 a.m.-4 p.m. ADDRESSES: Red Lion Hotel, 1101...

  10. 75 FR 19630 - Environmental Management Site-Specific Advisory Board Charter Renewal

    Science.gov (United States)

    2010-04-15

    ... Environmental Management Site-Specific Advisory Board Charter Renewal Pursuant to Section 14(a)(2)(A) of the...-Specific Advisory Board will be renewed for a two-year period beginning April 11, 2010. The Board provides... renewal of the Environmental Management Site- Specific Advisory Board has been determined to be...

  11. 77 FR 65374 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2012-10-26

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... National Laboratory/ICP Public Involvement/ Communications Public Participation: The EM SSAB,...

  12. 78 FR 58294 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2013-09-23

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... SSAB, Idaho National Laboratory, welcomes the attendance of the public at its advisory...

  13. 76 FR 39080 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2011-07-05

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... National Laboratory (INL) 101. INL EM Budget. Calcine Path Forward. Advanced Mixed Waste Treatment...

  14. 76 FR 10018 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2011-02-23

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... Public Participation: The EM SSAB, Idaho National Laboratory, welcomes the attendance of the public...

  15. 76 FR 66917 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2011-10-28

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... Environmental Assessment Public Participation: The EM SSAB, Idaho National Laboratory, welcomes the...

  16. 75 FR 24685 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2010-05-05

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... to Cleanup Idaho National Laboratory Site Wide Review--CERCLA Long-Term Ecological Program...

  17. 76 FR 53888 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2011-08-30

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... Participation: The EM SSAB, Idaho National Laboratory, welcomes the attendance of the public at its...

  18. 77 FR 10485 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2012-02-22

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act...--Future Work Plan Public Participation: The EM SSAB, Idaho National Laboratory, welcomes the attendance...

  19. 75 FR 39008 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2010-07-07

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... Laboratory, welcomes the attendance of the public at its advisory committee meetings and will make...

  20. 78 FR 12747 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2013-02-25

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... Participation: The EM SSAB, Idaho National Laboratory, welcomes the attendance of the public at its...

  1. 78 FR 30910 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2013-05-23

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act...: The EM SSAB, Idaho National Laboratory, welcomes the attendance of the public at its...

  2. 77 FR 53192 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2012-08-31

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act... Closeout Process Public Participation: The EM SSAB, Idaho National Laboratory, welcomes the attendance...

  3. 76 FR 25682 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2011-05-05

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National ] Laboratory. The Federal Advisory Committee Act... National Laboratory, welcomes the attendance of the public at its advisory committee meetings and will...

  4. 75 FR 56527 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2010-09-16

    ... Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy... Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory. The Federal Advisory Committee Act.... Overview Legacy Management--Long-Term Land Use at Idaho National Laboratory. Integrated Waste...

  5. A mathematical model for wild and sterile species in competition: immigration

    Science.gov (United States)

    Flores, J. C.

    2003-10-01

    We consider a mathematical model of eradication by competition between wild and sterile species when immigration is present. We discuss the case with constant and random immigration flux. In the first case, there is a threshold of eradication Mc related to the number M of sterile individuals. This threshold is evaluated analytically and depends on the niche capacity for instance . Under this threshold ( MCeratitis capitata) eradication and frontiers control program carried out in different regions of the world by the corresponding agricultural services.

  6. Generalized theory on the mechanism of site-specific DNA-protein interactions

    Science.gov (United States)

    Niranjani, G.; Murugan, R.

    2016-05-01

    We develop a generalized theoretical framework on the binding of transcription factor proteins (TFs) with specific sites on DNA that takes into account the interplay of various factors regarding overall electrostatic potential at the DNA-protein interface, occurrence of kinetic traps along the DNA sequence, presence of other roadblock protein molecules along DNA and crowded environment, conformational fluctuations in the DNA binding domains (DBDs) of TFs, and the conformational state of the DNA. Starting from a Smolochowski type theoretical framework on site-specific binding of TFs we logically build our model by adding the effects of these factors one by one. Our generalized two-step model suggests that the electrostatic attractive forces present inbetween the positively charged DBDs of TFs and the negatively charged phosphate backbone of DNA, along with the counteracting shielding effects of solvent ions, is the core factor that creates a fluidic type environment at the DNA-protein interface. This in turn facilitates various one-dimensional diffusion (1Dd) processes such as sliding, hopping and intersegmental transfers. These facilitating processes as well as flipping dynamics of conformational states of DBDs of TFs between stationary and mobile states can enhance the 1Dd coefficient on a par with three-dimensional diffusion (3Dd). The random coil conformation of DNA also plays critical roles in enhancing the site-specific association rate. The extent of enhancement over the 3Dd controlled rate seems to be directly proportional to the maximum possible 1Dd length. We show that the overall site-specific binding rate scales with the length of DNA in an asymptotic way. For relaxed DNA, the specific binding rate will be independent of the length of DNA as length increases towards infinity. For condensed DNA as in in vivo conditions, the specific binding rate depends on the length of DNA in a turnover way with a maximum. This maximum rate seems to scale with the

  7. FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Ding-Pei Long

    Full Text Available A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.

  8. An efficient procedure for marker-free mutagenesis of S. coelicolor by site-specific recombination for secondary metabolite overproduction.

    Science.gov (United States)

    Zhang, Bo; Zhang, Lin; Dai, Ruixue; Yu, Meiying; Zhao, Guoping; Ding, Xiaoming

    2013-01-01

    Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1 integrase-mediated multisite in vitro site-specific recombination. Four 'entry clones' were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four 'entry clones' contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by φC31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward φBT1 and φC31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes.

  9. An efficient procedure for marker-free mutagenesis of S. coelicolor by site-specific recombination for secondary metabolite overproduction.

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    Full Text Available Streptomyces bacteria are known for producing important natural compounds by secondary metabolism, especially antibiotics with novel biological activities. Functional studies of antibiotic-biosynthesizing gene clusters are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1 integrase-mediated multisite in vitro site-specific recombination. Four 'entry clones' were assembled into a circular plasmid to generate the destination gene-targeting vector by a one-step reaction. The four 'entry clones' contained two clones of the upstream and downstream flanks of the target gene, a selectable marker and an E. coli-Streptomyces shuttle vector. After targeted modification of the genome, the selectable markers were removed by φC31 integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Using this method, part of the calcium-dependent antibiotic (CDA and actinorhodin (Act biosynthetic gene clusters were deleted, and the rrdA encoding RrdA, a negative regulator of Red production, was also deleted. The final prodiginine production of the engineered strain was over five times that of the wild-type strain. This straightforward φBT1 and φC31 integrase-based strategy provides an alternative approach for rapid gene-targeting vector construction and marker removal in streptomycetes.

  10. Modeling the impact of climate change on wild Piper nigrum (Black Pepper) in Western Ghats, India using ecological niche models.

    Science.gov (United States)

    Sen, Sandeep; Gode, Ameya; Ramanujam, Srirama; Ravikanth, G; Aravind, N A

    2016-11-01

    The center of diversity of Piper nigrum L. (Black Pepper), one of the highly valued spice crops is reported to be from India. Black pepper is naturally distributed in India in the Western Ghats biodiversity hotspot and is the only known existing source of its wild germplasm in the world. We used ecological niche models to predict the potential distribution of wild P. nigrum in the present and two future climate change scenarios viz (A1B) and (A2A) for the year 2080. Three topographic and nine uncorrelated bioclim variables were used to develop the niche models. The environmental variables influencing the distribution of wild P. nigrum across different climate change scenarios were identified. We also assessed the direction and magnitude of the niche centroid shift and the change in niche breadth to estimate the impact of projected climate change on the distribution of P. nigrum. The study shows a niche centroid shift in the future climate scenarios. Both the projected future climate scenarios predicted a reduction in the habitat of P. nigrum in Southern Western Ghats, which harbors many wild accessions of P. nigrum. Our results highlight the impact of future climate change on P. nigrum and provide useful information for designing sound germplasm conservation strategies for P. nigrum.

  11. Model-based evaluation of highly and low pathogenic avian influenza dynamics in wild birds.

    Directory of Open Access Journals (Sweden)

    Viviane Hénaux

    Full Text Available There is growing interest in avian influenza (AI epidemiology to predict disease risk in wild and domestic birds, and prevent transmission to humans. However, understanding the epidemic dynamics of highly pathogenic (HPAI viruses remains challenging because they have rarely been detected in wild birds. We used modeling to integrate available scientific information from laboratory and field studies, evaluate AI dynamics in individual hosts and waterfowl populations, and identify key areas for future research. We developed a Susceptible-Exposed-Infectious-Recovered (SEIR model and used published laboratory challenge studies to estimate epidemiological parameters (rate of infection, latency period, recovery and mortality rates, considering the importance of age classes, and virus pathogenicity. Infectious contact leads to infection and virus shedding within 1-2 days, followed by relatively slower period for recovery or mortality. We found a shorter infectious period for HPAI than low pathogenic (LP AI, which may explain that HPAI has been much harder to detect than LPAI during surveillance programs. Our model predicted a rapid LPAI epidemic curve, with a median duration of infection of 50-60 days and no fatalities. In contrast, HPAI dynamics had lower prevalence and higher mortality, especially in young birds. Based on field data from LPAI studies, our model suggests to increase surveillance for HPAI in post-breeding areas, because the presence of immunologically naïve young birds is predicted to cause higher HPAI prevalence and bird losses during this season. Our results indicate a better understanding of the transmission, infection, and immunity-related processes is required to refine predictions of AI risk and spread, improve surveillance for HPAI in wild birds, and develop disease control strategies to reduce potential transmission to domestic birds and/or humans.

  12. Site-specific analysis of geothermal development-data files of prospective sites. Vol. III

    Energy Technology Data Exchange (ETDEWEB)

    Williams, F.; Cohen, A.; Pfundstein, R.; Pond, S.

    1978-02-01

    Development scenarios for 37 hydrothermal and geopressured prospects in the United States were analyzed. This third of three volumes presents site-specific data and sample development schedules for the first plant on line at each of the 37 prospects.

  13. Gated rotation mechanism of site-specific recombination by  C31 integrase

    National Research Council Canada - National Science Library

    Olorunniji, F. J; Buck, D. E; Colloms, S. D; McEwan, A. R; Smith, M. C. M; Stark, W. M; Rosser, S. J

    2012-01-01

    Integrases, such as that of the Streptomyces temperate bacteriophage [phi]C31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. [phi...

  14. 75 FR 17701 - Environmental Management Site-Specific Advisory Board Chairs

    Science.gov (United States)

    2010-04-07

    ... 28, 2010 EM Program Update, Priorities, and American Recovery and Reinvestment Act Discussion EM SSAB Chairs' Round Robin: Top Three Site-Specific Issues, EM SSAB Accomplishments, and Major Board...

  15. Design and integration of components for site specific control of fertilizer application

    NARCIS (Netherlands)

    Bergeijk, van J.

    2001-01-01

    Keywords: Precision Agriculture, Site Specific Agriculture, Global Positioning System, GPS, Fertilizer Application, Information System.

    Spatial and temporal variability in soil, crop and climate characteristics results in non optimal use of fertilizers when the application

  16. Mid-Season High-Resolution Satellite Imagery for Forecasting Site-Specific Corn Yield

    Directory of Open Access Journals (Sweden)

    Nahuel R. Peralta

    2016-10-01

    Full Text Available A timely and accurate crop yield forecast is crucial to make better decisions on crop management, marketing, and storage by assessing ahead and implementing based on expected crop performance. The objective of this study was to investigate the potential of high-resolution satellite imagery data collected at mid-growing season for identification of within-field variability and to forecast corn yield at different sites within a field. A test was conducted on yield monitor data and RapidEye satellite imagery obtained for 22 cornfields located in five different counties (Clay, Dickinson, Rice, Saline, and Washington of Kansas (total of 457 ha. Three basic tests were conducted on the data: (1 spatial dependence on each of the yield and vegetation indices (VIs using Moran’s I test; (2 model selection for the relationship between imagery data and actual yield using ordinary least square regression (OLS and spatial econometric (SPL models; and (3 model validation for yield forecasting purposes. Spatial autocorrelation analysis (Moran’s I test for both yield and VIs (red edge NDVI = NDVIre, normalized difference vegetation index = NDVIr, SRre = red-edge simple ratio, near infrared = NIR and green-NDVI = NDVIG was tested positive and statistically significant for most of the fields (p < 0.05, except for one. Inclusion of spatial adjustment to model improved the model fit on most fields as compared to OLS models, with the spatial adjustment coefficient significant for half of the fields studied. When selected models were used for prediction to validate dataset, a striking similarity (RMSE = 0.02 was obtained between predicted and observed yield within a field. Yield maps could assist implementing more effective site-specific management tools and could be utilized as a proxy of yield monitor data. In summary, high-resolution satellite imagery data can be reasonably used to forecast yield via utilization of models that include spatial adjustment to

  17. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

    OpenAIRE

    Lang, Kathrin; Davis, Lloyd; Torres-Kolbus, Jessica; Chou, Chungjung; Deiters, Alexander; Chin, Jason W.

    2012-01-01

    The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetr...

  18. Evaluation of site-specific lateral inclusion zone for vapor intrusion based on an analytical approach.

    Science.gov (United States)

    Yao, Yijun; Wu, Yun; Tang, Mengling; Wang, Yue; Wang, Jianjin; Suuberg, Eric M; Jiang, Lin; Liu, Jing

    2015-11-15

    In 2002, U.S. EPA proposed a general buffer zone of approximately 100 feet (30 m) laterally to determine which buildings to include in vapor intrusion (VI) investigations. However, this screening distance can be threatened by factors such as extensive surface pavements. Under such circumstances, EPA recommended investigating soil vapor migration distance on a site-specific basis. To serve this purpose, we present an analytical model (AAMLPH) as an alternative to estimate lateral VI screening distances at chlorinated compound-contaminated sites. Based on a previously introduced model (AAML), AAMLPH is developed by considering the effects of impervious surface cover and soil geology heterogeneities, providing predictions consistent with the three-dimensional (3-D) numerical simulated results. By employing risk-based and contribution-based screening levels of subslab concentrations (50 and 500 μg/m(3), respectively) and source-to-subslab attenuation factor (0.001 and 0.01, respectively), AAMLPH suggests that buildings greater than 30 m from a plume boundary can still be affected by VI in the presence of any two of the three factors, which are high source vapor concentration, shallow source and significant surface cover. This finding justifies the concern that EPA has expressed about the application of the 30 m lateral separation distance in the presence of physical barriers (e.g., asphalt covers or ice) at the ground surface.

  19. Improving probabilistic forecast skill by calibrating site-specific and gridded ensemble forecasts

    Science.gov (United States)

    Schuhen, Nina; Evans, Gavin; Jackson, Simon; Wright, Bruce

    2016-04-01

    While forecast ensembles allow for the design and usage of novel probabilistic forecast products, they still cannot capture all sources of uncertainty inherent to NWP forecasting. In particular they are often not calibrated, resulting in the fact that the probabilistic forecasts derived from ensembles are not statistically consistent with the corresponding observations. A number of statistical post-processing methods for the purpose of calibrating ensemble forecasts have been proposed over the last decade, with Bayesian Model Averaging and Ensemble Model Output Statistics (or Non-homogeneous Gaussian Regression) being among the most successful, as they can be applied to a variety of weather parameters. At the Met Office, the calibration of probabilistic forecasts has received more and more attention over the last few years and several calibration techniques based on BMA and EMOS are being trialled and assessed for their benefit over the raw ensemble forecasts. Challenges arise when addressing weather parameters which by nature don't exhibit a normal distribution. We present results for the calibration of site-specific and gridded forecasts, in the short- to medium-range, while highlighting the need for preserving the multivariate dependency structure inherent to the ensemble forecasts. We will also draw conclusions on the practicality of operational implementation and discuss the performance at individual sites.

  20. PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities

    Institute of Scientific and Technical Information of China (English)

    Yun-xia YANG; Yun FENG; Bo-yao WANG; Qi WU

    2004-01-01

    AIM: To construct PGEX-1λT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure. METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA- 1 cell mRNA and the prokaryotic expression vector PGEX- 1λT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells. RESULTS: Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseltdomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO2-4 containing Medium E. CONCLUSION: PCR-based mutagensis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping α-helical conformation of FALL-39 and increasing net positive charge can increase the

  1. Geospatial evaluation of lead bioaccessibility and distribution for site specific prediction of threshold limits.

    Science.gov (United States)

    Bower, Jennifer A; Lister, Sydney; Hazebrouck, Garrett; Perdrial, Nicolas

    2017-10-01

    Recent work identified the need for site-specific Pb bioaccessibility evaluation and scaled contaminant modeling. Pb heterogeneity has made bioaccessibility characterization difficult, and complicated distribution models. Using field testing, bioaccessibility measurement, Integrated Exposure Uptake and Biokinetic (IEUBK) modeling, and geospatial techniques, we propose a framework for conducting applied risk-based, multiscale assessment. This framework was tested and implemented in Burlington, VT, an area of old housing stock and high Pb burden (up to 15 000 mg kg(-1)) derived primarily from paint. After analyzing local soil samples for total and bioaccessible Pb, it was determined that bioaccessible and total Pb were well correlated in this area, through which an average bioaccessibility parameter was derived approximating Pb bioaccessibility for this soil type and Pb impact. This parameter was used with the IEUBK to recommend the local limit for residential soil Pb be reduced from 400 to 360 mg kg(-1), taking into consideration the lowering of the blood lead level threshold for Pb poisoning from 10 to 5 μg dL(-1) by the Centers for Disease Control (CDC). Geospatial investigation incorporated samples collected during this investigation and samples from a high school summer science academy, and relied on three techniques, used at different scales: kriging of total and background Pb alone, kriging of total and background Pb with housing age as a well-sampled, well-correlated secondary variable (cokriging), and inverse distance weighting of total and bioaccessible Pb. Modeling at different scales allowed for characterization of Pb impact at single sites as well as citywide. Model maps show positive correlation between areas of older housing and areas of high Pb burden, as well as potential at different scales for reducing the effects of Pb heterogeneity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Dynamic vs. static social networks in models of parasite transmission: predicting Cryptosporidium spread in wild lemurs.

    Science.gov (United States)

    Springer, Andrea; Kappeler, Peter M; Nunn, Charles L

    2016-12-14

    Social networks provide an established tool to implement heterogeneous contact structures in epidemiological models. Dynamic temporal changes in contact structure and ranging behaviour of wildlife may impact disease dynamics. A consensus has yet to emerge, however, concerning the conditions in which network dynamics impact model outcomes, as compared to static approximations that average contact rates over longer time periods. Furthermore, as many pathogens can be transmitted both environmentally and via close contact, it is important to investigate the relative influence of both transmission routes in real-world populations. Here, we use empirically derived networks from a population of wild primates, Verreaux's sifakas (Propithecus verreauxi), and simulated networks to investigate pathogen spread in dynamic vs. static social networks. First, we constructed a susceptible-exposed-infected-recovered model of Cryptosporidium spread in wild Verreaux's sifakas. We incorporated social and environmental transmission routes and parameterized the model for two different climatic seasons. Second, we used simulated networks and greater variation in epidemiological parameters to investigate the conditions in which dynamic networks produce larger outbreak sizes than static networks. We found that average outbreak size of Cryptosporidium infections in sifakas was larger when the disease was introduced in the dry season than in the wet season, driven by an increase in home range overlap towards the end of the dry season. Regardless of season, dynamic networks always produced larger average outbreak sizes than static networks. Larger outbreaks in dynamic models based on simulated networks occurred especially when the probability of transmission and recovery were low. Variation in tie strength in the dynamic networks also had a major impact on outbreak size, while network modularity had a weaker influence than epidemiological parameters that determine transmission and recovery

  3. Design of Visco-Elastic Dampers for RC Frame for Site-Specific Earthquake

    Science.gov (United States)

    Kamatchi, P.; Rama Raju, K.; Ravisankar, K.; Iyer, Nagesh R.

    2016-11-01

    Number of Reinforced Concrete (RC) framed buildings have got damaged at Ahmedabad city, India located at about 240 km away from epicenter during January 2001, 7.6 moment magnitude (Mw) Bhuj earthquake. In the present study, two dimensional nonlinear time history dynamic analyses of a typical 13 storey frame assumed to be located at Ahmedabad is carried out with the rock level and surface level site-specific ground motion for scenario earthquake of Mw 7.6 from Bhuj. Artificial ground motions are generated using extended finite source stochastic model with seismological parameters reported in literature for 2001 Bhuj earthquake. Surface level ground motions are obtained for a typical soil profile of 100 m depth reported in literature through one dimensional equivalent linear wave propagation analyses. From the analyses, failure of frame is observed for surface level ground motions which indicates that, in addition to the in-adequacy of the cross sections and reinforcement of the RC members of the frame chosen, the rich energy content of the surface level ground motion near the fundamental time period of the frame has also contributed for the failure of frame. As a part of retrofitting measure, five Visco-elastic Dampers (VED) in chevron bracing are added to frame. For the frame considered in the present study, provision of VED is found to be effective to mitigate damage for the soil site considered.

  4. Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

    Science.gov (United States)

    Keiderling, Timothy A

    2017-10-04

    Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

  5. Chromosomal manipulation by site-specific recombinases and fluorescent protein-based vectors.

    Directory of Open Access Journals (Sweden)

    Munehiro Uemura

    Full Text Available Feasibility of chromosomal manipulation in mammalian cells was first reported 15 years ago. Although this technique is useful for precise understanding of gene regulation in the chromosomal context, a limited number of laboratories have used it in actual practice because of associated technical difficulties. To overcome the practical hurdles, we developed a Cre-mediated chromosomal recombination system using fluorescent proteins and various site-specific recombinases. These techniques enabled quick construction of targeting vectors, easy identification of chromosome-rearranged cells, and rearrangement leaving minimum artificial elements at junctions. Applying this system to a human cell line, we successfully recapitulated two types of pathogenic chromosomal translocations in human diseases: MYC/IgH and BCR/ABL1. By inducing recombination between two loxP sites targeted into the same chromosome, we could mark cells harboring deletion or duplication of the inter-loxP segments with different colors of fluorescence. In addition, we demonstrated that the intrachromosomal recombination frequency is inversely proportional to the distance between two recombination sites, implicating a future application of this frequency as a proximity sensor. Our method of chromosomal manipulation can be employed for particular cell types in which gene targeting is possible (e.g. embryonic stem cells. Experimental use of this system would open up new horizons in genome biology, including the establishment of cellular and animal models of diseases caused by translocations and copy-number variations.

  6. Exploring site-specific chemical interactions at surfaces: a case study on highly ordered pyrolytic graphite

    Science.gov (United States)

    Dagdeviren, Omur E.; Götzen, Jan; Altman, Eric I.; Schwarz, Udo D.

    2016-12-01

    A material’s ability to interact with approaching matter is governed by the structural and chemical nature of its surfaces. Tailoring surfaces to meet specific needs requires developing an understanding of the underlying fundamental principles that determine a surface’s reactivity. A particularly insightful case occurs when the surface site exhibiting the strongest attraction changes with distance. To study this issue, combined noncontact atomic force microscopy and scanning tunneling microscopy experiments have been carried out, where the evolution of the local chemical interaction with distance leads to a contrast reversal in the force channel. Using highly ordered pyrolytic graphite surfaces and metallic probe tips as a model system, we find that at larger tip-sample distances, carbon atoms exhibit stronger attractions than hollow sites while upon further approach, hollow sites become energetically more favorable. For the tunneling current that is recorded at large tip-sample separations during acquisition of a constant-force image, the contrast is dominated by the changes in tip-sample distance required to hold the force constant (‘cross-talk’) at smaller separations the contrast turns into a convolution of this cross-talk and the local density of states. Analysis shows that the basic factors influencing the force channel contrast reversal are locally varying decay lengths and an onset of repulsive forces that occurs for distinct surface sites at different tip-sample distances. These findings highlight the importance of tip-sample distance when comparing the relative strength of site-specific chemical interactions.

  7. Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

    Science.gov (United States)

    Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

    2016-02-01

    Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence.

  8. Design of Visco-Elastic Dampers for RC Frame for Site-Specific Earthquake

    Science.gov (United States)

    Kamatchi, P.; Rama Raju, K.; Ravisankar, K.; Iyer, Nagesh R.

    2016-12-01

    Number of Reinforced Concrete (RC) framed buildings have got damaged at Ahmedabad city, India located at about 240 km away from epicenter during January 2001, 7.6 moment magnitude (Mw) Bhuj earthquake. In the present study, two dimensional nonlinear time history dynamic analyses of a typical 13 storey frame assumed to be located at Ahmedabad is carried out with the rock level and surface level site-specific ground motion for scenario earthquake of Mw 7.6 from Bhuj. Artificial ground motions are generated using extended finite source stochastic model with seismological parameters reported in literature for 2001 Bhuj earthquake. Surface level ground motions are obtained for a typical soil profile of 100 m depth reported in literature through one dimensional equivalent linear wave propagation analyses. From the analyses, failure of frame is observed for surface level ground motions which indicates that, in addition to the in-adequacy of the cross sections and reinforcement of the RC members of the frame chosen, the rich energy content of the surface level ground motion near the fundamental time period of the frame has also contributed for the failure of frame. As a part of retrofitting measure, five Visco-elastic Dampers (VED) in chevron bracing are added to frame. For the frame considered in the present study, provision of VED is found to be effective to mitigate damage for the soil site considered.

  9. Understanding site-specific residual strain and architecture in bovine cortical bone.

    Science.gov (United States)

    Giri, Bijay; Tadano, Shigeru; Fujisaki, Kazuhiro; Todoh, Masahiro

    2008-11-14

    Living bone is considered as adaptive material to the mechanical functions, which continually undergoes change in its histological arrangement with respect to external prolonged loading. Such remodeling phenomena within bone depend on the degree of stimuli caused by the mechanical loading being experienced, and therefore, are specific to the sites. In the attempts of understanding strain adaptive phenomena within bones, different theoretical models have been proposed. Also, the existing literatures mostly follow the measurement of surface strains using strain gauges to experimentally quantify the strains experienced in the functional environment. In this work, we propose a novel idea of understanding site-specific functional adaptation to the prolonged load in bone on the basis of inherited residual strains and structural organization. We quantified the residual strains and amount of apatite crystals distribution, i.e., the degree of orientation, using X-ray diffraction procedures. The sites of naturally existing hole in bone, called foramen, are considered from bovine femur and metacarpal samples. Significant values of residual strains are found to exist in the specimens. Trends of residual strains noted in the specimens are mostly consistent with the degree of orientation of the crystallites. These features explain the response behavior of bone to the mechanical loading history near the foramen sites. Preferential orientation of crystals mapped around a femoral foramen specimen showed furnished tailored arrangement of the crystals around the hole. Effect of external loading at the femoral foramen site is also explained by the tensile loading experiment.

  10. Site-specific chromosomal integration in mammalian cells: highly efficient CRE recombinase-mediated cassette exchange.

    Science.gov (United States)

    Feng, Y Q; Seibler, J; Alami, R; Eisen, A; Westerman, K A; Leboulch, P; Fiering, S; Bouhassira, E E

    1999-10-01

    Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis -DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100 % of negatively selected mouse erythroleukemia cells and ranges from 10 to 50 % in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis -regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases.

  11. Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

    Directory of Open Access Journals (Sweden)

    Bihao Cao

    2010-01-01

    Full Text Available This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38' was transformed with Cre gene and the inbred/pure line ('E-8' was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T0-plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T0-plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

  12. Rift Valley Fever Risk Map Model and Seroprevalence in Selected Wild Ungulates and Camels from Kenya

    Science.gov (United States)

    Britch, Seth C.; Binepal, Yatinder S.; Ruder, Mark G.; Kariithi, Henry M.; Linthicum, Kenneth J.; Anyamba, Assaf; Small, Jennifer L.; Tucker, Compton J.; Ateya, Leonard O.; Oriko, Abuu A.; hide

    2013-01-01

    Since the first isolation of Rift Valley fever virus (RVFV) in the 1930s, there have been multiple epizootics and epidemics in animals and humans in sub-Saharan Africa. Prospective climate-based models have recently been developed that flag areas at risk of RVFV transmission in endemic regions based on key environmental indicators that precede Rift Valley fever (RVF) epizootics and epidemics. Although the timing and locations of human case data from the 2006-2007 RVF outbreak in Kenya have been compared to risk zones flagged by the model, seroprevalence of RVF antibodies in wildlife has not yet been analyzed in light of temporal and spatial predictions of RVF activity. Primarily wild ungulate serum samples from periods before, during, and after the 2006-2007 RVF epizootic were analyzed for the presence of RVFV IgM and/or IgG antibody. Results show an increase in RVF seropositivity from samples collected in 2007 (31.8%), compared to antibody prevalence observed from 2000-2006 (3.3%). After the epizootic, average RVF seropositivity diminished to 5% in samples collected from 2008-2009. Overlaying maps of modeled RVF risk assessments with sampling locations indicated positive RVF serology in several species of wild ungulate in or near areas flagged as being at risk for RVF. Our results establish the need to continue and expand sero-surveillance of wildlife species Kenya and elsewhere in the Horn of Africa to further calibrate and improve the RVF risk model, and better understand the dynamics of RVFV transmission.

  13. Site-specific water quality guidelines: 1. Derivation approaches based on physicochemical, ecotoxicological and ecological data.

    Science.gov (United States)

    van Dam, R A; Humphrey, C L; Harford, A J; Sinclair, A; Jones, D R; Davies, S; Storey, A W

    2014-01-01

    Generic water quality guidelines (WQGs) are developed by countries/regions as broad scale tools to assist with the protection of aquatic ecosystems from the impacts of toxicants. However, since generic WQGs cannot adequately account for the many environmental factors that may affect toxicity at a particular site, site-specific WQGs are often needed, especially for high environmental value ecosystems. The Australian and New Zealand Guidelines for Fresh and Marine Water Quality provide comprehensive guidance on methods for refining or deriving WQGs for site-specific purposes. This paper describes three such methods for deriving site-specific WQGs, namely: (1) using local reference water quality data, (2) using biological effects data from laboratory-based toxicity testing, and (3) using biological effects data from field surveys. Two case studies related to the assessment of impacts arising from mining operations in northern Australia are used to illustrate the application of these methods. Finally, the potential of several emerging methods designed to assess thresholds of ecological change from field data for deriving site-specific WQGs is discussed. Ideally, multiple lines of evidence approaches, integrating both laboratory and field data, are recommended for deriving site-specific WQGs.

  14. Frequency-dependent selection by wild birds promotes polymorphism in model salamanders

    Directory of Open Access Journals (Sweden)

    Shook Kim

    2009-05-01

    Full Text Available Abstract Background Co-occurrence of distinct colour forms is a classic paradox in evolutionary ecology because both selection and drift tend to remove variation from populations. Apostatic selection, the primary hypothesis for maintenance of colour polymorphism in cryptic animals, proposes that visual predators focus on common forms of prey, resulting in higher survival of rare forms. Empirical tests of this frequency-dependent foraging hypothesis are rare, and the link between predator behaviour and maintenance of variation in prey has been difficult to confirm. Here, we show that predatory birds can act as agents of frequency-dependent selection on terrestrial salamanders. Polymorphism for presence/absence of a dorsal stripe is widespread in many salamander species and its maintenance is a long-standing mystery. Results We used realistic food-bearing model salamanders to test whether selection by wild birds maintains a stripe/no-stripe polymorphism. In experimental manipulations, whichever form was most common was most likely to be attacked by ground-foraging birds, resulting in a survival advantage for the rare form. Conclusion This experiment demonstrates that frequency-dependent foraging by wild birds can maintain colour polymorphism in cryptic prey.

  15. Microbial profile comparisons of saliva, pooled and site-specific subgingival samples in periodontitis patients

    DEFF Research Database (Denmark)

    Belstrøm, Daniel; Sembler-Møller, Maria Lynn; Grande, Maria Anastasia

    2017-01-01

    OBJECTIVES: The purpose of this study was to compare microbial profiles of saliva, pooled and site-specific subgingival samples in patients with periodontitis. We tested the hypotheses that saliva can be an alternative to pooled subgingival samples, when screening for presence of periopathogens....... DESIGN: Site specific subgingival plaque samples (n = 54), pooled subgingival plaque samples (n = 18) and stimulated saliva samples (n = 18) were collected from 18 patients with generalized chronic periodontitis. Subgingival and salivary microbiotas were characterized by means of HOMINGS (Human Oral...... to an AUC of 0.76 (sensitivity: 0.56, specificity: 0.94) in pooled subgingival samples. CONCLUSIONS: Site-specific presence of periodontal pathogens was detected with comparable accuracy in stimulated saliva samples and pooled subgingival plaque samples. Consequently, saliva may be a reasonable surrogate...

  16. Bifunctional chelating agent for the design and development of site specific radiopharmaceuticals and biomolecule conjugation strategy

    Science.gov (United States)

    Katti, Kattesh V.; Prabhu, Kandikere R.; Gali, Hariprasad; Pillarsetty, Nagavara Kishore; Volkert, Wynn A.

    2003-10-21

    There is provided a method of labeling a biomolecule with a transition metal or radiometal in a site specific manner to produce a diagnostic or therapeutic pharmaceutical compound by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radio metal or a transition metal, and covalently linking the resulting metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. Also provided is a method of synthesizing the --PR.sub.2 containing biomolecules by synthesizing a P.sub.2 N.sub.2 -bifunctional chelating agent intermediate, complexing the intermediate with a radiometal or a transition metal, and covalently linking the resulting radio metal-complexed bifunctional chelating agent with a biomolecule in a site specific manner. There is provided a therapeutic or diagnostic agent comprising a --PR.sub.2 containing biomolecule.

  17. A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

    DEFF Research Database (Denmark)

    Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B.; Goth, Christoffer K.

    2011-01-01

    Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition...... for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site...

  18. Geothermal investment analysis with site-specific applications to Roosevelt Hot Springs and Cove Fort-Sulphurdale, Utah

    Energy Technology Data Exchange (ETDEWEB)

    Cassel, T.A.V.; Edelstein, R.H.; Blair, P.D.

    1978-12-01

    The analysis and modeling of investment behavior in the development of hydrothermal electric power facilities are reported. This investment behavior reflects a degree of sensitivity to public policy alternatives concerning taxation and regulation of the resource and its related energy conversion facilities. The objective of the current research is to provide a realistic and theoretically sound means for estimating the impacts of such public policy alternatives. A stochastic simulation model was developed which offers an efficient means for site-specific investment analysis of private sector firms and investors. The results of the first year of work are discussed including the identification, analysis, quantification and modeling of: a decision tree reflecting the sequence of procedures, timing and stochastic elements of hydrothermal resource development projects; investment requirements, expenses and revenues incurred in the exploration, development and utilization of hydrothermal resources for electric power generation; and multiattribute investment decision criteria of the several types of firms in the geothermal industry. An application of the investment model to specific resource sites in the state of Utah is also described. Site specific data for the Known Geothermal Resource Areas of Roosevelt Hot Springs and Cove Fort-Sulphurdale are given together with hypothesized generation capacity growth rates.

  19. Evaluating the results of a site-specific PSHA from the perspective of a risk analyst

    Science.gov (United States)

    Klügel, Jens-Uwe

    2016-04-01

    From 1998 till 2015 Swiss Nuclear Power Plants sponsored a set of comprehensive site-specific PSHA-studies (PEGASOS, PEGASOS Refinement Project) to obtain the requested input for their plant specific probabilistic risk assessments following the US SSHAC procedures at their most elaborated level 4. The studies were performed by well-known earth scientists working completely independent from sponsors under participatory review of the Swiss Nuclear Safety Inspectorate. Risk analysts of Swiss Nuclear Power Plants recently have been mandated to implement the final results of the studies in their risk assessment studies. This triggered an in depth assessment of the results focussed on their practical applicability for risk studies. This assessment resulted in some important insights that are of interest for future PSHA studies performed for new nuclear power plants. The assessment included a review of the completeness of results with respect to risk applications as well as plausibility checks of hazard results based on Black Swan Theory and known historical events. The key lessons and recommendations for more detailed project output specifications for future projects are presented in the paper. It was established that future PSHA projects shall provide the joint probability distribution of ground motion hazard and the associated strong motion duration as the output to allow for a technically meaningful risk assessment. The recommendation of WENRA (West European Nuclear Regulators) published in their reference levels to perform natural hazard assessment preferably based on physical grounds (deterministic method) is also rationalized by recommending an holistic approach to hazard analysis comparing PSHA insights with the results of modelling deterministic Seismic Hazard Analysis.

  20. Understanding site-specific PSHA results by hazard deaggregation into site intensities

    Science.gov (United States)

    Klügel, Jens-Uwe

    2016-04-01

    From 1998 till 2015 Swiss Nuclear Power Plants sponsored a set of comprehensive site-specific PSHA-studies (PEGASOS, PEGASOS Refinement Project) to define review level earthquakes as well as the input for their plant specific probabilistic risk assessments. The studies were performed following the US SSHAC procedures at their most elaborated level 4. Safety experts and risk analysts of Swiss Nuclear Power Plants recently have been mandated to implement the final results of the studies in their risk assessment studies. For an in depth understanding of the consequences of the hazard on practical decision making it is reasonable to compare the new studies with the original hazard assessment studies used for the development of the seismic design basis of the plants. These studies were performed in terms of intensity. For the comparison a hazard deaggregation methodology was developed that allows for the conversion of standard uniform hazard spectra (UHS) into site-intensity (factors) hazard curves. The method was applied for the nuclear power plant Goesgen using the PEGASOS hazard. The results were compared with the results of earlier hazard studies as well as with actual deterministic and probabilistic hazard studies performed independently from the PEGASOS study in terms of EMS-98 intensities. The comparison revealed that the results of the PEGASOS study led to site intensity factors comparable with the results of studies from the 1970-ies. The study may have under predicted the safety importance of historical large earthquakes like the Basel earthquake of 1356. Therefore, an important conclusion is that probabilistic hazard studies for critical infrastructures have to be accompanied by an independent physics-based study (modelling hazard assessment) that allows to perform a safety evaluation of historical earthquakes. The paper presents the deaggregation methodology and the results of its application.

  1. Site-specific, adult bone benefits attributed to loading during youth: A preliminary longitudinal analysis.

    Science.gov (United States)

    Scerpella, Tamara A; Bernardoni, Brittney; Wang, Sijian; Rathouz, Paul J; Li, Quefeng; Dowthwaite, Jodi N

    2016-04-01

    We examined site-specific bone development in relation to childhood and adolescent artistic gymnastics exposure, comparing up to 10years of prospectively acquired longitudinal data in 44 subjects, including 31 non-gymnasts (NON) and 13 gymnasts (GYM) who participated in gymnastics from pre-menarche to ≥1.9years post-menarche. Subjects underwent annual regional and whole-body DXA scans; indices of bone geometry and strength were calculated. Anthropometrics, physical activity, and maturity were assessed annually, coincident with DXA scans. Non-linear mixed effect models centered growth in bone outcomes at menarche and adjusted for menarcheal age, height, and non-bone fat-free mass to evaluate GYM-NON differences. A POST-QUIT variable assessed the withdrawal effect of quitting gymnastics. Curves for bone area, mass (BMC), and strength indices were higher in GYM than NON at both distal radius metaphysis and diaphysis (pGYM BMC (pGYM endosteal diameter (pGYM advantages in narrow neck cortical thickness and buckling ratio (both pGYM than NON (pGYM slopes increased for distal radius diaphysis parameters (p≤0.01) and for narrow neck BR (p=0.02). At the distal radius metaphysis, GYM BMC and compressive strength slopes decreased, as did slopes for lumbar spine BMC, femoral neck BMC, and narrow neck cortical thickness (p<0.02). In conclusion, advantages in bone mass, geometry, and strength at multiple skeletal sites were noted across growth and into young adulthood in girls who participated in gymnastics loading to at least 1.9years post-menarche. Following gymnastics cessation, advantages at cortical bone sites improved or stabilized, while advantages at corticocancellous sites stabilized or diminished. Additional longitudinal observation is necessary to determine whether residual loading benefits enhance lifelong skeletal strength.

  2. Site specific endonucleases for human genome mapping. Final report, April 1, 1992--March 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Knoche, K.; Selman, S.; Hung, L. [and others

    1994-06-01

    Current large scale genome mapping methodology suffers from a lack of tools for generating specific DNA fragments in the megabase size range. While technology such as pulsed field gel electrophoresis can resolve DNA fragments greater than 10 megabases in size, current methods for cleaving mammalian DNA using bacterial restriction enzymes are incapable of producing such fragments. Though several multidimensional approaches are underway to overcome this limitation, there currently is no single step procedure to generate specific DNA fragments in the 2-100 megabase size range. In order to overcome these limitations, we proposed to develop a family of site-specific endonucleases capable of generating DNA fragments in the 2-100 megabase size range in a single step. Additionally, we proposed to accomplish this by relaxing the specificity of a very-rare cutting intron-encoded endonucleases, I-Ppo I, and potentially using the process as a model for development of other enzymes. Our research has uncovered a great deal of information about intron-encoded endonucleases. We have found that I-Ppo I has a remarkable ability to tolerate degeneracy within its recognition sequence, and we have shown that the recognition sequence is larger than 15 base pairs. These findings suggest that a detailed study of the mechanism by which intron-encoded endonucleases recognize their target sequences should provide new sights into DNA-protein interactions; this had led to a continuation of the study of I-Ppo I in Dr. Raines` laboratory and we expect a more detailed understanding of the mechanism of I-Ppo I action to result.

  3. Development of Unmanned Aerial Vehicles for Site-Specific Crop Production Management

    Science.gov (United States)

    Unmanned Aerial Vehicles (UAV) have been developed and applied to support the practice of precision agriculture. Compared to piloted aircrafts, an Unmanned Aerial Vehicle can focus on much smaller crop fields with much lower flight altitude than regular airplanes to perform site-specific management ...

  4. Microinjection of cre recombinase RNA induces site-specific recombination of a transgene in mouse oocytes

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); F.G. Grosveld (Frank); T.P.M. de Wit (Ton)

    1998-01-01

    textabstractWe have developed a strategy for producing single copy transgenic mouse lines using Cre-loxP site specific recombination. The method is based on transient expression of the recombinase after injection of in vitro transcribed mRNA into the cytoplasm of fertil

  5. Summary of some feasibility studies for site-specific solar industrial process heat

    Energy Technology Data Exchange (ETDEWEB)

    None

    1982-01-01

    Some feasibility studies for several different site specific solar industrial process heat applications are summarized. The followng applications are examined. Leather Tanning; Concrete Production: Lumber and Paper Processing; Milk Processing; Molding, Curing or Drying; Automobile Manufacture; and Food Processing and Preparation. For each application, site and process data, system design, and performance and cost estimates are summarized.

  6. 76 FR 18713 - Malheur National Forest; Oregon; Malheur National Forest Site-Specific Invasive Plants Treatment...

    Science.gov (United States)

    2011-04-05

    ... Forest Service Malheur National Forest; Oregon; Malheur National Forest Site- Specific Invasive Plants... invasive plant inventory and proposed action have been updated since then resulting in this correction. The... contain invasive plants within the Malheur National Forest. The Proposed Action is to treat invasive...

  7. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    Science.gov (United States)

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA.

  8. Stoichiometry of Site-specific Lysine Acetylation in an Entire Proteome*♦

    Science.gov (United States)

    Baeza, Josue; Dowell, James A.; Smallegan, Michael J.; Fan, Jing; Amador-Noguez, Daniel; Khan, Zia; Denu, John M.

    2014-01-01

    Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD+-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism. PMID:24917678

  9. Hellsgate Big Game Winter Range Wildlife Mitigation Site Specific Management Plan for the Hellsgate Project.

    Energy Technology Data Exchange (ETDEWEB)

    Berger, Matthew T.; Judd, Steven L.

    1999-01-01

    This report contains a detailed site-specific management plan for the Hellsgate Winter Range Wildlife Mitigation Project. The report provides background information about the mitigation process, the review process, mitigation acquisitions, Habitat Evaluation Procedures (HEP) and mitigation crediting, current habitat conditions, desired future habitat conditions, restoration/enhancements efforts and maps.

  10. A method for site-specific labeling of multiple protein thiols

    NARCIS (Netherlands)

    Kuiper, Johanna M.; Pluta, Radek; Huibers, Wim H. C.; Fusetti, Fabrizia; Geertsma, Eric R.; Poolman, Bert

    We present a generic method for the site-specific and differential labeling of multiple cysteine residues in one protein. Phenyl arsenic oxide has been employed as a protecting group of two closely spaced thiols, allowing first labeling of a single thiol. Subsequently, the protecting group is

  11. 30 CFR 46.11 - Site-specific hazard awareness training.

    Science.gov (United States)

    2010-07-01

    ... TRAINING TRAINING AND RETRAINING OF MINERS ENGAGED IN SHELL DREDGING OR EMPLOYED AT SAND, GRAVEL, SURFACE STONE, SURFACE CLAY, COLLOIDAL PHOSPHATE, OR SURFACE LIMESTONE MINES. § 46.11 Site-specific hazard... environmental conditions, recognition and avoidance of hazards such as electrical and powered-haulage...

  12. Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

    NARCIS (Netherlands)

    Witte, Martin D.; Wu, Tongfei; Guimaraes, Carla P.; Theile, Christopher S.; Blom, Annet E. M.; Ingram, Jessica R.; Li, Zeyang; Kundrat, Lenka; Goldberg, Shalom D.; Ploegh, Hidde L.

    2015-01-01

    Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes

  13. Selective pressures to maintain attachment site specificity of integrative and conjugative elements.

    Directory of Open Access Journals (Sweden)

    Kayla L Menard

    Full Text Available Integrative and conjugative elements (ICEs are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.

  14. 76 FR 18540 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2011-04-04

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico (known locally as the Northern New Mexico Citizens....m.-4 p.m. ADDRESSES: Holiday Inn Express and Suites, 60 Entrada Drive, Los Alamos, New Mexico...

  15. 76 FR 11772 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2011-03-03

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico (known locally as the Northern New Mexico Citizens... Norte, Espanola, New Mexico 87532. FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern...

  16. 78 FR 38305 - Environmental Management Site-Specific Advisory Board, Northern New Mexico

    Science.gov (United States)

    2013-06-26

    ... Environmental Management Site-Specific Advisory Board, Northern New Mexico AGENCY: Department of Energy. ACTION...-Specific Advisory Board (EM SSAB), Northern New Mexico. The Federal Advisory Committee Act (Pub. L. 92-463... 87544. ] FOR FURTHER INFORMATION CONTACT: Menice Santistevan, Northern New Mexico Citizens'...

  17. Appreciating "Thirdspace": An Alternative Way of Viewing and Valuing Site-Specific Dance Performance

    Science.gov (United States)

    Munjee, Tara

    2014-01-01

    Site-specific dance performance involves the presentation of choreography in connection with a site. The context of the site combined with a viewer's personal history, beliefs, and identity impact the reading and appreciation of the performance. Although both stage and site dance performance valuing elicit multiple interpretations of artistic…

  18. Site-Specific Earthquake Response Analysis for Portsmouth Gaseous Diffusion Plant, Portsmouth, Ohio

    Science.gov (United States)

    1993-08-01

    Paducah Gaseous Diffusion Plant (PGDP), located near Paducah , Kentucky, under the same IAG and is reported under...Specific Earthquake Response Analysis for Portsmouth Gaseous Diffusion Plant , Portsmouth, Ohio by David W. Sykora, Jennifer J. Davis Geotechnical Laboratory...PAPER Miscellaneous Paper GL-93-13 August 1993 Site-Specific Earthquke Response Analysis for Portsmouth Gaseous Diffusion Plant , Portsmouth, Ohio by

  19. Site-Specific Earthquake Response Analysis for Paducah Gaseous Diffusion Plant, Paducah, Kentucky

    Science.gov (United States)

    1993-08-01

    Paducah Gaseous Diffusion Plant , Paducah , Kentucky by David W. Sykora, Jennifer J. Davis Geotechnical Laboratory Approved For Public Release...1993 Site-Specific Earthquake Response Analysis for Paducah Gaseous Diffusion Plant , Paducah , Kentucky by David W. Sykora, Jennifer J. Davis... Gaseous diffusion plants -- Kentucky -- Paducah . 3. Nuclear facilities - Kentucky -- Paducah . 1. Davis, Jennifer J. 11. United

  20. 77 FR 4799 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2012-01-31

    ... Gaseous Diffusion Plant Site Public Comments Adjourn Breaks Taken As Appropriate Public Participation: The... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION...-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86...

  1. 76 FR 80355 - Environmental Management Site-Specific Advisory Board, Paducah

    Science.gov (United States)

    2011-12-23

    ... for Paducah Gaseous Diffusion Plant Site. Public Comments. Adjourn. Breaks Taken As Appropriate... Environmental Management Site-Specific Advisory Board, Paducah AGENCY: Department of Energy (DOE). ACTION...-Specific Advisory Board (EM SSAB), Paducah. The Federal Advisory Committee Act (Pub. L. 92-463, 86...

  2. Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins

    DEFF Research Database (Denmark)

    Lambertsen, L.; Sternberg, Claus; Molin, Søren

    2004-01-01

    The mini-Tn7 transposon system is a convenient tool for site-specific tagging of bacteria in which the tagging DNA is inserted at a unique and neutral chromosomal site. We have expanded the panel of mini-Tn7 delivery plasmids expressing different fluorescent proteins (stable and unstable) from th...

  3. Integration of aerial imaging and variable-rate technology for site-specific aerial herbicide application

    Science.gov (United States)

    As remote sensing and variable rate technology are becoming more available for aerial applicators, practical methodologies on effective integration of these technologies are needed for site-specific aerial applications of crop production and protection materials. The objectives of this study were to...

  4. Site-specific dynamics in remnant populations of Northern Wheatears Oenanthe oenanthe in the Netherlands

    NARCIS (Netherlands)

    Oosten, van H.H.; Turnhout, van C.; Hallmann, C.A.; Majoor, F.; Roodbergen, M.; Schekkerman, H.; Versluijs, R.; Waasdorp, S.; Siepel, H.

    2015-01-01

    Dynamics of populations may be synchronized at large spatial scales, indicating driving forces acting beyond local scales, but may also vary locally as a result of site-specific conditions. Conservation measures for fragmented and declining populations may need to address such local effects to avoid

  5. Appreciating "Thirdspace": An Alternative Way of Viewing and Valuing Site-Specific Dance Performance

    Science.gov (United States)

    Munjee, Tara

    2014-01-01

    Site-specific dance performance involves the presentation of choreography in connection with a site. The context of the site combined with a viewer's personal history, beliefs, and identity impact the reading and appreciation of the performance. Although both stage and site dance performance valuing elicit multiple interpretations of artistic…

  6. Environmental Restoration Site-Specific Plan for the Portsmouth Gaseous Diffusion Plant, FY 93

    Energy Technology Data Exchange (ETDEWEB)

    1993-01-15

    The purpose of this Site-Specific Plan (SSP) is to describe past, present, and future activities undertaken to implement Environmental Restoration and Waste Management goals at the Portsmouth Gaseous Diffusion Plant (PORTS). The SSP is presented in sections emphasizing Environmental Restoration description of activities, resources, and milestones.

  7. 40 CFR 170.232 - Knowledge of labeling and site-specific information.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Knowledge of labeling and site... (CONTINUED) PESTICIDE PROGRAMS WORKER PROTECTION STANDARD Standard for Pesticide Handlers § 170.232 Knowledge of labeling and site-specific information. (a) Knowledge of labeling information. (1) The...

  8. Germinal transmission of site-specific excised genomic DNA by the bacterial ParA resolvase

    Science.gov (United States)

    Genome engineering is an essential tool in research and product development. Behind some of the recent advances in plant gene transfer is the development of site-specific recombination systems that enable the precise manipulation of DNA, e.g. the deletion, integration or translocation of DNA. DNA ...

  9. Stoichiometry of site-specific lysine acetylation in an entire proteome.

    Science.gov (United States)

    Baeza, Josue; Dowell, James A; Smallegan, Michael J; Fan, Jing; Amador-Noguez, Daniel; Khan, Zia; Denu, John M

    2014-08-01

    Acetylation of lysine ϵ-amino groups influences many cellular processes and has been mapped to thousands of sites across many organisms. Stoichiometric information of acetylation is essential to accurately interpret biological significance. Here, we developed and employed a novel method for directly quantifying stoichiometry of site-specific acetylation in the entire proteome of Escherichia coli. By coupling isotopic labeling and a novel pairing algorithm, our approach performs an in silico enrichment of acetyl peptides, circumventing the need for immunoenrichment. We investigated the function of the sole NAD(+)-dependent protein deacetylase, CobB, on both site-specific and global acetylation. We quantified 2206 peptides from 899 proteins and observed a wide distribution of acetyl stoichiometry, ranging from less than 1% up to 98%. Bioinformatic analysis revealed that metabolic enzymes, which either utilize or generate acetyl-CoA, and proteins involved in transcriptional and translational processes displayed the highest degree of acetylation. Loss of CobB led to increased global acetylation at low stoichiometry sites and induced site-specific changes at high stoichiometry sites, and biochemical analysis revealed altered acetyl-CoA metabolism. Thus, this study demonstrates that sirtuin deacetylase deficiency leads to both site-specific and global changes in protein acetylation stoichiometry, affecting central metabolism.

  10. Site-Specific Differentiation of Fibroblasts in Normal and Scleroderma Skin

    Science.gov (United States)

    2009-06-01

    noncoding RNA expression in scleroderma ● Construction of skin tissue microarrays ● Use of skin diversity TMA to interrogate site-specific gene...disrupted during disease. Tissue microarray: genomics and proteo- mics in the context of skin architecture Gene and tiling microarrays are useful for

  11. 76 FR 5365 - Environmental Management Site-Specific Advisory Board, Nevada

    Science.gov (United States)

    2011-01-31

    ..., waste management, and related activities. Purpose of the Industrial Sites Committee: The purpose of the... Environmental Management Site-Specific Advisory Board, Nevada AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Industrial Sites and Soils Committees of...

  12. Recent advances in covalent, site-specific protein immobilization [version 1; referees

    DEFF Research Database (Denmark)

    Meldal, Morten Peter; Schoffelen, Sanne

    2016-01-01

    The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that...

  13. Use of GIS-based Site-specific Nitrogen Management for Improving Energy Efficiency

    Science.gov (United States)

    To our knowledge, geographical information system (GIS)-based site-specific nitrogen management (SSNM) techniques have not been used to assess agricultural energy costs and efficiency. This chapter uses SSNM case studies for corn (Zea mays L.) grown in Missouri and cotton (Gossypium hirsutum L.) gro...

  14. 75 FR 11872 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2010-03-12

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF ENERGY Environmental Management Site-Specific Advisory Board, Idaho National Laboratory AGENCY: Department of Energy..., Idaho National Laboratory to be held on March 16, 2010 75 FR 9590. In that notice, the meeting...

  15. 75 FR 346 - Environmental Management Site-Specific Advisory Board, Idaho National Laboratory

    Science.gov (United States)

    2010-01-05

    ... Laboratory AGENCY: Department of Energy. ACTION: Notice of open meeting. SUMMARY: This notice announces a meeting of the Environmental Management Site-Specific Advisory Board (EM SSAB), Idaho National Laboratory... Calcine Record of Decision. Public Participation: The EM SSAB, Idaho National Laboratory, welcomes...

  16. The evolutionary forces maintaining a wild polymorphism of Littorina saxatilis: model selection by computer simulations.

    Science.gov (United States)

    Pérez-Figueroa, A; Cruz, F; Carvajal-Rodríguez, A; Rolán-Alvarez, E; Caballero, A

    2005-01-01

    Two rocky shore ecotypes of Littorina saxatilis from north-west Spain live at different shore levels and habitats and have developed an incomplete reproductive isolation through size assortative mating. The system is regarded as an example of sympatric ecological speciation. Several experiments have indicated that different evolutionary forces (migration, assortative mating and habitat-dependent selection) play a role in maintaining the polymorphism. However, an assessment of the combined contributions of these forces supporting the observed pattern in the wild is absent. A model selection procedure using computer simulations was used to investigate the contribution of the different evolutionary forces towards the maintenance of the polymorphism. The agreement between alternative models and experimental estimates for a number of parameters was quantified by a least square method. The results of the analysis show that the fittest evolutionary model for the observed polymorphism is characterized by a high gene flow, intermediate-high reproductive isolation between ecotypes, and a moderate to strong selection against the nonresident ecotypes on each shore level. In addition, a substantial number of additive loci contributing to the selected trait and a narrow hybrid definition with respect to the phenotype are scenarios that better explain the polymorphism, whereas the ecotype fitnesses at the mid-shore, the level of phenotypic plasticity, and environmental effects are not key parameters.

  17. Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.

    Science.gov (United States)

    Massa, Sam; Xavier, Catarina; De Vos, Jens; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Devoogdt, Nick

    2014-05-21

    Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.

  18. Too packed to change: side-chain packing and site-specific substitution rates in protein evolution

    Directory of Open Access Journals (Sweden)

    María Laura Marcos

    2015-04-01

    Full Text Available In protein evolution, due to functional and biophysical constraints, the rates of amino acid substitution differ from site to site. Among the best predictors of site-specific rates are solvent accessibility and packing density. The packing density measure that best correlates with rates is the weighted contact number (WCN, the sum of inverse square distances between a site’s Cα and the Cα of the other sites. According to a mechanistic stress model proposed recently, rates are determined by packing because mutating packed sites stresses and destabilizes the protein’s active conformation. While WCN is a measure of Cα packing, mutations replace side chains. Here, we consider whether a site’s evolutionary divergence is constrained by main-chain packing or side-chain packing. To address this issue, we extended the stress theory to model side chains explicitly. The theory predicts that rates should depend solely on side-chain contact density. We tested this prediction on a data set of structurally and functionally diverse monomeric enzymes. We compared side-chain contact density with main-chain contact density measures and with relative solvent accessibility (RSA. We found that side-chain contact density is the best predictor of rate variation among sites (it explains 39.2% of the variation. Moreover, the independent contribution of main-chain contact density measures and RSA are negligible. Thus, as predicted by the stress theory, site-specific evolutionary rates are determined by side-chain packing.

  19. Theory on the mechanism of site-specific DNA-protein interactions in the presence of traps

    Science.gov (United States)

    Niranjani, G.; Murugan, R.

    2016-08-01

    The speed of site-specific binding of transcription factor (TFs) proteins with genomic DNA seems to be strongly retarded by the randomly occurring sequence traps. Traps are those DNA sequences sharing significant similarity with the original specific binding sites (SBSs). It is an intriguing question how the naturally occurring TFs and their SBSs are designed to manage the retarding effects of such randomly occurring traps. We develop a simple random walk model on the site-specific binding of TFs with genomic DNA in the presence of sequence traps. Our dynamical model predicts that (a) the retarding effects of traps will be minimum when the traps are arranged around the SBS such that there is a negative correlation between the binding strength of TFs with traps and the distance of traps from the SBS and (b) the retarding effects of sequence traps can be appeased by the condensed conformational state of DNA. Our computational analysis results on the distribution of sequence traps around the putative binding sites of various TFs in mouse and human genome clearly agree well the theoretical predictions. We propose that the distribution of traps can be used as an additional metric to efficiently identify the SBSs of TFs on genomic DNA.

  20. Too packed to change: side-chain packing and site-specific substitution rates in protein evolution.

    Science.gov (United States)

    Marcos, María Laura; Echave, Julian

    2015-01-01

    In protein evolution, due to functional and biophysical constraints, the rates of amino acid substitution differ from site to site. Among the best predictors of site-specific rates are solvent accessibility and packing density. The packing density measure that best correlates with rates is the weighted contact number (WCN), the sum of inverse square distances between a site's C α and the C α of the other sites. According to a mechanistic stress model proposed recently, rates are determined by packing because mutating packed sites stresses and destabilizes the protein's active conformation. While WCN is a measure of C α packing, mutations replace side chains. Here, we consider whether a site's evolutionary divergence is constrained by main-chain packing or side-chain packing. To address this issue, we extended the stress theory to model side chains explicitly. The theory predicts that rates should depend solely on side-chain contact density. We tested this prediction on a data set of structurally and functionally diverse monomeric enzymes. We compared side-chain contact density with main-chain contact density measures and with relative solvent accessibility (RSA). We found that side-chain contact density is the best predictor of rate variation among sites (it explains 39.2% of the variation). Moreover, the independent contribution of main-chain contact density measures and RSA are negligible. Thus, as predicted by the stress theory, site-specific evolutionary rates are determined by side-chain packing.

  1. On the Exploration of the Viewpoints of Oscar Wilde in Model Millionaire --With the Approach of Appraisal Theory%On the Exploration of the Viewpoints of Oscar Wilde in Model Millionaire --With the Approach of Appraisal Theory

    Institute of Scientific and Technical Information of China (English)

    窦瑞芳

    2011-01-01

    Oscar Wilde has always been regarded as a controversial writer. A variety of literary criticism has been made on his works in the pure literary way. In this paper, the writer tends to obtain his viewpoints from analyzing Wilde's piece of works Model Millionaire with the approach of Appraisal Theory, which is part of Systemic-Functional Grammar.

  2. Egalitarian despots: hierarchy steepness, reciprocity and the grooming-trade model in wild chimpanzees, Pan troglodytes.

    Science.gov (United States)

    Kaburu, Stefano S K; Newton-Fisher, Nicholas E

    2015-01-01

    Biological market theory models the action of natural selection as a marketplace in which animals are viewed as traders with commodities to offer and exchange. Studies of female Old World monkeys have suggested that grooming might be employed as a commodity to be reciprocated or traded for alternative services, yet previous tests of this grooming-trade model in wild adult male chimpanzees have yielded mixed results. Here we provide the strongest test of the model to date for male chimpanzees: we use data drawn from two social groups (communities) of chimpanzees from different populations and give explicit consideration to variation in dominance hierarchy steepness, as such variation results in differing conditions for biological markets. First, analysis of data from published accounts of other chimpanzee communities, together with our own data, showed that hierarchy steepness varied considerably within and across communities and that the number of adult males in a community aged 20-30 years predicted hierarchy steepness. The two communities in which we tested predictions of the grooming-trade model lay at opposite extremes of this distribution. Second, in accord with the grooming-trade model, we found evidence that male chimpanzees trade grooming for agonistic support where hierarchies are steep (despotic) and consequent effective support is a rank-related commodity, but not where hierarchies are shallow (egalitarian). However, we also found that grooming was reciprocated regardless of hierarchy steepness. Our findings also hint at the possibility of agonistic competition, or at least exclusion, in relation to grooming opportunities compromising the free market envisioned by biological market theory. Our results build on previous findings across chimpanzee communities to emphasize the importance of reciprocal grooming exchanges among adult male chimpanzees, which can be understood in a biological markets framework if grooming by or with particular individuals is a

  3. Three-Dimensional Geometrical Modelling of Wild Boar Head by Reverse Engineering Technology

    Institute of Scientific and Technical Information of China (English)

    Liang Xu; Min-xu Lin; Jian-qiao Li; Zhao-liang Wang; B. Chirende

    2008-01-01

    In this paper, a wild boar head was taken as the bionic research object for the development of new ridgers, a kind of plough."The reverse engineering technology was adopted to obtain the surface geometrical information of the head. Several three-dimensional (3D) point clouds of the head were captured first using a non-touch laser scanner, and an integrated point cloud was generated by aligning these point clouds using UG/Imageware. Then, the digital surface model of the head was rebuilt by means of CATIA. The characteristic curves of the surface model were analyzed. The results show that the average error between the rebuilt surface and the point cloud is -0.431 mm. The max curvature of the ridge on the neb of the head is 0.187 mm-1, and the max and rain Gauss curvatures on the surface are 0.008 mm-2 and -0.002 mm-2. These geometrical information are the essential parameters for biomimetics study of the ridger.

  4. Site-specific and compensatory mutations imply unexpected pathways for proton delivery to the QB binding site of the photosynthetic reaction center.

    Science.gov (United States)

    Hanson, D K; Tiede, D M; Nance, S L; Chang, C H; Schiffer, M

    1993-01-01

    In photosynthetic reaction centers, a quinone molecule, QB, is the terminal acceptor in light-induced electron transfer. The protonatable residues Glu-L212 and Asp-L213 have been implicated in the binding of QB and in proton transfer to QB anions generated by electron transfer from the primary quinone QA. Here we report the details of the construction of the Ala-L212/Ala-L213 double mutant strain by site-specific mutagenesis and show that its photosynthetic incompetence is due to an inability to deliver protons to the QB anions. We also report the isolation and biophysical characterization of a collection of revertant and suppressor strains that have regained the photosynthetic phenotype. The compensatory mutations that restore function are diverse and show that neither Glu-L212 nor Asp-L213 is essential for efficient light-induced electron or proton transfer in Rhodobacter capsulatus. Second-site mutations, located within the QB binding pocket or at more distant sites, can compensate for mutations at L212 and L213 to restore photocompetence. Acquisition of a single negatively charged residue (at position L213, across the binding pocket at position L225, or outside the pocket at M43) or loss of a positively charged residue (at position M231) is sufficient to restore proton transfer activity to the complex. The proton transport pathways in the suppressor strains cannot, in principle, be identical to that of the wild type. The apparent mutability of this pathway suggests that the reaction center can serve as a model system to study the structural basis of protein-mediated proton transport. PMID:8105468

  5. Site-specific hyperphosphorylation of pRb in HIV-induced neurotoxicity.

    Science.gov (United States)

    Akay, C; Lindl, K A; Wang, Y; White, M G; Isaacman-Beck, J; Kolson, D L; Jordan-Sciutto, K L

    2011-06-01

    nonphosphorylatable pRb (ppRb S795A) attenuated HIV-MDM-induced neurotoxicity. These findings indicate that HIV-infection in the brain is associated with site-specific hyperphosphorylation of pRb at serine795, which is not induced by other tested stimuli, and that this phosphorylation contributes to neuronal death in this disease, demonstrating that specific pRb sites are differentially targeted and may have diverse impacts on the viability of post-mitotic neurons.

  6. Site-specific seismic probabilistic tsunami hazard analysis: performances and potential applications

    Science.gov (United States)

    Tonini, Roberto; Volpe, Manuela; Lorito, Stefano; Selva, Jacopo; Orefice, Simone; Graziani, Laura; Brizuela, Beatriz; Smedile, Alessandra; Romano, Fabrizio; De Martini, Paolo Marco; Maramai, Alessandra; Piatanesi, Alessio; Pantosti, Daniela

    2017-04-01

    Seismic Probabilistic Tsunami Hazard Analysis (SPTHA) provides probabilities to exceed different thresholds of tsunami hazard intensity, at a specific site or region and in a given time span, for tsunamis caused by seismic sources. Results obtained by SPTHA (i.e., probabilistic hazard curves and inundation maps) represent a very important input to risk analyses and land use planning. However, the large variability of source parameters implies the definition of a huge number of potential tsunami scenarios, whose omission could lead to a biased analysis. Moreover, tsunami propagation from source to target requires the use of very expensive numerical simulations. At regional scale, the computational cost can be reduced using assumptions on the tsunami modeling (i.e., neglecting non-linear effects, using coarse topo-bathymetric meshes, empirically extrapolating maximum wave heights on the coast). On the other hand, moving to local scale, a much higher resolution is required and such assumptions drop out, since detailed inundation maps require significantly greater computational resources. In this work we apply a multi-step method to perform a site-specific SPTHA which can be summarized in the following steps: i) to perform a regional hazard assessment to account for both the aleatory and epistemic uncertainties of the seismic source, by combining the use of an event tree and an ensemble modeling technique; ii) to apply a filtering procedure which use a cluster analysis to define a significantly reduced number of representative scenarios contributing to the hazard of a specific target site; iii) to perform high resolution numerical simulations only for these representative scenarios and for a subset of near field sources placed in very shallow waters and/or whose coseismic displacements induce ground uplift or subsidence at the target. The method is applied to three target areas in the Mediterranean located around the cities of Milazzo (Italy), Thessaloniki (Greece) and

  7. Modelling biological control with wild-type and genetically modified baculoviruses in the Helicoverpa armigera-cotton system

    NARCIS (Netherlands)

    Sun, X.; Werf, van der W.; Bianchi, F.J.J.A.; Hu, Z.; Vlak, J.M.

    2006-01-01

    A comprehensive model was developed to simulate virus epizootics in a stage structured insect population and analyse scenarios for the biological control of cotton bollworm (CBW), Helicoverpa armigera, in cotton, using wild-type or genetically modified baculoviruses. In simulations on dosage and tim

  8. Wild-type mouse models to screen antisense oligonucleotides for exon-skipping efficacy in Duchenne muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Limin Cao

    Full Text Available A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD, a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.

  9. Identification of highly reactive cysteine residues at less exposed positions in the Fab constant region for site-specific conjugation.

    Science.gov (United States)

    Shiraishi, Yasuhisa; Muramoto, Takashige; Nagatomo, Kazutaka; Shinmi, Daisuke; Honma, Emiko; Masuda, Kazuhiro; Yamasaki, Motoo

    2015-06-17

    Engineered cysteine residues are currently used for the site-specific conjugation of antibody-drug conjugates (ADC). In general, positions on the protein surface have been selected for substituting a cysteine as a conjugation site; however, less exposed positions (with less than 20% of accessible surface area [ASA]) have not yet been evaluated. In this study, we engineered original cysteine positional variants of a Fab fragment, with less than 20% of ASA, and evaluated their thiol reactivities through conjugation with various kinds of payloads. As a result, we have identified three original cysteine positional variants (heavy chain: Hc-A140C, light chain: Lc-Q124C and Lc-L201C), which exhibited similar monomer content, thermal stability, and antigen binding affinity in comparison to the wild-type Fab. In addition, the presence of cysteine in these positions made it possible for the Fab variants to react with variable-sized molecules with high efficiency. The favorable physical properties of the cysteine positional variants selected in our study suggest that less exposed positions, with less than 20% of ASA, provide an alternative for creating conjugation sites.

  10. Site-specific proteolysis of the transcriptional coactivator HCF-1 can regulate its interaction with protein cofactors.

    Science.gov (United States)

    Vogel, Jodi L; Kristie, Thomas M

    2006-05-01

    Limited proteolytic processing is an important transcriptional regulatory mechanism. In various contexts, proteolysis controls the cytoplasmic-to-nuclear transport of important transcription factors or removes domains to produce factors with altered activities. The transcriptional coactivator host cell factor-1 (HCF-1) is proteolytically processed within a unique domain consisting of 20-aa reiterations. Site-specific cleavage within one or more repeats generates a family of amino- and carboxyl-terminal subunits that remain tightly associated. However, the consequences of HCF-1 processing have been undefined. In this study, it was determined that the HCF-1-processing domain interacts with several proteins including the transcriptional coactivator/corepressor four-and-a-half LIM domain-2 (FHL2). Analysis of this interaction has uncovered specificity with both sequence and context determinants within the reiterations of this processing domain. In cells, FHL2 interacts exclusively with the nonprocessed coactivator and costimulates transcription of an HCF-1-dependent target gene. The functional interaction of HCF-1 with FHL2 supports a model in which site-specific proteolysis regulates the interaction of HCF-1 with protein partners and thus can modulate the activity of this coactivator. This paradigm expands the biological significance of limited proteolytic processing as a regulatory mechanism in gene transcription.

  11. Generating site-specifically modified proteins via a versatile and stable nucleophilic carbon ligation.

    Science.gov (United States)

    Kudirka, Romas; Barfield, Robyn M; McFarland, Jesse; Albers, Aaron E; de Hart, Gregory W; Drake, Penelope M; Holder, Patrick G; Banas, Stefanie; Jones, Lesley C; Garofalo, Albert W; Rabuka, David

    2015-02-19

    There is a need for facile chemistries that allow for chemo- and regioselectivity in bioconjugation reactions. To address this need, we are pioneering site-specific bioconjugation methods that use formylglycine as a bioorthogonal handle on a protein surface. Here we introduce aldehyde-specific bioconjugation chemistry, the trapped-Knoevenagel ligation. The speed and stability of the trapped-Knoevenagel ligation further advances the repertoire of aldehyde-based bioconjugations and expands the toolbox for site-specific protein modifications. The trapped-Knoevenagel ligation reaction can be run at near neutral pH in the absence of catalysts to produce conjugates that are stable under physiological conditions. Using this new ligation, we generated an antibody-drug conjugate that demonstrates excellent efficacy in vitro and in vivo.

  12. Potentials for site-specific design of MW sized wind turbines

    DEFF Research Database (Denmark)

    Thomsen, K.; Fuglsang, P.; Schepers, G.

    2001-01-01

    The potential for site specific design of MW sized wind turbines is quantified by comparing design loads for wind turbines installed at a range of different sites. The sites comprise on-shore normal flat terrain stand-alone conditions and wind farm conditions together with offshore and mountainous...... complex terrain wind farms. The design loads are established for a 1 MW active stall regulated wind turbine with the aeroelastic code HAWC. The load analysis is limited to fatigue loads. We do not consider ultimate loads in this paper. The results illustrate the differences in design wind conditions...... for different sites and the related differences in design loads for the 1 MW wind turbine. Based on the difference in the design loads, the potentials for site specific design of the wind turbine main components are identified. The results show that the variation in aerodynamically driven loads and energy...

  13. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2014-01-01

    The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies...... have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding...... yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells....

  14. Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system.

    Science.gov (United States)

    Baum, J A; Kakefuda, M; Gawron-Burke, C

    1996-12-01

    The cry genes of Bacillus thuringiensis encode a diverse group of crystal-forming proteins that exhibit insecticidal activity, particularly against the larvae of lepidopteran, coleopteran, and dipteran insects. The efficacy of B. thuringiensis-based biopesticides may be improved through the genetic manipulation of these genes. A gene transfer system has been developed for the introduction and maintenance of cloned insecticidal cry genes on small plasmids in B. thuringiensis. This vector system combines a B. thuringiensis plasmid replicon and an indigenous site-specific recombination system that allows for the selective removal of ancillary or foreign DNA from the recombinant bacterium after introduction of the Cry-encoding plasmid. The site-specific recombination system is useful for engineering strains with unique combinations of cry genes, resulting in new active ingredients with improved insecticidal properties.

  15. Site-specific growth of Au particles on ZnO nanopyramids under ultraviolet illumination

    KAUST Repository

    Yao, Kexin

    2011-01-01

    In this work, wurtzite ZnO nanocrystals with unique "pyramid" morphology were firstly prepared via solvothermal synthesis. It was determined that the ZnO nanopyramids are grown along the polar c-axis with the vertexes pointing to the [001] direction. When the mixture of ZnO nanopyramids and Au precursor (HAuCl4) was exposed to ultraviolet (UV) illumination, Au particles were site-specifically formed on the vertexes of ZnO nanopyramids. The obtained Au/ZnO nanocomposite showed significantly enhanced photocatalytic activity as compared to the bare ZnO nanopyramids. First-principles based calculations well explained the formation of ZnO nanopyramids as well as the site-specific growth of Au, and revealed that during the photocatalysis process the Au particles can accommodate photoelectrons and thus facilitate the charge separation. © 2011 The Royal Society of Chemistry.

  16. Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2007-02-21

    Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.

  17. The site-specific deoxyribonuclease from Bacillus pumilus (endonuclease R.Bpu1387).

    Science.gov (United States)

    Ikawa, S; Shibata, T; Ando, T

    1976-12-01

    A new site-specific endonuclease (DNase) was isolated from the cells of Bacillus pumilus AHU 1387 strain. This enzyme (endonuclease R.Bpu 1387) introduced double-stranded scissions at unique sites on DNA's of coli phage lambda, lambdadvl, coli phage T7, Bacillus phage phi105C, Bacillus phage SP10, and Simian Virus 40, in the presence of magnesium ion. The activity was stimulated by the presence of NaCl.

  18. Site-specific cancer risk in the Baltic cohort of Chernobyl cleanup workers, 1986–2007

    Science.gov (United States)

    Rahu, Kaja; Hakulinen, Timo; Smailyte, Giedre; Stengrevics, Aivars; Auvinen, Anssi; Inskip, Peter D.; Boice, John D.; Rahu, Mati

    2013-01-01

    Objective To assess site-specific cancer risk in the Baltic cohort of Chernobyl cleanup workers 1986–2007. Methods The Baltic cohort includes 17,040 men from Estonia, Latvia and Lithuania who participated in the environmental cleanup after the accident at the Chernobyl Nuclear Power Station in 1986–1991, and who were followed for cancer incidence until the end of 2007. Cancer cases diagnosed in the cohort and in the male population of each country were identified from the respective national cancer registers. The proportional incidence ratio (PIR) with 95% confidence interval (CI) was used to estimate the site-specific cancer risk in the cohort. For comparison and as it was possible, the site-specific standardized incidence ratio (SIR) was calculated for the Estonian sub-cohort, which was not feasible for the other countries. Results Overall, 756 cancer cases were reported during 1986–2007. A higher proportion of thyroid cancers in relation to the male population was found (PIR=2.76; 95%CI 1.63–4.36), especially among those who started their mission shortly after the accident, in April–May 1986 (PIR=6.38; 95% CI 2.34–13.89). Also, an excess of oesophageal cancers was noted (PIR=1.52; 95% CI 1.06–2.11). No increased PIRs for leukaemia or radiation-related cancer sites combined were observed. PIRs and SIRs for the Estonian sub-cohort demonstrated the same site-specific cancer risk pattern. Conclusion Consistent evidence of an increase in radiation-related cancers in the Baltic cohort was not observed with the possible exception of thyroid cancer, where conclusions are hampered by known medical examination including thyroid screening among cleanup workers. PMID:23683549

  19. A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila

    OpenAIRE

    Gao, Guanjun; McMahon, Conor; Chen, Jie; Rong, Yikang S.

    2008-01-01

    Gene targeting provides a powerful tool for dissecting gene function. However, repeated targeting of a single locus remains a practice mostly limited to unicellular organisms that afford simple targeting methodologies. We developed an efficient method to repeatedly target a single locus in Drosophila. In this method, which we term “site-specific integrase mediated repeated targeting” (SIRT), an attP attachment site for the phage phiC31 integrase is first targeted to the vicinity of the gene o...

  20. Drainage filters and constructed wetlands to mitigate site-specific nutrient losses

    DEFF Research Database (Denmark)

    Kjærgaard, Charlotte; Hoffmann, Carl Christian; Iversen, Bo Vangsø

    options targeting subsurface drainage are lacking. An end-of-pipe drainage filter solution offers the benefits of a targeted measure typically applied to point sources. This calls for a shift of paradigm towards the development of new, cost-efficient technologies to mitigate site-specific nutrient losses...... drainage. The project studies different approaches of implementing the filter technologies including drainage well or drainage pipe filters as well as surface-flow and sub-surface flow constructed wetlands....

  1. Rapid Subtyping of Dengue Virus Serotypes 1 and 4 by Restriction Site-Specific PCR

    OpenAIRE

    Marize P Miagostovich; dos Santos, Flavia B.; Gutiérrez, C. Milena; Riley, Lee W.; Harris, Eva

    2000-01-01

    We previously reported a simple subtyping method, restriction site-specific PCR (RSS-PCR), for dengue virus serotypes 2 and 3; here we describe its application for subtyping dengue virus serotypes 1 and 4. Three major RSS-PCR types were observed for dengue virus serotype 1 and two types were observed for dengue virus serotype 4, in agreement with previous strain classifications based on sequence analysis. Because of its simplicity, this method is amenable to rapid subtyping and application to...

  2. Site-specific mutagenesis of histidine residues in the lac permease of Escherichia coli.

    OpenAIRE

    Padan, E; Sarkar, H K; Viitanen, P V; Poonian, M S; Kaback, H. R.

    1985-01-01

    The lacY gene of Escherichia coli, which encodes the lac permease, has been modified by oligonucleotide-directed, site-specific mutagenesis such that each of the four histidine residues in the molecule is replaced with an arginine residue. Replacement of histidine-35 and histidine-39 with arginine has no apparent effect on permease activity. In contrast, replacement of either histidine-205 or histidine-322 by arginine causes a dramatic loss of transport activity, although the cells contain a ...

  3. Site-specific herbicide splitting in field carrots based on camera detected weed infestation

    Directory of Open Access Journals (Sweden)

    Dammer, Karl-Heinz

    2014-02-01

    Full Text Available The production of carrots for the German market comes mainly from domestic production. To ensure the efficiency of chemical weed control and to minimize the damage of the crop a splitting of the recommended dosage up to three times is often practiced. Because of large field areas of the carrot cropping and processing enterprises, the potential to save herbicides by practicing an herbicide application adapted to the weed occurrence is high. The efficiency of a site-specific herbicide splitting on the late weed occurrence as well as on yield parameters was tested in field strip trials. Weed species and abundance were determined manually by raster sampling using a counting frame in spring before spraying. Afterwards in the three leaf growth stages of the carrots the weed coverage level was detected online using a camera sensor developed by the Leibniz Institute for Agricultural Engineering (ATB. Based on weed coverage level maps three application zones (200 L ha-1, 300 L ha-1, 400 L ha-1 were defined. On both sides of the site specific splitting strip a uniform splitting strip (400 L ha-1 was applied. The position of the application zones were the same during site-specific splitting at the second and third herbicide spraying respectively. Compared to a conventional uniform splitting herbicide savings were 16% (2005 und 20% (2006 at each spraying time. To evaluate the efficacy of the site-specific splitting on the yield manually harvesting were performed at opposite points in both treatments. The yield parameters fresh weight and numbers of carrots “total” and “marketable” were determined. Assuming a significance level of α = 5% the difference method for controlled treatment comparison in large scale field trials (t-test resulted in 19 of the 24 tests in total no differences between the treatments. The late weed occurrence in both treatments was low.

  4. Intruder scenarios for site-specific low-level radioactive waste classification

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, W.E. Jr.; Peloquin, R.A.

    1988-09-01

    The US Department of Energy (DOE) has revised its low-level radioactive waste (LLW) management requirements and guidelines for waste generated at its facilities supporting defense missions. Specifically, draft DOE Order 5820.2A, Chapter 3 describes the purpose, policy, and requirements necessary for the management of defense LLW. The draft DOE policy calls for LLW operations to be managed to protect the health and safety of the public, preserve the environment, and ensure that no remedial action will be necessary after termination of operations. The basic approach used by DOE is to establish overall performance objectives, in terms of groundwater protection and public radiation dose limits, and to require site-specific performance assessments to determine compliance. As a result of these performance assessments, each site will develop waste acceptance criteria that define the allowable quantities and concentrations of specific radioisotopes. Additional limitations on waste disposal design, waste form, and waste treatment will also be developed on a site-specific basis. As a key step in the site-specific performance assessments, an evaluation must be conducted of potential radiation doses to intruders who may inadvertently move onto a closed DOE LLW disposal site after loss of institutional controls. This report (1) describes the types of intruder scenarios that should be considered when performing this step of the site-specific performance assessment, (2) provides the results of generic calculations performed using unit concentrations of various radionuclides as a comparison of the magnitude of importance of the various intruder scenarios, and (3) shows the relationship between the generic doses and waste classification limits for defense wastes.

  5. Wide band gap tunability in complex transition metal oxides by site-specific substitution

    OpenAIRE

    Choi, Woo Seok; Chisholm, Matthew F.; Singh, David J.; Choi, Taekjib; Jellison Jr, Gerald E.; Lee, Ho Nyung

    2012-01-01

    Fabricating complex transition metal oxides with a tuneable band gap without compromising their intriguing physical properties is a longstanding challenge. Here we examine the layered ferroelectric bismuth titanate and demonstrate that, by site-specific substitution with the Mott insulator lanthanum cobaltite, its band gap can be narrowed as much as one electron volt, while remaining strongly ferroelectric. We find that when a specific site in the host material is preferentially substituted, ...

  6. Landscape ecology and wild rabbit (Oryctolagus cuniculus habitat modeling in the Mediterranean region

    Directory of Open Access Journals (Sweden)

    Narce, M.

    2012-01-01

    Full Text Available Landscape modification is one of the reasons for the decrease in rabbit populations. The objective of this study was to model wild rabbit habitat using landscape ecology to create a diagnosis method able to assess habitat quality at a large scale. Rabbit presence/absence was recorded on 536 plots of 1 ha. Spotlight transect counts indicated a low relative abundance (KIA = 2.3 rabbits/km. We produced a land use map with metric precision using remote sensing. Water, bare soil, herbaceous, shrubs and trees were identified. Landscape structure and diversity were evaluated using variables available in FRAGSTATS. A logistic regression was performed to assess the link between rabbit presence/absence and landscape structure. Our results indicate that a suitable habitat has a high diversity, a medium number of patches and a small proportion of shrubs. These results could be used to diagnose the landscape prior to any management action to enhance rabbit populations and conversely be helpful as a tool of integrated control in the cases of local outbreaks with agricultural damages.

  7. Site-specific unfolding thermodynamics of a helix-turn-helix protein.

    Science.gov (United States)

    Amunson, Krista E; Ackels, Loren; Kubelka, Jan

    2008-07-01

    The thermal unfolding of a 40-residue helix-turn-helix subdomain of the P22 viral coat protein was investigated using circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) with site-specific 13C isotopic labeling. Helix-turn-helix is the simplest alpha-helical structural motif that combines both secondary and tertiary structural elements. The CD of individual helical fragments reveals that the P22 subdomain is stabilized by tertiary interhelical interactions. Overall the temperature-dependent CD and FTIR data can be described by a three-state process with a partially folded intermediate. However, the analysis of the site-specific 13C IR signals reveals distinct unfolding thermodynamics for each of the labeled sites. The thermodynamic parameters of the thermal unfolding of each of the labeled segments were obtained using singular value decomposition in combination with target transformation and global fitting. The P22 subdomain unfolds from the N-terminus toward the helical segments near the turn. Our results show that as few as two 13C labeled residues can be detected in a 40 residue protein and provide local, site-specific structural information about protein unfolding, which is not resolved by standard, nonsite-specific spectroscopic probes.

  8. Site-Specific N-Glycosylation of Endothelial Cell Receptor Tyrosine Kinase VEGFR-2.

    Science.gov (United States)

    Chandler, Kevin Brown; Leon, Deborah R; Meyer, Rosana D; Rahimi, Nader; Costello, Catherine E

    2017-02-03

    Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of (18)O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.

  9. Cancer registries in Japan: National Clinical Database and site-specific cancer registries.

    Science.gov (United States)

    Anazawa, Takayuki; Miyata, Hiroaki; Gotoh, Mitsukazu

    2015-02-01

    The cancer registry is an essential part of any rational program of evidence-based cancer control. The cancer control program is required to strategize in a systematic and impartial manner and efficiently utilize limited resources. In Japan, the National Clinical Database (NCD) was launched in 2010. It is a nationwide prospective registry linked to various types of board certification systems regarding surgery. The NCD is a nationally validated database using web-based data collection software; it is risk adjusted and outcome based to improve the quality of surgical care. The NCD generalizes site-specific cancer registries by taking advantage of their excellent organizing ability. Some site-specific cancer registries, including pancreatic, breast, and liver cancer registries have already been combined with the NCD. Cooperation between the NCD and site-specific cancer registries can establish a valuable platform to develop a cancer care plan in Japan. Furthermore, the prognosis information of cancer patients arranged using population-based and hospital-based cancer registries can help in efficient data accumulation on the NCD. International collaboration between Japan and the USA has recently started and is expected to provide global benchmarking and to allow a valuable comparison of cancer treatment practices between countries using nationwide cancer registries in the future. Clinical research and evidence-based policy recommendation based on accurate data from the nationwide database may positively impact the public.

  10. Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene- Deoxyadenosine Lesions

    Science.gov (United States)

    Wickramaratne, Susith; Seiler, Christopher L.

    2016-01-01

    Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1, N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen, 1,3-butadiene (BD). The resulting oligomers containing site specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis. PMID:26344227

  11. Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases.

    Science.gov (United States)

    Raeeszadeh-Sarmazdeh, Maryam; Parthasarathy, Ranganath; Boder, Eric T

    2015-04-01

    We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.

  12. Wild Soul

    Institute of Scientific and Technical Information of China (English)

    Marina; C

    2005-01-01

    There’s a wild soul inside me waiting to show its self to the world There's a wild soul inside me with fire in it’s eyes, and danger in it’s claws. There’s a wild soul in side me with a voice that no one can’t hear and a presence no one can’t feel There’S a wild soul inside me waiting for me to let it run to let it turn into, wind,fire,and rain There’s a wild soul inside me that is coming ou to ignight a fire in us a11 it will spread like a brush fire. My wild soul, Your wild soul, a flame that can not die....

  13. Modelling growth of, and removal of Zn and Hg by a wild microalgal consortium

    Energy Technology Data Exchange (ETDEWEB)

    Monteiro, Cristina M.; Brandao, Teresa R.S.; Castro, Paula M.L. [Universidade Catolica Portuguesa, Porto (Portugal). CBQF/Escola Superior de Biotecnologia; Malcata, F. Xavier [ISMAI - Instituto Superior da Maia, Avioso S. Pedro (Portugal); CIMAR/CIIMAR - Centro Interdisciplinar de Investigacao Marinha e Ambiental, Porto (Portugal)

    2012-04-15

    Microorganisms isolated from sites contaminated with heavy metals usually possess a higher removal capacity than strains from regular cultures. Heavy metal-containing soil samples from an industrial dumpsite in Northern Portugal were accordingly collected; following enrichment under metal stress, a consortium of wild microalgae was obtained. Their ability to grow in the presence of, and their capacity to recover heavy metals was comprehensively studied; the datasets thus generated were fitted to by a combined model of biomass growth and metal uptake, derived from first principles. After exposure to 15 and 25 mg/L Zn{sup 2+} for 6 days, the microalgal consortium reached similar, or higher cell density than the control; however, under 50 and 65 mg/L Zn{sup 2+}, 71% to 84% inhibition was observed. Growth in the presence of Hg{sup 2+} was significantly inhibited, even at a concentration as low as 25 {mu}g/L, and 90% inhibition was observed above 100 {mu}g/L. The maximum amount of Zn{sup 2+} removed was 21.3 mg/L, upon exposure to 25 mg/L for 6 day, whereas the maximum removal of Hg{sup 2+} was 335 {mu}g/L, upon 6 day in the presence of 350 {mu}g/L. The aforementioned mechanistic model was built upon Monod assumptions (including heavy metal inhibition), coupled with Leudeking-Piret relationships between the rates of biomass growth and metal removal. The overall fits were good under all experimental conditions tested, thus conveying a useful tool for rational optimisation of microalga-mediated bioremediation. (orig.)

  14. Remote Sensing and Geospatial Technological Applications for Site-specific Management of Fruit and Nut Crops: A Review

    Directory of Open Access Journals (Sweden)

    Joel O. Paz

    2010-08-01

    Full Text Available Site-specific crop management (SSCM is one facet of precision agriculture which is helping increase production with minimal input. It has enhanced the cost-benefit scenario in crop production. Even though the SSCM is very widely used in row crop agriculture like corn, wheat, rice, soybean, etc. it has very little application in cash crops like fruit and nut. The main goal of this review paper was to conduct a comprehensive review of advanced technologies, including geospatial technologies, used in site-specific management of fruit and nut crops. The review explores various remote sensing data from different platforms like satellite, LIDAR, aerial, and field imaging. The study analyzes the use of satellite sensors, such as Quickbird, Landsat, SPOT, and IRS imagery as well as hyperspectral narrow-band remote sensing data in study of fruit and nut crops in blueberry, citrus, peach, apple, etc. The study also explores other geospatial technologies such as GPS, GIS spatial modeling, advanced image processing techniques, and information technology for suitability study, orchard delineation, and classification accuracy assessment. The study also provides an example of a geospatial model developed in ArcGIS ModelBuilder to automate the blueberry production suitability analysis. The GIS spatial model is developed using various crop characteristics such as chilling hours, soil permeability, drainage, and pH, and land cover to determine the best sites for growing blueberry in Georgia, U.S. The study also provides a list of spectral reflectance curves developed for some fruit and nut crops, blueberry, crowberry, redblush citrus, orange, prickly pear, and peach. The study also explains these curves in detail to help researchers choose the image platform, sensor, and spectrum wavelength for various fruit and nut crops SSCM.

  15. Analysis of High Frequency Site-Specific Nitrogen and Oxygen Isotopic Composition of Atmospheric Nitrous Oxide at Mace Head, Ireland

    Science.gov (United States)

    McClellan, M. J.; Harris, E. J.; Olszewski, W.; Ono, S.; Prinn, R. G.

    2014-12-01

    Atmospheric nitrous oxide (N2O) significantly impacts Earth's climate due to its dual role as an inert potent greenhouse gas in the troposphere and as a reactive source of ozone-destroying nitrogen oxides in the stratosphere. However, there remain significant uncertainties in the global budget of this gas. The marked spatial divide in its reactivity means that all stages in the N2O life cycle—emission, transport, and destruction—must be examined to understand the overall effect of N2O on climate. Source and sink processes of N2O lead to varying concentrations of N2O isotopologues (14N14N16O, 14N15N16O, 15N14N16O, and 14N14N18O being measured) due to preferential isotopic production and elimination in different environments. Estimation of source and sink fluxes can be improved by combining isotopically resolved N2O observations with simulations using a chemical transport model with reanalysis meteorology and treatments of isotopic signatures of specific surface sources and stratospheric intrusions. We present the first few months of site-specific nitrogen and oxygen isotopic composition data from the Stheno-TILDAS instrument (Harris et al, 2013) at Mace Head, Ireland and compare these to results from MOZART-4 (Model for Ozone and Related Chemical Tracers, version 4) chemical transport model runs including N2O isotopic fractionation processes and reanalysis meterological fields (NCEP/NCAR, MERRA, and GEOS-5). This study forms the basis for future inverse modeling experiments that will improve the accuracy of isotopically differentiated N2O emission and loss estimates. Ref: Harris, E., D. Nelson, W. Olszewski, M. Zahniser, K. Potter, B. McManus, A. Whitehill, R. Prinn, and S. Ono, Development of a spectroscopic technique for continuous online monitoring of oxygen and site-specific nitrogen isotopic composition of atmospheric nitrous oxide, Analytical Chemistry, 2013; DOI: 10.1021/ac403606u.

  16. Site-specific electronic structure of Pr in $Pr_{1+x} Ba_{2-x} Cu _{3} O_{7-} \\delta$

    CERN Document Server

    Staub, U; O'Conner, A G; Kramer, M J; Knapp, M

    2001-01-01

    We report on resonant X-ray powder-diffraction measurements in the vicinity of the Pr L/sub 3/ absorption edge of Pr/sub 1+x/Ba/sub 2-x /Cu/sub 3/O/sub 7- delta /. The Pr site occupation has been accurately determined: 6+or-2% of the Pr is on the Ba site. From an analysis of the energy dependence of selected Bragg reflections, the site-specific oxidation state of Pr has been obtained. These results are discussed in the context of models describing the suppression of superconductivity by Pr, and they indicate that Pr on the R site is responsible for T/sub c/ suppression. (34 refs).

  17. Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo

    DEFF Research Database (Denmark)

    Mikkelsen, J G; Lund, Anders Henrik; Duch, M

    2000-01-01

    synthesis in newly infected cells. We have previously shown that template shifts within the 5' leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single......-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site...... specificity of recombination within the highly structured 5' leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA...

  18. Direct site-specific glycoform identification and quantitative comparison of glycoprotein therapeutics: imiglucerase and velaglucerase alfa.

    Science.gov (United States)

    Ye, Hongping; Hill, John; Gucinski, Ashley C; Boyne, Michael T; Buhse, Lucinda F

    2015-03-01

    Gaucher disease, the most common lysosomal metabolic disorder, can be treated with enzyme replacement therapy (ERT). Recombinant human glucocerebrosidase imiglucerase (Cerezyme(®)), produced in Chinese hamster ovary cells, has been used for ERT of Gaucher disease for 20 years. Another recombinant glucocerebrosidase velaglucerase alfa (VPRIV), expressed in a human fibroblast cell line, was approved by the US Food and Drug Administration in 2010. The amino acid sequence difference at residue 495 of these two products is well documented. The overall N-linked qualitative glycan composition of these two products has also been reported previously. Herein, employing our recently developed approach utilizing isobaric tandem mass tag (TMT) labeling and an LTQ Orbitrap XL electron transfer dissociation (ETD) hybrid mass spectrometer, the site-specific glycoforms of these products were identified with ETD and collision-induced dissociation (CID) spectra. The quantitative comparison of site-specific glycans was achieved utilizing higher-energy collisional dissociation (HCD) spectra with a NanoMate used as both a fraction collector and a sample introduction device. From the trypsin-digested mixture of these two products, over 90 glycopeptides were identified by accurate mass matching. In addition to those previously reported, additional glycopeptides were detected with moderate abundance. The relative amount of each glycoform at a specific glycosylation site was determined based on reporter signal intensities of the TMT labeling reagents. This is the first report of site-specific simultaneous qualitative and quantitative comparison of glycoforms for Cerezyme(®) and VPRIV. The results demonstrate that this method could be utilized for biosimilarity determination and counterfeit identification of glycoproteins.

  19. Spatial Distribution and Site-Specific Spraying of Main Sucking Pests of Elm Trees.

    Science.gov (United States)

    Karimzadeh, R; Iranipour, S

    2016-11-09

    Elm trees are important landscape trees and sucking insects weaken the elm trees and produce large amounts of honeydew. The main objectives of this study were to identify main honeydew-producing pests of elm trees and do site-specific spraying against these pests. To map the spatial distribution of the sucking pests in the large scale, the study area was divided into 40 × 40 m grids and one tree was chosen randomly from each grid (a total of 55 trees). These trees were sampled twice a year in 2011 and 2012. Each sample was a 30-cm branch terminal. Eight samples were taken from each tree in four cardinal directions and two canopy levels. The number of sucking insects and leaves of each sample were counted and recorded. Spatial analysis of the data was carried out using geostatistics. Kriging was used for producing prediction maps. Insecticide application was restricted to the regions with populations higher than threshold. To identify within-tree distribution of the honeydew-producing pests, six and four elm trees were chosen in 2011 and 2012 respectively, and sampled weekly. These trees were sampled as described previously. European elm scale (EES), Gossyparia spuria (Modeer) and two species of aphids were the dominant honeydew-producing pests. The results revealed that the effects of direction, canopy level and their interactions on insect populations were not statistically significant (P < 0.05). Site-specific spraying decreased the amount of insecticides used by ca. 20%, while satisfactory control of the sucking pests and honeydew excretion was obtained. Considering the environmental and economic benefits of site-specific spraying, it is worth doing more complementary works in this area.

  20. Site-specific modification of ED-B-targeting antibody using intein-fusion technology

    Directory of Open Access Journals (Sweden)

    Greven Simone

    2011-07-01

    Full Text Available Abstract Background A promising new approach in cancer therapy is the use of tumor specific antibodies coupled to cytotoxic agents. Currently these immunoconjugates are prepared by rather unspecific coupling chemistries, resulting in heterogeneous products. As the drug load is a key parameter for the antitumor activity, site-specific strategies are desired. Expressed protein ligation (EPL and protein trans-splicing (PTS are methods for the specific C-terminal modification of a target protein. Both include the expression as an intein fusion protein, followed by the exchange of the intein for a functionalized moiety. Results A full-length IgG specific for fibronectin ED-B was expressed as fusion protein with an intein (Mxe GyrA or Npu DnaE attached to each heavy chain. In vitro protocols were established to site-specifically modify the antibodies in high yields by EPL or PTS, respectively. Although reducing conditions had to be employed during the process, the integrity or affinity of the antibody was not affected. The protocols were used to prepare immunoconjugates containing two biotin molecules per antibody, attached to the C-termini of the heavy chains. Conclusion Full-length antibodies can be efficiently and site-specifically modified at the C-termini of their heavy chains by intein-fusion technologies. The described protocols can be used to prepare immunoconjugates of high homogeneity and with a defined drug load of two. The attachment to the C-termini is expected to retain the affinity and effector functions of the antibodies.

  1. How to awaken your nanomachines: Site-specific activation of focal adhesion kinases through ligand interactions

    KAUST Repository

    Walkiewicz, Katarzyna Wiktoria

    2015-06-17

    The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein ‘nanomachines’ to become activated in a site-specific manner.

  2. Study on Site Specific Design Earthquake Ground Motion of Nuclear Power Plants in China1

    Institute of Scientific and Technical Information of China (English)

    Zhou Bochang; Li Xiaojun; Li Yaqi

    2008-01-01

    The main technical backgrounds and requirements are introduced with regard to earthquake ground motion design parameters in several domestic and American standards,codes and guides involved in the seismic analysis and design activities of nuclear power plants in China.Based on the research results from site seismic safety evaluation of domestic nuclear power plant projects in the last years,characteristics and differences of site specific design spectra are analyzed in comparison with standard response spectra,and the suitability of standard response spectra for domestic nuclear power plant projects is discussed.

  3. Environmental restoration and waste management Site-Specific Plan for the Oak Ridge Reservation. FY 1993

    Energy Technology Data Exchange (ETDEWEB)

    1993-01-15

    The United States Department of Energy (DOE) is committed to achieving and maintaining environmental regulatory compliance while responding to public concerns and emphasizing waste minimization. DOE publishes the Environmental Restoration and Waste Management Five-Year Plan (FYP) annually to document its progress towards these goals. The purpose of this Site-Specific Plan (SSP) is to describe the activities undertaken to implement the FYP goals at the DOE Oak Ridge Field Office (DOE/OR) installations and programs specifically for the Oak Ridge Reservation (ORR) and surrounding areas. This SSP addresses activities and goals to be accomplished during FY93 even through the FYP focuses on FY94.

  4. Physical activity and cancer risk: dose-response and cancer, all sites and site-specific.

    Science.gov (United States)

    Thune, I; Furberg, A S

    2001-06-01

    The association between physical activity and overall and site-specific cancer risk is elaborated in relation to whether any observed dose-response association between physical activity and cancer can be interpreted in terms of how much physical activity (type, intensity, duration, frequency) is needed to influence site- and gender-specific cancer risk. Observational studies were reviewed that have examined the independent effect of the volume of occupational physical activity (OPA) and/or leisure time physical activity (LPA) on overall and site-specific cancer risk. The evidence of cohort and case-control studies suggests that both leisure time and occupational physical activity protect against overall cancer risk, with a graded dose-response association suggested in both sexes. Confounding effects such as diet, body weight, and parity are often included as a covariate in the analyses, with little influence on the observed associations. A crude graded inverse dose-response association was observed between physical activity and colon cancer in 48 studies including 40,674 colon/colorectal cancer cases for both sexes. A dose-response effect of physical activity on colon cancer risk was especially observed, when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed as MET-hours per week. An observed inverse association with a dose-response relationship between physical activity and breast cancer was also identified in the majority of the 41 studies including 108,031 breast cancer cases. The dose-response relationship was in particular observed in case-control studies and supported by observations in cohort studies when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed by MET-hours per week. This association between physical activity and breast cancer risk is possibly dependent on age at exposure, age at diagnosis, menopausal status and other effect

  5. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    Science.gov (United States)

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  6. Basewide Engineering Evaluation-Cost Analysis for Soil Vapor Extraction. Site Specific Document IC 1

    Science.gov (United States)

    1993-11-01

    7, BASEW= ENGPRMMG EVALUAnON- COff ANAIYSIS FOR SOLL VAPOR EXTRAmON, SITE SPECIFIC DOCUMENT IC 1 93-2886 McCleflan Air Force Bwe DISCLAIMER NOTICE...Antalisis Secton 2 ~I&LLT Site 47, Electroplating Shop * ~DmMt of bonngs 0 0eញft o > 60 feea 0 0 C30 10I ILcto Z=g Figure 2-1 ZU 13S" Maps and Site...storage removed by parking (1946-1949) 1981 Open air storage Most structures razed area (after 1957) by 1982 Site 47 Building 666 Electroplating shop

  7. Site Specific Synthesis and in-situ Immobilization of Fluorescent Silver Nanoclusters on DNA Nanoscaffolds Using Tollens Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Suchetan [Arizona State Univ., Tempe, AZ (United States); Varghese, R. [Arizona State Univ., Tempe, AZ (United States); Deng, Z. [Arizona State Univ., Tempe, AZ (United States); Zhao, Z. [Arizona State Univ., Tempe, AZ (United States); Kumar, A. [Arizona State Univ., Tempe, AZ (United States); Yan, Hao [Arizona State Univ., Tempe, AZ (United States); Liu, Yan [Arizona State Univ., Tempe, AZ (United States)

    2011-04-06

    DNA strands with specific sequences and covalently attached sugar moieties were used for the site-specific incorporation of the sugar units on a DNA origami scaffold. This approach enabled the subsequent site-specific synthesis and in situ immobilization of fluorescent Ag clusters at predefined positions on the DNA nanoscaffold by treatment with the Tollens reagent.

  8. 40 CFR Table 9 to Subpart Jjj of... - Site-specific Compliance Schedules and Increments of Progress

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Site-specific Compliance Schedules and Increments of Progress 9 Table 9 to Subpart JJJ of Part 62 Protection of Environment ENVIRONMENTAL PROTECTION... or Before August 30, 1999 Pt. 62, Subpt. JJJ, Table 9 Table 9 to Subpart JJJ of Part 62—Site-specific...

  9. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

    Directory of Open Access Journals (Sweden)

    Hui Jian

    2016-07-01

    Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

  10. IFN-{gamma} enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease

    DEFF Research Database (Denmark)

    Baron, Rona; Nemirovsky, Anna; Harpaz, Idan

    2008-01-01

    the spatial learning and memory performance of the animals. In older mice, the effect of IFN-gamma is more pronounced in both wild-type mice and mice with Alzheimer's-like disease and is associated with neuroprotection. In addition, IFN-gamma reverses the increase in oligodendrogenesis observed in a mouse...... model of Alzheimer's disease. We demonstrate that limited amounts of IFN-gamma in the brain shape the neuropoietic milieu to enhance neurogenesis, possibly representing the normal function of the immune system in controlling brain inflammation and repair.-Baron, R., Nemirovsky, A., Harpaz, I., Cohen, H......., Owens, T., Monsonego, A. IFN-gamma enhances neurogenesis in wild-type mice and in a mouse model of Alzheimer's disease....

  11. A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

    Science.gov (United States)

    Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

    2014-05-21

    Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions.

  12. Applying nitrogen site-specifically using soil electrical conductivity maps and precision agriculture technology.

    Science.gov (United States)

    Lund, E D; Wolcott, M C; Hanson, G P

    2001-10-16

    Soil texture varies significantly within many agricultural fields. The physical properties of soil, such as soil texture, have a direct effect on water holding capacity, cation exchange capacity, crop yield, production capability, and nitrogen (N) loss variations within a field. In short, mobile nutrients are used, lost, and stored differently as soil textures vary. A uniform application of N to varying soils results in a wide range of N availability to the crop. N applied in excess of crop usage results in a waste of the grower"s input expense, a potential negative effect on the environment, and in some crops a reduction of crop quality, yield, and harvestability. Inadequate N levels represent a lost opportunity for crop yield and profit. The global positioning system (GPS)-referenced mapping of bulk soil electrical conductivity (EC) has been shown to serve as an effective proxy for soil texture and other soil properties. Soils with a high clay content conduct more electricity than coarser textured soils, which results in higher EC values. This paper will describe the EC mapping process and provide case studies of site-specific N applications based on EC maps. Results of these case studies suggest that N can be managed site-specifically using a variety of management practices, including soil sampling, variable yield goals, and cropping history.

  13. ADMA/SDMA in Elderly Subjects with Asymptomatic Carotid Atherosclerosis: Values and Site-Specific Association

    Directory of Open Access Journals (Sweden)

    Graziano Riccioni

    2014-04-01

    Full Text Available Asymmetric dimethylarginine (ADMA is an endogenous nitric oxide synthase (NOS inhibitor known as a mediator of endothelial dysfunction and atherosclerosis. Circulating ADMA levels are correlated with cardiovascular risk factors such as hypercholesterolemia, arterial hypertension, diabetes mellitus, hyperhomocysteinemia, age and smoking. We assessed the relationship between ADMA values and site-specific association of asymptomatic carotid atherosclerosis (intima-media thickness (CIMT and plaque in elderly subjects. One hundred and eighty subjects underwent a complete history and physical examination, determination of serum chemistries and ADMA levels, and carotid ultrasound investigation (CUI. All subjects had no acute or chronic symptoms of carotid atherosclerosis. Statistical analyses showed that high plasma levels of ADMA/SDMA were positively correlated to carotid atherosclerosis (CIMT and plaque (p < 0.001, with significant site-specific association. Total cholesterol, low density lipoprotein cholesterol, triglycerides and C-reactive protein plasma concentrations were significantly associated with asymptomatic carotid atherosclerosis (p < 0.001. High serum concentrations of ADMA and SDMA were associated with carotid atherosclerotic lesions as measured by CIMT ad plaque and may represent a new marker of asymptomatic carotid atherosclerosis in elderly subjects.

  14. Site-Specific Incorporation of Functional Components into RNA by an Unnatural Base Pair Transcription System

    Directory of Open Access Journals (Sweden)

    Rie Kawai

    2012-03-01

    Full Text Available Toward the expansion of the genetic alphabet, an unnatural base pair between 7-(2-thienylimidazo[4,5-b]pyridine (Ds and pyrrole-2-carbaldehyde (Pa functions as a third base pair in replication and transcription, and provides a useful tool for the site-specific, enzymatic incorporation of functional components into nucleic acids. We have synthesized several modified-Pa substrates, such as alkylamino-, biotin-, TAMRA-, FAM-, and digoxigenin-linked PaTPs, and examined their transcription by T7 RNA polymerase using Ds-containing DNA templates with various sequences. The Pa substrates modified with relatively small functional groups, such as alkylamino and biotin, were efficiently incorporated into RNA transcripts at the internal positions, except for those less than 10 bases from the 3′-terminus. We found that the efficient incorporation into a position close to the 3′-terminus of a transcript depended on the natural base contexts neighboring the unnatural base, and that pyrimidine-Ds-pyrimidine sequences in templates were generally favorable, relative to purine-Ds-purine sequences. The unnatural base pair transcription system provides a method for the site-specific functionalization of large RNA molecules.

  15. Applying Nitrogen Site-Specifically Using Soil Electrical Conductivity Maps and Precision Agriculture Technology

    Directory of Open Access Journals (Sweden)

    E.D. Lund

    2001-01-01

    Full Text Available Soil texture varies significantly within many agricultural fields. The physical properties of soil, such as soil texture, have a direct effect on water holding capacity, cation exchange capacity, crop yield, production capability, and nitrogen (N loss variations within a field. In short, mobile nutrients are used, lost, and stored differently as soil textures vary. A uniform application of N to varying soils results in a wide range of N availability to the crop. N applied in excess of crop usage results in a waste of the grower’s input expense, a potential negative effect on the environment, and in some crops a reduction of crop quality, yield, and harvestability. Inadequate N levels represent a lost opportunity for crop yield and profit. The global positioning system (GPS-referenced mapping of bulk soil electrical conductivity (EC has been shown to serve as an effective proxy for soil texture and other soil properties. Soils with a high clay content conduct more electricity than coarser textured soils, which results in higher EC values. This paper will describe the EC mapping process and provide case studies of site-specific N applications based on EC maps. Results of these case studies suggest that N can be managed site-specifically using a variety of management practices, including soil sampling, variable yield goals, and cropping history.

  16. New developments for the site-specific attachment of protein to surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2005-05-12

    Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

  17. Intein-mediated site-specific conjugation of Quantum Dots to proteins in vivo

    Directory of Open Access Journals (Sweden)

    Skourides Paris A

    2009-12-01

    Full Text Available Abstract We describe an intein based method to site-specifically conjugate Quantum Dots (QDs to target proteins in vivo. This approach allows the covalent conjugation of any nanostructure and/or nanodevice to any protein and thus the targeting of such material to any intracellular compartment or signalling complex within the cells of the developing embryo. We genetically fused a pleckstrin-homology (PH domain with the N-terminus half of a split intein (IN. The C-terminus half (IC of the intein was conjugated to QDs in vitro. IC-QD's and RNA encoding PH-IN were microinjected into Xenopus embryos. In vivo intein-splicing resulted in fully functional QD-PH conjugates that could be monitored in real time within live embryos. Use of Near Infra Red (NIR-emitting QDs allowed monitoring of QD-conjugates within the embryo at depths where EGFP is undetectable demonstrating the advantages of QD's for this type of experiment. In conclusion, we have developed a novel in vivo methodology for the site-specific conjugation of QD's and other artificial structures to target proteins in different intracellular compartments and signaling complexes.

  18. Wild harvest

    NARCIS (Netherlands)

    Cruz-Garcia, G.S.; Struik, P.C.; Johnson, D.E.

    2016-01-01

    Rice fields provide not only a staple food but are also bio-diverse and multi-functional ecosystems. Wild food plants are important elements of biodiversity in rice fields and are critical components to the subsistence of poor farmers. The spatial and seasonal distribution of wild food plants wer

  19. Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

    Directory of Open Access Journals (Sweden)

    Paul Majkut

    Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

  20. Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

    Science.gov (United States)

    Majkut, Paul; Claußnitzer, Iris; Merk, Helmut; Freund, Christian; Hackenberger, Christian P R; Gerrits, Michael

    2013-01-01

    The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

  1. Controlling disease outbreaks in wildlife using limited culling: modelling classical swine fever incursions in wild pigs in Australia.

    Science.gov (United States)

    Cowled, Brendan D; Garner, M Graeme; Negus, Katherine; Ward, Michael P

    2012-01-16

    Disease modelling is one approach for providing new insights into wildlife disease epidemiology. This paper describes a spatio-temporal, stochastic, susceptible- exposed-infected-recovered process model that simulates the potential spread of classical swine fever through a documented, large and free living wild pig population following a simulated incursion. The study area (300 000 km2) was in northern Australia. Published data on wild pig ecology from Australia, and international Classical Swine Fever data was used to parameterise the model. Sensitivity analyses revealed that herd density (best estimate 1-3 pigs km-2), daily herd movement distances (best estimate approximately 1 km), probability of infection transmission between herds (best estimate 0.75) and disease related herd mortality (best estimate 42%) were highly influential on epidemic size but that extraordinary movements of pigs and the yearly home range size of a pig herd were not. CSF generally established (98% of simulations) following a single point introduction. CSF spread at approximately 9 km2 per day with low incidence rates (wild pig area). The low incidence rate indicates that surveillance for wildlife disease epidemics caused by short lived infections will be most efficient when surveillance is based on detection and investigation of clinical events, although this may not always be practical. Epidemics could be contained and eradicated with culling (aerial shooting) or vaccination when these were adequately implemented. It was apparent that the spatial structure, ecology and behaviour of wild populations must be accounted for during disease management in wildlife. An important finding was that it may only be necessary to cull or vaccinate relatively small proportions of a population to successfully contain and eradicate some wildlife disease epidemics.

  2. Common cancer in a wild animal: the California sea lion (Zalophus californianus) as an emerging model for carcinogenesis.

    Science.gov (United States)

    Browning, Helen M; Gulland, Frances M D; Hammond, John A; Colegrove, Kathleen M; Hall, Ailsa J

    2015-07-19

    Naturally occurring cancers in non-laboratory species have great potential in helping to decipher the often complex causes of neoplasia. Wild animal models could add substantially to our understanding of carcinogenesis, particularly of genetic and environmental interactions, but they are currently underutilized. Studying neoplasia in wild animals is difficult and especially challenging in marine mammals owing to their inaccessibility, lack of exposure history, and ethical, logistical and legal limits on experimentation. Despite this, California sea lions (Zalophus californianus) offer an opportunity to investigate risk factors for neoplasia development that have implications for terrestrial mammals and humans who share much of their environment and diet. A relatively accessible California sea lion population on the west coast of the USA has a high prevalence of urogenital carcinoma and is regularly sampled during veterinary care in wildlife rehabilitation centres. Collaborative studies have revealed that genotype, persistent organic pollutants and a herpesvirus are all associated with this cancer. This paper reviews research to date on the epidemiology and pathogenesis of urogenital carcinoma in this species, and presents the California sea lion as an important and currently underexploited wild animal model of carcinogenesis.

  3. Mercury speciation modeling using site specific chemical and redox data from the TNXOD OU

    Energy Technology Data Exchange (ETDEWEB)

    Kaplan, D.I.

    2000-03-22

    The objective of this study was to evaluate mercury speciation under reducing conditions expected in sediments at the TNX Outfall Delta Operable Unit. These changes in speciation would then be used to infer whether mercury toxicity and mobility would be expected to be significantly altered under reducing conditions. The results from this work suggest that mercury would likely become more strongly retained by the solid phase under reducing conditions than under oxidizing conditions at the TNX Outfall Delta Site. Considering that experimental results indicate that mercury is extremely tightly bound to the solid phase under oxidizing conditions, little mercury mobility would therefore be expected under reducing conditions.

  4. Rough Set Model for Nutrition Management in Site Specific Rice Growing Areas

    Directory of Open Access Journals (Sweden)

    K. Lavanya

    2014-09-01

    Full Text Available The optimized fertilizer usage for better yield of rice cultivation is influenced by key factors like soil fertility, crop variety, duration, season, nutrient content of the fertilizer, time of application etc., It is observed that 60 percent of yield gap in tamilnadu is due to farmers lack of knowledge on key factors and informal sources of information by pesticide dealers. In this study the major contributing factors for fertilizer requirement and optimum crop yield were analyzed based on rough set theory. In data analytics perspective the nutrient plan is sort of multiple attribute decision-making processes. To reduce the complexity of decision making, key factors that are indiscernible to conclusion are eliminated. Our rough set based approach improved the quality of agricultural data through removal of missing and redundant attributes. After pretreatment the data formed as target information, then attribute reduction algorithm was used to derive rules. The generated rules were used to structure the nutrition management decision-making. The precision was above 88% and experiments proved the feasibility of the developed decision support system for nutrient management.

  5. Evaluating the All-Ages Lead Model Using SiteSpecific Data: Approaches and Challenges

    Science.gov (United States)

    Lead (Pb) exposure continues to be a problem in the United States. Even after years of progress in reducing environmental levels, CDC estimates at least 500,000 U.S. children ages 1-5 years have blood Pb levels (BLL) above the CDC reference level of 5 µg/dL. Childhood Pb ex...

  6. A Model to Estimate Willingness to Pay for Harvest Permits for Wild Edible Mushrooms: Application to Andalusian Forests

    Directory of Open Access Journals (Sweden)

    Pablo de Frutos

    2016-11-01

    Full Text Available Public demand for harvesting wild edible mushrooms has risen in recent decades and currently affects many forested areas around the world. The idea of introducing permits for users has been conceived as a tool for ecosystem management. The problem is that policy-makers lack the necessary means to help guide them when establishing prices for such harvesting permits. Valuing the recreational benefits which mushroom harvesters derive from harvesting wild edible mushrooms may provide certain guidelines as to how much people would be willing to pay and may also justify future payments levied on harvesters. The aim of the present article is to estimate a model for determining citizens’ willingness to pay for a harvesting permit in a forest in Andalusia (Spain using contingent valuation methods. Results show that mean willingness to pay is 22.61 Euros (USD28.18 per harvester and season. This amount depends on several socioeconomic factors and preferences related to harvesters’ experiences.

  7. Development of remote sensing based site specific weed management for Midwest mint production

    Science.gov (United States)

    Gumz, Mary Saumur Paulson

    Peppermint and spearmint are high value essential oil crops in Indiana, Michigan, and Wisconsin. Although the mints are profitable alternatives to corn and soybeans, mint production efficiency must improve in order to allow industry survival against foreign produced oils and synthetic flavorings. Weed control is the major input cost in mint production and tools to increase efficiency are necessary. Remote sensing-based site-specific weed management offers potential for decreasing weed control costs through simplified weed detection and control from accurate site specific weed and herbicide application maps. This research showed the practicability of remote sensing for weed detection in the mints. Research was designed to compare spectral response curves of field grown mint and weeds, and to use these data to develop spectral vegetation indices for automated weed detection. Viability of remote sensing in mint production was established using unsupervised classification, supervised classification, handheld spectroradiometer readings and spectral vegetation indices (SVIs). Unsupervised classification of multispectral images of peppermint production fields generated crop health maps with 92 and 67% accuracy in meadow and row peppermint, respectively. Supervised classification of multispectral images identified weed infestations with 97% and 85% accuracy for meadow and row peppermint, respectively. Supervised classification showed that peppermint was spectrally distinct from weeds, but the accuracy of these measures was dependent on extensive ground referencing which is impractical and too costly for on-farm use. Handheld spectroradiometer measurements of peppermint, spearmint, and several weeds and crop and weed mixtures were taken over three years from greenhouse grown plants, replicated field plots, and production peppermint and spearmint fields. Results showed that mints have greater near infrared (NIR) and lower green reflectance and a steeper red edge slope than

  8. Site specific passive acoustic detection and densities of humpback whale calls off the coast of California

    Science.gov (United States)

    Helble, Tyler Adam

    Passive acoustic monitoring of marine mammal calls is an increasingly important method for assessing population numbers, distribution, and behavior. Automated methods are needed to aid in the analyses of the recorded data. When a mammal vocalizes in the marine environment, the received signal is a filtered version of the original waveform emitted by the marine mammal. The waveform is reduced in amplitude and distorted due to propagation effects that are influenced by the bathymetry and environment. It is important to account for these effects to determine a site-specific probability of detection for marine mammal calls in a given study area. A knowledge of that probability function over a range of environmental and ocean noise conditions allows vocalization statistics from recordings of single, fixed, omnidirectional sensors to be compared across sensors and at the same sensor over time with less bias and uncertainty in the results than direct comparison of the raw statistics. This dissertation focuses on both the development of new tools needed to automatically detect humpback whale vocalizations from single-fixed omnidirectional sensors as well as the determination of the site-specific probability of detection for monitoring sites off the coast of California. Using these tools, detected humpback calls are "calibrated" for environmental properties using the site-specific probability of detection values, and presented as call densities (calls per square kilometer per time). A two-year monitoring effort using these calibrated call densities reveals important biological and ecological information on migrating humpback whales off the coast of California. Call density trends are compared between the monitoring sites and at the same monitoring site over time. Call densities also are compared to several natural and human-influenced variables including season, time of day, lunar illumination, and ocean noise. The results reveal substantial differences in call densities

  9. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

    Directory of Open Access Journals (Sweden)

    Rajeevan Mangalathu S

    2009-06-01

    Full Text Available Abstract Background Differential expression of perforin (PRF1, a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction. Findings We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA or DNA extracted from peripheral blood mononuclear cells (PBMC DNA from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18% to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92% in the distal enhancer region (CpG sites 1–10, a variable methylation (range 49%–83% in the methylation sensitive region (CpG sites 11–17, and a progressively declining methylation level (range 12%–80% in the proximal promoter region (CpG sites 18–32 of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA. Conclusion This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune

  10. Salmon Life Cycle Models Illuminate Population Consequences of Disparate Survival and Behavior Between Hatchery- and Wild-Origin Fish

    Science.gov (United States)

    Beakes, M.; Satterthwaite, W.; Petrik, C.; Hendrix, N.; Danner, E.; Lindley, S. T.

    2016-02-01

    In past decades there has been a heavy reliance on the production of hatchery-reared fish to supplement declining population numbers of Pacific salmon. In some cases, the benefits of hatchery supplementation have been negligible despite concerted long-term stocking efforts. The management and conservation of depressed salmon populations, via hatchery practices or otherwise, can be improved by expanding our understanding of the dissimilarities between hatchery and wild salmon and how each interacts with the environment. In this study we use a stage-structured salmon life-cycle model to explore the population consequences of disparate survival and behavior between hatchery and wild-origin fall-run Chinook Salmon (Oncorhynchus tshawytscha) in the California Central Valley. We couple empirically-based statistical functions with deterministic theoretical models to identify how environmental conditions (e.g., water temperature, flow) and habitat drive the survival and abundance of both hatchery and wild salmon as they integrate across riverscapes and cross marine and freshwater ecosystem boundaries during their life cycle. Results from this study suggest that hatchery practices can lead to dissimilar interactions between hatchery and wild salmon and the environmental conditions they experience. As such, the population dynamics of fall-run Chinook Salmon in the California Central Valley are partly dependent on the composition of individuals that make up their populations. In total, this study improves out ability to conserve imperiled salmonids by identifying mechanistic linkages between the natal origin of salmon, survival and behavior, and the environment at spatiotemporal scales relevant to salmon populations and fisheries management.

  11. Simulation of Wild oat (Avena ludoviciana L. Competition on Winter Wheat (Triticum astivum Growth and Yield. I: Model Description and Validation

    Directory of Open Access Journals (Sweden)

    F Mondani

    2015-09-01

    Full Text Available Crop growth models could stimulate growth and development based on science principles and mathematical equations. They also able to evaluate effects of climate, soil, water and agronomic management practices on crop yield. In the present study, an eco-physiological simulation model developed to assess wild oat damage to winter wheat growth and yield. The general structure of this model is derived from LINTUL1 model which modified to wild oat competition against winter wheat. LINTUL1 model was developed for simulation of spring wheat potential production level. In this study, first, we added development stage (DVS and vernalization to LINTUL1 for simulation of winter wheat growth and development and then the model calibrated for potential production level. Finally, we incorporate harmful effects of wild oat to winter wheat growth and yield. Weather data used as input were average daily minimum and maximum temperature (°C and daily global radiation (MJ m-2 in Mashhad, Iran. Parameter values were derived from the literature. The model is written in Fortran Simulation Translator (FST programming language and then validated based on an experiment data. For these purposes different wild oat plant densities were arranged. The data of this experiment does not use for calibration. The results showed that this model was in general able to simulate the temporal changes in DVS of winter wheat and wild oat, total dry matter (TDM of winter wheat and wild oat and yield loss of wheat due to wild oat competition in all treatments, satisfactorily. Root mean square error (RMSE for winter wheat DVS, wild oat DVS, average winter wheat TDM, average wild oat TDM, and yield loss of winter wheat was 10.4, 14.5, 5.8, 7.6 and 7.5, respectively.

  12. Thermal denaturation of beta-galactosidase and of two site-specific mutants.

    Science.gov (United States)

    Edwards, R A; Jacobson, A L; Huber, R E

    1990-12-11

    The thermal denaturation of wild-type beta-galactosidase and two beta-galactosidases with substitutions at the active site was studied by kinetics, differential scanning calorimetry, electrophoresis, molecular exclusion chromatography, and circular dichroism. From the results, a model is developed for thermal denaturation of beta-galactosidase which includes the reversible dissociation of ligands, reversible formation of an inactive tetramer, irreversible dissociation of the inactive tetramer to inactive monomers, and subsequent aggregation of inactive monomers to dimers and larger aggregates. Under some conditions, partial reversibility of the activity loss could be demonstrated, and several intermediates in the thermal denaturation process were trapped by quenching and observed by electrophoresis and molecular exclusion chromatography. The ligands Mg2+ and phenylethyl thio-beta-D-galactoside increase the stability of beta-galactosidase to heat denaturation by shifting the ligand binding equilibrium according to Le Chatelier's principle, thus decreasing the concentration of the ligand-free tetramer which can proceed to subsequent steps. Circular dichroism results indicated that beta-galactosidase is dominated by beta-sheet with lower amounts of alpha-helix. Large changes in secondary structure begin to occur only after activity has been lost. Single amino acid changes at the active site can have significant effects on thermal stability of beta-galactosidases. Some of the effects result from increased thermal stability of the ligand-free enzyme itself. Other effects result from changes in ligand binding, but the magnitude of the resulting changes in stability is not related to the strength of ligand binding in a simple fashion.

  13. Site-specific functionalization of anisotropic nanoparticles: from colloidal atoms to colloidal molecules

    DEFF Research Database (Denmark)

    Li, Fan; Yoo, Won Cheol; Beernink, Molly B

    2009-01-01

    Multipodal nanoparticles (NPs) with controlled tethers are promising principal building blocks, useful for constructing more complex materials, much like atoms are connected into more complex molecules. Here we report colloidal sphere templating as a viable means to create tetrapodal NPs with site......-specific tethers. Amorphous sol-gel materials were molded by the template into shaped NPs that mimic tetravalent atoms but on the length scale of colloids. Synthetic methods were developed to modify only the tips of the tetrapods with a range of possible functional groups to generate anisotropic NPs capable...... of directional bonding to other NPs. We also illustrate that sets of tethered "colloidal atoms" can assemble themselves into "colloidal molecules" with precise placement of the modifying colloids. The templating and tethering approaches to these anisotropic colloidal building blocks and the assembly methods...

  14. Assessment of Wind Turbine for Site-Specific Conditions using Probabilistic Methods

    DEFF Research Database (Denmark)

    Heras, Enrique Gómez de las; Gutiérrez, Roberto; Azagra, Elena

    2013-01-01

    turbines, helping to the decision making during the site assessment phase of wind farm designs. First, the design equation for the failure mode of interest is defined, where the loads associated to the site-specific wind conditions are compared with the design limits of the structural component. A limit......This paper describes a new approach to assess the structural integrity of wind turbines for sitespecific conditions using probabilistic methods, taking into account the particular uncertainties associated to each site. This new approach intends to improve the site suitability analysis of wind...... state equation is defined making the loads and resistance depending on a set of stochastic variables representing the uncertainties. In this paper, special focus is put on the uncertainties related to the assessment of wind data, which is the main input for the sitespecific load assessment, and can...

  15. Impairments in site-specific AS160 phosphorylation and effects of exercise training

    DEFF Research Database (Denmark)

    Consitt, Leslie A; Van Meter, Jessica; Newton, Christopher A

    2013-01-01

    The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult lifespan (18 to 84 years) and if endurance- and/or strength-oriented exercise training...... in whole-body insulin action were associated with impairments in insulin-induced phosphorylation of skeletal muscle AS160 on sites Ser-588, Thr-642, Ser-666 and phospho-Akt substrate (PAS), but not Ser-318 or Ser-751. Twelve weeks of either endurance- or strength-oriented exercise training increased whole...... population and that exercise training is an effective intervention for treating these impairments....

  16. Site-specific integration by the adeno-associated virus rep protein.

    Science.gov (United States)

    Recchia, Alessandra; Mavilio, Fulvio

    2011-10-01

    Inserting genetic information at precise locations into the human genome has been the goal of gene transfer technology for almost two decades. The spectacular progress of mammalian genetics has led to the development of technology for genome editing and homologous recombination in human somatic cells that is finally approaching efficiency compatible with clinical application. Site-specific integration, or the insertion of genes at known locations by enzymes with target recognition capacity, has progressed slowly but steadily in recent years, and could very well be the basis of the next generation of gene transfer technology. This review focuses on the use of Rep, the replicase/integrase of the adeno-associated virus (AAV), to insert genes at the natural AAV integration site on human chromosome 19. This region (AAVS1) has characteristics that make it an ideal target for somatic transgenesis.

  17. Precision farming - Technology assessment of site-specific input application in cereals

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus

    About 400 Danish farmers have within the large 10 years adopted some precision farming technologies on their farms. Precision farming is a management system, in which the use of variable inputs (fertilisers, lime and pesticides) is applied according to the spatial soil and crop variation at a sub...... but it seems possible to obtain a socio-economic benefits from lime, variable rate herbicide and possibly nitrogen application • The technology may improve farm logistics, planning and crop quality (e.g. protein content) - but • The costs of implementing PF-practices are high and • Technical functionality...... and hardware-compatibility is a concern among farmers Precisions farming is primarily economic viable on relative large farm holdings and site-specific application is only economic attractive if some degree of soil texture variation is present on the field. There is a need for additional information about...

  18. Precision farming - Technology assessment of site-specific input application in cereals

    DEFF Research Database (Denmark)

    Pedersen, Søren Marcus

    About 400 Danish farmers have within the large 10 years adopted some precision farming technologies on their farms. Precision farming is a management system, in which the use of variable inputs (fertilisers, lime and pesticides) is applied according to the spatial soil and crop variation at a sub......-field level. Most precision farming systems uses the GPS-system for geographical positioning. This study assesses the technical, environmental and economic barriers and potentials of precision farming in Denmark. The project is based on a combined participatory technology assessment approach with businesses...... economic and socio-economic analysis. The current status of precision farming in Denmark is as follows: • The technology is primarily applicable for large farm holdings • Economic viability depends on site-specific yield variation • So far, the business economic benefits from most PF-practices are modest...

  19. A site-specific curated database for the microorganisms of activated sludge and anaerobic digesters

    DEFF Research Database (Denmark)

    McIlroy, Simon Jon; Kirkegaard, Rasmus Hansen; McIlroy, Bianca

    the composition and dynamics of the most abundant organisms. However, to understand the relationship between the population dynamics and operational parameters of the system, a functional role must be attributed to each organism. The Microbial Database for Activated Sludge (MiDAS) and Anaerobic Digesters (AD......) presented here provides a site specific curated taxonomy for abundant and important microorganisms and integrates it into a community knowledge web platform about the microbes in activated sludge (AS) and their associated ADs (www.midasfieldguide.org). The MiDAS taxonomy, a manual curation of the SILVA......, to improve the classification of unknown organisms and link these names to the wealth of present and future functional information about their ecology....

  20. Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in vitro

    Science.gov (United States)

    Cooney, Michael; Czernuszewicz, Graznya; Postel, Edith H.; Flint, S. Jane; Hogan, Michael E.

    1988-07-01

    A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

  1. Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

    Science.gov (United States)

    Zhang, Shaofeng; Basile, Franco

    2011-01-01

    A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

  2. A general method for site-specific incorporation of unnatural amino acids into proteins

    Energy Technology Data Exchange (ETDEWEB)

    Noren, C.J.; Anthony-Cahill, S.J; Griffith, M.C.; Schultz, P.G. (Lawrence Berkeley Lab., CA (USA))

    1989-04-14

    A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme {beta}-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity. The catalytic properties of several mutants at the conserved Phe{sup 66} were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function. 45 refs., 7 figs., 1 tab.

  3. Proteomic Approaches for Site-specific O-GlcNAcylation Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Sheng; Yang, Feng; Camp, David G.; Rodland, Karin D.; Qian, Weijun; Liu, Tao; Smith, Richard D.

    2014-10-01

    O-GlcNAcylation is a dynamic protein post-translational modification of serine or threonine residues by an O-linked monosaccharide N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation was discovered three decades ago, and it has been shown to contribute to various disease states, such as metabolic diseases, cancer and neurological diseases. Yet it remains technically difficult to characterize comprehensively and quantitatively, due to its exceptionally low abundance and extremely labile nature under conventional tandem mass spectrometry conditions. Herein, we review the recent efforts for tackling these challenges in developing proteomic approaches for site-specific O-GlcNAcylation analysis, such as specific enrichment of O-GlcNAc peptides/proteins, unambiguous site-determination of O-GlcNAc modification, and quantitative analysis of O-GlcNAcylation.

  4. Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition.

    Science.gov (United States)

    Kim, Sanggil; Ko, Wooseok; Park, Hyunji; Lee, Hyun Soo

    2016-12-23

    There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

  5. Site-specific glycan-Peptide analysis for determination of N-glycoproteome heterogeneity

    DEFF Research Database (Denmark)

    Parker, Benjamin L; Thaysen-Andersen, Morten; Solis, Nestor;

    2013-01-01

    -tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7......A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography.......9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation...

  6. Access to site-specific Fc-cRGD peptide conjugates through streamlined expressed protein ligation.

    Science.gov (United States)

    Frutos, S; Jordan, J B; Bio, M M; Muir, T W; Thiel, O R; Vila-Perelló, M

    2016-10-12

    An ideal drug should be highly effective, non-toxic and be delivered by a convenient and painless single dose. We are still far from such optimal treatment but peptides, with their high target selectivity and low toxicity profiles, provide a very attractive platform from which to strive towards it. One of the major limitations of peptide drugs is their high clearance rates, which limit dosage regimen options. Conjugation to antibody Fc domains is a viable strategy to improve peptide stability by increasing their hydrodynamic radius and hijacking the Fc recycling pathway. We report the use of a split-intein based semi-synthetic approach to site-specifically conjugate a synthetic integrin binding peptide to an Fc domain. The strategy described here allows conjugating synthetic peptides to Fc domains, which is not possible via genetic methods, fully maintaining the ability of both the Fc domain and the bioactive peptide to interact with their binding partners.

  7. Use of Site-Specifically Tethered Chemical Nucleases to Study Macromolecular Reactions

    Directory of Open Access Journals (Sweden)

    Mukherjee Srabani

    2003-01-01

    Full Text Available During a complex macromolecular reaction multiple changes in molecular conformation and interactions with ligands may occur. X-ray crystallography may provide only a limited set of snapshots of these changes. Solution methods can augment such structural information to provide a more complete picture of a macromolecular reaction. We analyzed the changes in protein conformation and protein:nucleic acid interactions which occur during transcription initiation by using a chemical nuclease tethered to cysteines introduced site-specifically into the RNA polymerase of bacteriophage T7 (T7 RNAP. Changes in cleavage patterns as the polymerase steps through transcription reveal a series of structural transitions which mediate transcription initiation. Cleavage by tethered chemical nucleases is seen to be a powerful method for revealing the conformational dynamics of macromolecular reactions, and has certain advantages over cross-linking or energy transfer approaches.

  8. Hepatic DNA hydroxymethylation is site-specifically altered by chronic alcohol consumption and aging.

    Science.gov (United States)

    Tammen, Stephanie A; Park, Lara K; Dolnikowski, Gregory G; Ausman, Lynne M; Friso, Simonetta; Choi, Sang-Woon

    2017-03-01

    Global DNA hydroxymethylation is markedly decreased in human cancers, including hepatocellular carcinoma, which is associated with chronic alcohol consumption and aging. Because gene-specific changes in hydroxymethylcytosine may affect gene transcription, giving rise to a carcinogenic environment, we determined genome-wide site-specific changes in hepatic hydroxymethylcytosine that are associated with chronic alcohol consumption and aging. Young (4 months) and old (18 months) male C57Bl/6 mice were fed either an ethanol-containing Lieber-DeCarli liquid diet or an isocaloric control diet for 5 weeks. Genomic and gene-specific hydroxymethylcytosine patterns were determined through hydroxymethyl DNA immunoprecipitation array in hepatic DNA. Hydroxymethylcytosine patterns were more perturbed by alcohol consumption in young mice than in old mice (431 differentially hydroxymethylated regions, DhMRs, in young vs 189 DhMRs in old). A CpG island ~2.5 kb upstream of the glucocorticoid receptor gene, Nr3c1, had increased hydroxymethylation as well as increased mRNA expression (p = 0.015) in young mice fed alcohol relative to the control group. Aging alone also altered hydroxymethylcytosine patterns, with 331 DhMRs, but alcohol attenuated this effect. Aging was associated with a decrease in hydroxymethylcytosine ~1 kb upstream of the leptin receptor gene, Lepr, and decreased transcription of this gene (p = 0.029). Nr3c1 and Lepr are both involved in hepatic lipid homeostasis and hepatosteatosis, which may create a carcinogenic environment. These results suggest that the location of hydroxymethylcytosine in the genome is site specific and not random, and that changes in hydroxymethylation may play a role in the liver's response to aging and alcohol.

  9. Early precursors of site-specific cancers in college men and women.

    Science.gov (United States)

    Whittemore, A S; Paffenbarger, R S; Anderson, K; Lee, J E

    1985-01-01

    Physical and social characteristics recorded at college physical examination and reported in subsequent questionnaires to alumni in 1962 or 1966 by 50,000 former students from Harvard University and the University of Pennsylvania were reviewed for their relationship to major site-specific cancer occurrence. The records of 1,359 subjects who died with a major site-specific cancer in a 16- to 50-year follow-up period and of 672 subjects who reported such a cancer by mail questionnaire in 1976 or 1977 were compared with those of 8,084 matched classmates who were known to be alive and free of cancer at the time subjects with cancer had died or had been diagnosed. Cigarette smoking, as reported both in student years and years as alumni, predicted increased risk for cancers of the respiratory tract, pancreas, and bladder. Student coffee consumption was associated with elevated risk for leukemia, but it was unrelated to cancers of the pancreas and bladder. Male students with a record of proteinuria at college physical examination experienced increased risk of kidney cancer, and those with a history of tonsillectomy experienced increased risk of prostate cancer. Students who at college entrance reported occasional vague abdominal pain were at elevated risk for pancreatic and colorectal cancers in later years. Increased body weight during college was associated with increased risks of kidney and bladder cancers, whereas for alumni this index was associated only with kidney cancer. Increased weight-for-height during college (but not in 1962 or 1966) predicted increased occurrence of female breast cancer. Jewish students experienced elevated risk for subsequent cancers of the female breast, colon, and combined colorectum. These and other findings are presented as clues deserving further exploration for any etiologic significance that they may hold for the cancer sites studied.

  10. Novel therapeutic approaches for pulmonary arterial hypertension: Unique molecular targets to site-specific drug delivery.

    Science.gov (United States)

    Vaidya, Bhuvaneshwar; Gupta, Vivek

    2015-08-10

    Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by increased blood pressure in the small arterioles supplying blood to lungs for oxygenation. Advances in understanding of molecular and cellular biology techniques have led to the findings that PAH is indeed a cascade of diseases exploiting multi-faceted complex pathophysiology, with cellular proliferation and vascular remodeling being the key pathogenic events along with several cellular pathways involved. While current therapies for PAH do provide for amelioration of disease symptoms and acute survival benefits, their full therapeutic potential is hindered by patient incompliance and off-target side effects. To overcome the issues related with current therapy and to devise a more selective therapy, various novel pathways are being investigated for PAH treatment. In addition, inability to deliver anti-PAH drugs to the disease site i.e., distal pulmonary arterioles has been one of the major challenges in achieving improved patient outcomes and improved therapeutic efficacy. Several novel carriers have been explored to increase the selectivity of currently approved anti-PAH drugs and to act as suitable carriers for the delivery of investigational drugs. In the present review, we have discussed potential of various novel molecular pathways/targets including RhoA/Rho kinase, tyrosine kinase, endothelial progenitor cells, vasoactive intestinal peptide, and miRNA in PAH therapeutics. We have also discussed various techniques for site-specific drug delivery of anti-PAH therapeutics so as to improve the efficacy of approved and investigational drugs. This review will provide gainful insights into current advances in PAH therapeutics with an emphasis on site-specific drug payload delivery.

  11. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  12. phiC31 integrase-mediated site-specific recombination in barley.

    Directory of Open Access Journals (Sweden)

    Eszter Kapusi

    Full Text Available The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP, which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

  13. Site-Specific Conjugation to Native and Engineered Lysines in Human Immunoglobulins by Microbial Transglutaminase.

    Science.gov (United States)

    Spidel, Jared L; Vaessen, Benjamin; Albone, Earl F; Cheng, Xin; Verdi, Arielle; Kline, J Bradford

    2017-09-20

    The use of microbial transglutaminase (MTG) to produce site-specific antibody-drug conjugates (ADCs) has thus far focused on the transamidation of engineered acyl donor glutamine residues in an antibody based on the hypothesis that the lower specificity of MTG for acyl acceptor lysines may result in the transamidation of multiple native lysine residues, thereby yielding heterogeneous products. We investigated the utilization of native IgG lysines as acyl acceptor sites for glutamine-based acyl donor substrates. Of the approximately 80 lysines in multiple recombinant IgG monoclonal antibodies (mAbs), none were transamidated. Because recombinant mAbs lack the C-terminal Lys447 due to cleavage by carboxypeptidase B in the production cell host, we explored whether blocking the cleavage of Lys447 by the addition of a C-terminal amino acid could result in transamidation of Lys447 by a variety of acyl donor substrates. MTG efficiently transamidated Lys447 in the presence of any nonacidic, nonproline amino acid residue at position 448. Lysine scanning mutagenesis throughout the antibody further revealed several transamidation sites in both the heavy- and light-chain constant regions. Additionally, scanning mutagenesis of the hinge region in a Fab' fragment revealed sites of transamidation that were not reactive in the context of the full-length mAb. Here, we demonstrate the utility of single lysine substitutions and the C-terminal Lys447 for engineering efficient acyl acceptor sites suitable for site-specific conjugation to a range of glutamine-based acyl donor substrates.

  14. Wild yam

    Science.gov (United States)

    ... laboratory into various steroids, such as estrogen and dehydroepiandrosterone (DHEA). The root and the bulb of the plant ... wild yam and diosgenin promoted as a “natural DHEA.” This is because in the laboratory DHEA is ...

  15. Site-specific Protocol for Monitoring Marsh Birds : Don Edwards San Francisco Bay and San Pablo Bay National Wildlife Refuges

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This site-specific survey protocol provides standardized methods for monitoring marsh birds and was designed for use by the U.S. Fish and Wildlife Service (USFWS) on...

  16. A regulatory network modeled from wild-type gene expression data guides functional predictions in Caenorhabditis elegans development

    Directory of Open Access Journals (Sweden)

    Stigler Brandilyn

    2012-06-01

    Full Text Available Abstract Background Complex gene regulatory networks underlie many cellular and developmental processes. While a variety of experimental approaches can be used to discover how genes interact, few biological systems have been systematically evaluated to the extent required for an experimental definition of the underlying network. Therefore, the development of computational methods that can use limited experimental data to define and model a gene regulatory network would provide a useful tool to evaluate many important but incompletely understood biological processes. Such methods can assist in extracting all relevant information from data that are available, identify unexpected regulatory relationships and prioritize future experiments. Results To facilitate the analysis of gene regulatory networks, we have developed a computational modeling pipeline method that complements traditional evaluation of experimental data. For a proof-of-concept example, we have focused on the gene regulatory network in the nematode C. elegans that mediates the developmental choice between mesodermal (muscle and ectodermal (skin cell fates in the embryonic C lineage. We have used gene expression data to build two models: a knowledge-driven model based on gene expression changes following gene perturbation experiments, and a data-driven mathematical model derived from time-course gene expression data recovered from wild-type animals. We show that both models can identify a rich set of network gene interactions. Importantly, the mathematical model built only from wild-type data can predict interactions demonstrated by the perturbation experiments better than chance, and better than an existing knowledge-driven model built from the same data set. The mathematical model also provides new biological insight, including a dissection of zygotic from maternal functions of a key transcriptional regulator, PAL-1, and identification of non-redundant activities of the T-box genes

  17. A regulatory network modeled from wild-type gene expression data guides functional predictions in Caenorhabditis elegans development.

    Science.gov (United States)

    Stigler, Brandilyn; Chamberlin, Helen M

    2012-06-26

    Complex gene regulatory networks underlie many cellular and developmental processes. While a variety of experimental approaches can be used to discover how genes interact, few biological systems have been systematically evaluated to the extent required for an experimental definition of the underlying network. Therefore, the development of computational methods that can use limited experimental data to define and model a gene regulatory network would provide a useful tool to evaluate many important but incompletely understood biological processes. Such methods can assist in extracting all relevant information from data that are available, identify unexpected regulatory relationships and prioritize future experiments. To facilitate the analysis of gene regulatory networks, we have developed a computational modeling pipeline method that complements traditional evaluation of experimental data. For a proof-of-concept example, we have focused on the gene regulatory network in the nematode C. elegans that mediates the developmental choice between mesodermal (muscle) and ectodermal (skin) cell fates in the embryonic C lineage. We have used gene expression data to build two models: a knowledge-driven model based on gene expression changes following gene perturbation experiments, and a data-driven mathematical model derived from time-course gene expression data recovered from wild-type animals. We show that both models can identify a rich set of network gene interactions. Importantly, the mathematical model built only from wild-type data can predict interactions demonstrated by the perturbation experiments better than chance, and better than an existing knowledge-driven model built from the same data set. The mathematical model also provides new biological insight, including a dissection of zygotic from maternal functions of a key transcriptional regulator, PAL-1, and identification of non-redundant activities of the T-box genes tbx-8 and tbx-9. This work provides a strong

  18. Piv site-specific invertase requires a DEDD motif analogous to the catalytic center of the RuvC Holliday junction resolvases.

    Science.gov (United States)

    Buchner, John M; Robertson, Anne E; Poynter, David J; Denniston, Shelby S; Karls, Anna C

    2005-05-01

    Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.

  19. Assessment of Site Specific Mutational Effect on Transcription Initiation at Escherichia coli Promoter

    Directory of Open Access Journals (Sweden)

    S. Kannan

    2009-01-01

    Full Text Available Problem statement: It is widely accepted thought that the weak promoters control the RNA synthesis and play regulatory role in complex genetic networks in bacterial system. An experiment had been designed to address whether mutations in the -16/-17 region affect the rate of transcription at an activator-independent promoter in E. coli or not? Approach: The aim of this study was to determine whether mutations in the -16/-17 region affect the rate of expression at an activator-dependent promoter in JM109 strain of E. coli. Primers were constructed to amplify the mutant promoter genes through PCR. The amplified PCR product was checked and then inserted into the MCS region of pAA128 plasmid. Further the plasmid vector was transformed into JM109 strain of E. coli and then cloned the selected transformats. Finally, the plasmid from each mutant colony was then sequenced using the protocol supplied with the Amersham Pharmacia Biotech T7 sequencing Kit. The JM109 cultures for which the sequences were determined, then assayed for ß-galactosidase activity to assess the rate of gene expression from the altered promoters. Results: The present investigation revealed that the extended-10 promoter region has a substantial effect on the rate of transcription at weak promoter sequence and also bearing little resemblance to the consensus sequence recognized by RNA. The expression of the genetically engineered plasmid proved that the 2 bps (-16 and -17 base pair found adjacently upstream of the extended-10 promoter have an effect on the level of transcription. This was achieved by site specific base substitutions into the weak promoter of a modified lac operon lacking any activator or repressor binding sites. The results from gene expression assays of several mutants showed a distinct preference for either GG or TT located adjacently upstream of the extended promoter element. Thus the present study emphasized that

  20. Environmental Management Waste Management Facility (EMWMF) Site-Specific Health and Safety Plan, Oak Ridge, Tennessee

    Energy Technology Data Exchange (ETDEWEB)

    Flynn, N.C. Bechtel Jacobs

    2008-04-21

    The Bechtel Jacobs Company LLC (BJC) policy is to provide a safe and healthy workplace for all employees and subcontractors. The implementation of this policy requires that operations of the Environmental Management Waste Management Facility (EMWMF), located one-half mile west of the U.S. Department of Energy (DOE) Y-12 National Security Complex, be guided by an overall plan and consistent proactive approach to environment, safety and health (ES&H) issues. The BJC governing document for worker safety and health, BJC/OR-1745, 'Worker Safety and Health Program', describes the key elements of the BJC Safety and Industrial Hygiene (IH) programs, which includes the requirement for development and implementation of a site-specific Health and Safety Plan (HASP) where required by regulation (refer also to BJC-EH-1012, 'Development and Approval of Safety and Health Plans'). BJC/OR-1745, 'Worker Safety and Health Program', implements the requirements for worker protection contained in Title 10 Code of Federal Regulations (CFR) Part 851. The EMWMF site-specific HASP requirements identifies safe operating procedures, work controls, personal protective equipment, roles and responsibilities, potential site hazards and control measures, site access requirements, frequency and types of monitoring, site work areas, decontamination procedures, and outlines emergency response actions. This HASP will be available on site for use by all workers, management and supervisors, oversight personnel and visitors. All EMWMF assigned personnel will be briefed on the contents of this HASP and will be required to follow the procedures and protocols as specified. The policies and procedures referenced in this HASP apply to all EMWMF operations activities. In addition the HASP establishes ES&H criteria for the day-to-day activities to prevent or minimize any adverse effect on the environment and personnel safety and health and to meet standards that define acceptable

  1. Streaming Clumps Ejection Model and the Heterogeneous Inner Coma of Comet Wild 2

    Science.gov (United States)

    Clark, B. C.; Economou, T. E.; Green, S. F.; Sandford, S. A.; Zolensky, M. E.

    2004-01-01

    The conventional concept of cometary comae is that they are dominated by fine particulates released individually by sublimation of surface volatiles and subsequent entrainment in the near-surface gas. It has long been recognized that such particulates could be relatively large, with early estimates that objects perhaps up to one meter in size may be levitated from the surface of the typical cometary nucleus. However, the general uniformity and small average particulate size of observed comae and the relatively smooth, monotonic increases and decreases in particle density during the Giotto flythrough of comet Halley s coma in 1986 reinforced the view that the bulk of the particles are released at the surface, are fine-sized and inert. Jets have been interpreted as geometrically constrained release of these particulates. With major heterogeneities observed during the recent flythrough of the inner coma of comet Wild 2, these views deserve reconsideration.

  2. Streaming Clumps Ejection Model and the Heterogeneous Inner Coma of Comet Wild 2

    Science.gov (United States)

    Clark, B. C.; Economou, T. E.; Green, S. F.; Sandford, S. A.; Zolensky, M. E.

    2004-01-01

    The conventional concept of cometary comae is that they are dominated by fine particulates released individually by sublimation of surface volatiles and subsequent entrainment in the near-surface gas. It has long been recognized that such particulates could be relatively large, with early estimates that objects perhaps up to one meter in size may be levitated from the surface of the typical cometary nucleus. However, the general uniformity and small average particulate size of observed comae and the relatively smooth, monotonic increases and decreases in particle density during the Giotto flythrough of comet Halley s coma in 1986 reinforced the view that the bulk of the particles are released at the surface, are fine-sized and inert. Jets have been interpreted as geometrically constrained release of these particulates. With major heterogeneities observed during the recent flythrough of the inner coma of comet Wild 2, these views deserve reconsideration.

  3. Assessing display variability in wild brown anoles Anolis sagrei using a mechanical lizard model

    Directory of Open Access Journals (Sweden)

    Sarah R. PARTAN, Peter OTOVIC, Virginia L. PRICE, Scott E. BROWN

    2011-04-01

    Full Text Available Signals used for species identity ought to be highly stereotyped so as to facilitate immediate recognition by conspecifics. It is surprising therefore to find variability in putative species signature displays. The brown anole Anolis sagrei has a high degree of variability in its signature bobbing display. In this study we collected descriptive data on variability in the temporal structure of wild brown anole bobbing patterns, finding that no two displays analyzed had the same temporal structure, and we also tested whether wild brown anoles prefer the signature display over an alternate display pattern by using mechanical robot playbacks in the field. As a response metric we assessed whether or not the lizards showed social responses (pushup, dewlap extension, or head-nod in response to the robotic presentations. We found that the lizards responded slightly more to the signature than to the alternate pattern, providing support for the idea that despite the variability seen in displays, the signature pattern is meaningful to them. We tested two other independent variables: speed of the display and elevation of the robot during its display, neither of which was significant. Dewlap extensions were given predominantly by adult males and were more likely to be given in the breeding season than the nonbreeding season. Pushups and head-nods were given equally by males and a combined class of females and juveniles, and were not seasonal. Head-nods increased after the robot turned off, suggesting that they may be used in a conversational turn-taking style during communication [Current Zoology 57 (2: 140–152, 2011].

  4. Assessing display variability in wild brown anoles Anolis sagrei using a mechanical lizard model

    Institute of Scientific and Technical Information of China (English)

    Sarah R.PARTAN; Peter OTOVIC; Virginia L.PRICE; Scott E.BROWN

    2011-01-01

    Signals used for species identity ought to be highly stereotyped so as to facilitate immediate recognition by conspecifics.It is surprising therefore to find variability in putative species signature displays.The brown anole Anolis sasrei has a high degree of variability in its signature bobbing display.In this study we collected descriptive data on variability in the temporal stiucture of wild brown anole bobbing patterns,finding that no two displays analyzed had the same temporal structure,and we also tested whether wild brown anoles prefer the signature display over an alternate display pattern by using mechanical robot playbacks in the field.As a response metric we assessed whether or not the lizards showed social responses(pushup,dewlap extension,or head-nod)in response to the robotic presentations.We found that the lizards responded slightly more to the signature than to the alternate pattern,providing support for the idea that despite the variability seen in displays,the signature pattern is meaningful to them.We tested two other independent variables:speed of the display and elevation of the robot during its display,neither of which was significant.Dewlap extensions were given predominantly by adult males and were more likely to be given in the breeding season than the nonbreeding season.Pushups and head-nods were given equally by males and a combined class of females and juveniles,and were not seasonal.Head-nods increased after the robot turned off,suggesting that they may be used in a conversational turn-taking style during communication.

  5. Embracing the emerging precision agriculture technologies for site-specific management of yield-limiting factors.

    Science.gov (United States)

    Melakeberhan, H

    2002-09-01

    Precision agriculture (PA) is providing an information revolution using Global Positioning (GPS) and Geographic Information (GIS) systems and Remote Sensing (RS). These technologies allow better decision making in the management of crop yield-limiting biotic and abiotic factors and their interactions on a site-specific (SSM) basis in a wide range of production systems. Characterizing the nature of the problem(s) and public education are among the challenges that scientists, producers, and industry face when adapting PA technologies. To apply SSM, spatio-temporal characteristics of the problem(s) need to be determined and variations within a field demonstrated. Spatio-temporal characteristics of a given pathogen or pest problem may be known but may not be the only or primary cause of the problem. Hence, exact cause-and-effect relationships need to be established by incorporating GIS, GPS, and RS-generated data as well as possible interactions. Exploiting the potential of PA technologies in sustainable ways depends on whether or not we first ask ''Are we doing the right thing?'' (strategic) as opposed to ''Are we doing it right?'' (tactical).

  6. Site-Specific Zwitterionic Polymer Conjugates of a Protein Have Long Plasma Circulation.

    Science.gov (United States)

    Bhattacharjee, Somnath; Liu, Wenge; Wang, Wei-Han; Weitzhandler, Isaac; Li, Xinghai; Qi, Yizhi; Liu, Jinyao; Pang, Yan; Hunt, Donald F; Chilkoti, Ashutosh

    2015-11-01

    Many proteins suffer from suboptimal pharmacokinetics (PK) that limit their utility as drugs. The efficient synthesis of polymer conjugates of protein drugs with tunable PK to optimize their in vivo efficacy is hence critical. We report here the first study of the in vivo behavior of a site-specific conjugate of a zwitterionic polymer and a protein. To synthesize the conjugate, we first installed an initiator for atom-transfer radical polymerization (ATRP) at the N terminus of myoglobin (Mb-N-Br). Subsequently, in situ ATRP was carried out in aqueous buffer to grow an amine-functionalized polymer from Mb-N-Br. The cationic polymer was further derivatized to two zwitterionic polymers by treating the amine groups of the cationic polymer with iodoacetic acid to obtain poly(carboxybetaine methacrylate) with a one-carbon spacer (PCBMA; C1 ), and sequentially with 3-iodopropionic acid and iodoacetic acid to obtain PCBMA(mix) with a mixture of C1 and C2 spacers. The Mb-N-PCBMA polymer conjugates had a longer in vivo plasma half-life than a PEG-like comb polymer conjugate of similar molecular weights (MW). The structure of the zwitterion plays a role in controlling the in vivo behavior of the conjugate, as the PCBMA conjugate with a C1 spacer had significantly longer plasma circulation than the conjugate with a mixture of C1 and C2 spacers.

  7. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

    Science.gov (United States)

    Onouchi, H; Yokoi, K; Machida, C; Matsuzaki, H; Oshima, Y; Matsuoka, K; Nakamura, K; Machida, Y

    1991-12-11

    Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

  8. Site-specific function and regulation of Osterix in tooth root formation

    Science.gov (United States)

    He, Y. D.; Sui, B. D.; Li, M.; Huang, J.; Chen, S.; Wu, L. A.

    2016-01-01

    Congenital diseases of tooth roots, in terms of developmental abnormalities of short and thin root phenotypes, can lead to loss of teeth. A more complete understanding of the genetic molecular pathways and biological processes controlling tooth root formation is required. Recent studies have revealed that Osterix (Osx), a key mesenchymal transcriptional factor participating in both the processes of osteogenesis and odontogenesis, plays a vital role underlying the mechanisms of developmental differences between root and crown. During tooth development, Osx expression has been identified from late embryonic to postnatal stages when the tooth root develops, particularly in odontoblasts and cementoblasts to promote their differentiation and mineralization. Furthermore, the site-specific function of Osx in tooth root formation has been confirmed, because odontoblastic Osx-conditional knockout mice demonstrate primarily short and thin root phenotypes with no apparent abnormalities in the crown (Journal of Bone and Mineral Research 30, 2014 and 742, Journal of Dental Research 94, 2015 and 430). These findings suggest that Osx functions to promote odontoblast and cementoblast differentiation and root elongation only in root, but not in crown formation. Mechanistic research shows regulatory networks of Osx expression, which can be controlled through manipulating the epithelial BMP signalling, mesenchymal Runx2 expression and cellular phosphorylation levels, indicating feasible routes of promoting Osx expression postnatally (Journal of Cellular Biochemistry 114, 2013 and 975). In this regard, a promising approach might be available to regenerate the congenitally diseased root and that regenerative therapy would be the best choice for patients with developmental tooth diseases. PMID:26599722

  9. A Site-Specific Index Based on Weathering Forms Visible in Central Oxford, UK

    Directory of Open Access Journals (Sweden)

    Mary J. Thornbush

    2012-11-01

    Full Text Available The authenticity of much of the stone-work along Queen’s Lane in central Oxford, UK presented an opportunity to produce a photographic survey from which a weathering index could be established. This represents a site-specific approach to devising a weathering form. Because it is photo-based, weathering forms are visible for comparison and classification purposes across disciplines. Limestone pertaining to building ashlar and plinths along this roadway, which mainly belong to Queen’s College, St Edmund Hall, New College, and Hertford College, was classified according to this newly introduced weathering index, the size-extent (S-E index, through consideration of type, size, extent, impact, and trigger. This size- (range and extent-based classification system enables for the assessment of weathering forms of various types, including soiling and decay features as well as those potentially expected in the presence of vegetation and animals. Weathering forms of a range of sizes were present, with a slightly greater abundance of small types (mm-cm in the micro- to mesoscale and more discrete types with a low extent. For this location in central Oxford, chemical weathering was found to be the predominant type of soiling and decay.

  10. Gated rotation mechanism of site-specific recombination by ϕC31 integrase.

    Science.gov (United States)

    Olorunniji, Femi J; Buck, Dorothy E; Colloms, Sean D; McEwan, Andrew R; Smith, Margaret C M; Stark, W Marshall; Rosser, Susan J

    2012-11-27

    Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

  11. A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance.

    Science.gov (United States)

    Sandersjöö, Lisa; Kostallas, George; Löfblom, John; Samuelson, Patrik

    2014-01-01

    Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

  12. Site-specific integration and tailoring of cassette design for sustainable gene transfer.

    Science.gov (United States)

    Lombardo, Angelo; Cesana, Daniela; Genovese, Pietro; Di Stefano, Bruno; Provasi, Elena; Colombo, Daniele F; Neri, Margherita; Magnani, Zulma; Cantore, Alessio; Lo Riso, Pietro; Damo, Martina; Pello, Oscar M; Holmes, Michael C; Gregory, Philip D; Gritti, Angela; Broccoli, Vania; Bonini, Chiara; Naldini, Luigi

    2011-08-21

    Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

  13. Site-specific genetic engineering of the Anopheles gambiae Y chromosome.

    Science.gov (United States)

    Bernardini, Federica; Galizi, Roberto; Menichelli, Miriam; Papathanos, Philippos-Aris; Dritsou, Vicky; Marois, Eric; Crisanti, Andrea; Windbichler, Nikolai

    2014-05-27

    Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line-specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.

  14. Magnetic norbornene polymer as multiresponsive nanocarrier for site specific cancer therapy.

    Science.gov (United States)

    Rao N, Vijayakameswara; Ganivada, Mutyala Naidu; Sarkar, Santu; Dinda, Himadri; Chatterjee, Koushik; Dalui, Tanmoy; Das Sarma, Jayasri; Shunmugam, Raja

    2014-02-19

    A site-specific, stimuli-responsive nanocarrier has been synthesized by conjugating folate, magnetic particles and doxorubicin to the backbone of norbornene polymer. Monomers, namely, cis-5-norbornene-6-(diethoxyphosphoryl)hexanote (mono 1), norbornene grafted poly(ethyleneglycol)-folate (mono 2), and norbornene derived doxorubicin (mono 3) are carefully designed to demonstrate the smart nanorcarrier capabilities. The synthesis and complete characterization of all three monomers are elaborately discussed. Their copolymerization is done by controlled/living ring-opening metathesis polymerization (ROMP) to get the triblock copolymer PHOS-FOL-DOX. NMR spectroscopy and gel permeation chromatography confirm the formation of the triblock copolymer, while FT-IR spectroscopy, thermogravimetric analysis, along with transmission electron microscope confirm the anchoring of iron particle (Fe3O4) to the PHOS-FOL-DOX. Drug release profile shows the importance of having the hydrazone linker that helps to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. The newly designed nanocarrier shows greater internalization (about 8 times) due to magnetic field. Also, increased intracellular DOX release is observed due to the folate receptor. From these results, it is clear that PHOS-FOL-DOX has the potential to act as a smart nanoreservoir with the magnetic field guidance, folate receptor targeting, and finally pH stimulation.

  15. Site Specific Effect of Tobacco Addiction in Upper Aerodigestive Tract Tumors: A Retrospective Clinicopathological Study

    Directory of Open Access Journals (Sweden)

    Ashok Kumar

    2014-01-01

    Full Text Available An institutional study was carried out in 102 patients to investigate the site specific effect of addictions, that is, tobacco smoking and tobacco chewing (smokeless, both independently and synergistically in development of malignancies in upper aerodigestive tract through retrograde questionnaire. The histopathologically proven cases were interviewed regarding different forms of addictions followed by clinical examination and investigations for grading (according to Modified Broadmann’s method and TNM staging (according to UICC according to the tumor site. Statistical analysis was done by Pearson test. Out of all proven cases of cancers, 29.4% were only tobacco chewers (smokeless, 25.5% were only smokers, 42.2% were having both types of tobacco addictions (smoke and smokeless, and only 2.9% were having no addiction. Out of only tobacco chewers (smokeless, 83.3% were of oral cavity cancers, 6.7% were of oro- and hypopharynx and the rest were of others. Among only smokers, 69.2% cases were of laryngeal and oro- and hypopharynx as compared to 11.5% of oral cavity cancers (nearly 6 times. Tobacco (smokeless chewing is associated with oral cancers whereas tobacco smoking is associated with laryngeal and hypopharyngeal carcinoma. Both smoking and smokeless tobacco act in synergy with each other.

  16. Site-Specific Analysis of Geothermal Development-Data Files of Prospective Sites

    Energy Technology Data Exchange (ETDEWEB)

    Williams, F.; Cohen, A.; Pfundstein, R.; Pond, S.

    1977-10-01

    This document presents site-specific data and sample development schedules for the first plant on line at 30 hydrothermal and 7 geopressured prospective sites (prospects) that are believed to be suited for supporting the geothermal generation of electricity. This report includes many prospects from an earlier METREK report on geothermal development scenarios. The list has been augmented with other sites chosen as development prospects by the division of Geothermal Energy (DGE) of the Energy Research and Development Administration (ERDA). The DGE additions include a general area called ''Cascade Range''. METREK has chosen the following specific Cascade Range Sites in place of that general area: Baker Hot Springs, Mount Hood, Lassen and Glass Mountain/ Diablo. All the prospects have been selected on the basis of current knowledge of hydrothermal and geopressured resources. The selection is intended for program planning purposes. Neither METREK nor the Federal government warrants that any of these sites will necessarily be developed, nor does their selection necessarily imply any commitment on the part of the Federal government to their development. [DJE-2005

  17. Site specific seismic hazard analysis and determination of response spectra of Kolkata for maximum considered earthquake

    Science.gov (United States)

    Shiuly, Amit; Sahu, R. B.; Mandal, Saroj

    2017-06-01

    This paper presents site specific seismic hazard analysis of Kolkata city, former capital of India and present capital of state West Bengal, situated on the world’s largest delta island, Bengal basin. For this purpose, peak ground acceleration (PGA) for a maximum considered earthquake (MCE) at bedrock level has been estimated using an artificial neural network (ANN) based attenuation relationship developed on the basis of synthetic ground motion data for the region. Using the PGA corresponding to the MCE, a spectrum compatible acceleration time history at bedrock level has been generated by using a wavelet based computer program, WAVEGEN. This spectrum compatible time history at bedrock level has been converted to the same at surface level using SHAKE2000 for 144 borehole locations in the study region. Using the predicted values of PGA and PGV at the surface, corresponding contours for the region have been drawn. For the MCE, the PGA at bedrock level of Kolkata city has been obtained as 0.184 g, while that at the surface level varies from 0.22 g to 0.37 g. Finally, Kolkata has been subdivided into eight seismic subzones, and for each subzone a response spectrum equation has been derived using polynomial regression analysis. This will be very helpful for structural and geotechnical engineers to design safe and economical earthquake resistant structures.

  18. Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA

    Science.gov (United States)

    Birgisdottir, Åsa B.; Johansen, Steinar

    2005-01-01

    The wide, but scattered distribution of group I introns in nature is a result of two processes; the vertical inheritance of introns with or without losses, and the occasional transfer of introns across species barriers. Reversal of the group I intron self-splicing reaction, termed reverse splicing, coupled with reverse transcription and genomic integration potentially mediate an RNA-based intron mobility pathway. Compared to the well characterized endonuclease-mediated intron homing, reverse splicing is less specific and represents a likely explanation for many intron transpositions into new genomic sites. However, the frequency and general role of an RNA-based mobility pathway in the spread of natural group I introns is still unclear. We have used the twin-ribozyme intron (Dir.S956-1) from the myxomycete Didymium iridis to test how a mobile group I intron containing a homing endonuclease gene (HEG) selects between potential insertion sites in the small subunit (SSU) rRNA in vitro, in Escherichia coli and in yeast. Surprisingly, the results show a site-specific RNA-based targeting of Dir.S956-1 into its natural (S956) SSU rRNA site. Our results suggest that reverse splicing, in addition to the established endonuclease-mediated homing mechanism, potentially accounts for group I intron spread into the homologous sites of different strains and species. PMID:15817568

  19. Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

    Science.gov (United States)

    Liu, Tao; Tian, Chang Fu; Chen, Wen Xin

    2015-01-01

    Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis), ActR (pH stress and microaerobic adaption), SupA (potassium stress), chaperonin GroEL2 (viability and potentially symbiosis), and ExoP (succinoglycan synthesis and secretion). These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

  20. Site-Specific Ser/Thr/Tyr Phosphoproteome of Sinorhizobium meliloti at Stationary Phase.

    Directory of Open Access Journals (Sweden)

    Tao Liu

    Full Text Available Sinorhizobium meliloti, a facultative microsymbiont of alfalfa, should fine-tune its cellular processes to live saprophytically in soils characterized with limited nutrients and diverse stresses. In this study, TiO2 enrichment and LC-MS/MS were used to uncover the site-specific Ser/Thr/Tyr phosphoproteome of S. meliloti in minimum medium at stationary phase. There are a total of 96 unique phosphorylated sites, with a Ser/Thr/Tyr distribution of 63:28:5, in 77 proteins. Phosphoproteins identified in S. meliloti showed a wide distribution pattern regarding to functional categories, such as replication, transcription, translation, posttranslational modification, transport and metabolism of amino acids, carbohydrate, inorganic ion, succinoglycan etc. Ser/Thr/Tyr phosphosites identified within the conserved motif in proteins of key cellular function indicate a crucial role of phosphorylation in modulating cellular physiology. Moreover, phosphorylation in proteins involved in processes related to rhizobial adaptation was also discussed, such as those identified in SMa0114 and PhaP2 (polyhydroxybutyrate synthesis, ActR (pH stress and microaerobic adaption, SupA (potassium stress, chaperonin GroEL2 (viability and potentially symbiosis, and ExoP (succinoglycan synthesis and secretion. These Ser/Thr/Tyr phosphosites identified herein would be helpful for our further investigation and understanding of the role of phosphorylation in rhizobial physiology.

  1. Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

    Science.gov (United States)

    Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

    Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

  2. Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion

    DEFF Research Database (Denmark)

    Zhen, W P; Buchardt, O; Nielsen, Henrik

    1986-01-01

    A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair...... fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen...... was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of the pi-pi electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA....

  3. Design of a soil cutting resistance sensor for application in site-specific tillage.

    Science.gov (United States)

    Agüera, Juan; Carballido, Jacob; Gil, Jesús; Gliever, Chris J; Perez-Ruiz, Manuel

    2013-05-10

    One objective of precision agriculture is to provide accurate information about soil and crop properties to optimize the management of agricultural inputs to meet site-specific needs. This paper describes the development of a sensor equipped with RTK-GPS technology that continuously and efficiently measures soil cutting resistance at various depths while traversing the field. Laboratory and preliminary field tests verified the accuracy of this prototype soil strength sensor. The data obtained using a hand-operated soil cone penetrometer was used to evaluate this field soil compaction depth profile sensor. To date, this sensor has only been tested in one field under one gravimetric water content condition. This field test revealed that the relationships between the soil strength profile sensor (SSPS) cutting force and soil cone index values are assumed to be quadratic for the various depths considered: 0-10, 10-20 and 20-30 cm (r2 = 0.58, 0.45 and 0.54, respectively). Soil resistance contour maps illustrated its practical value. The developed sensor provides accurate, timely and affordable information on soil properties to optimize resources and improve agricultural economy.

  4. Design of a Soil Cutting Resistance Sensor for Application in Site-Specific Tillage

    Directory of Open Access Journals (Sweden)

    Manuel Perez-Ruiz

    2013-05-01

    Full Text Available One objective of precision agriculture is to provide accurate information about soil and crop properties to optimize the management of agricultural inputs to meet site-specific needs. This paper describes the development of a sensor equipped with RTK-GPS technology that continuously and efficiently measures soil cutting resistance at various depths while traversing the field. Laboratory and preliminary field tests verified the accuracy of this prototype soil strength sensor. The data obtained using a hand-operated soil cone penetrometer was used to evaluate this field soil compaction depth profile sensor. To date, this sensor has only been tested in one field under one gravimetric water content condition. This field test revealed that the relationships between the soil strength profile sensor (SSPS cutting force and soil cone index values are assumed to be quadratic for the various depths considered: 0–10, 10–20 and 20–30 cm (r2 = 0.58, 0.45 and 0.54, respectively. Soil resistance contour maps illustrated its practical value. The developed sensor provides accurate, timely and affordable information on soil properties to optimize resources and improve agricultural economy.

  5. Site-specific conjugation of bifunctional chelator BAT to mouse IgG1 Fab' fragment

    Institute of Scientific and Technical Information of China (English)

    Jun LI; Xue-hao WANG; Xiao-ming WANG; Zhao-lai CHEN

    2006-01-01

    Aim: To perform a site-specific conjugation of Fab' fragments of a mouse monoclonal antibody(MoAb) B43(of IgG1 subtype) to a bifunctional chelator 6-[p-(bromoacetamido) benzyl]-l,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (BAT) via the thiol groups in the hinge distal to the antigenbinding site of the Fab'. Methods: B43 was cleaved using a simple 2-step method.First, stable F(ab')2 was produced by pepsin treatment. Fab' with free thiol in the hinge region was then obtained by cysteine reduction of F(ab')2. Second, a sitespecific conjugation of Fab' to thiol-specific BAT was performed in a one-step reaction. Results: The Fab' fragment had approximately 1.8 free thiol groups per molecule after cysteine reduction. The conjugation efficiency and the chemical yield were approximately 1.28 moles chelator/Fab' and 74% of the initial concentration of Fab', respectively. The F(ab')2, Fab' and Fab'-BAT all maintained reasonable antigen-binding properties. 67Cu labeling of the conjugate under standard conditions did not impair the immunoreactivity of Fab'-BAT. Conclusion: This is a simple and efficient method for producing immunoreactive conjugates of Fab'-BAT, which can be used to make radiometal-labeled conjugates for further diagnostic and therapeutic applications.

  6. Applications of the site-specific recombinase Cre to the study of genomic imprinting.

    Science.gov (United States)

    Oh-McGinnis, Rosemary; Jones, Meaghan J; Lefebvre, Louis

    2010-07-01

    The development of gene targeting approaches has had a tremendous impact on the functional analysis of the mouse genome. A specific application of this technique has been the adaptation of the bacteriophage P1 Cre/loxP site-specific recombinase system which allows for the precise recombination between two loxP sites, resulting in deletion or inversion of the intervening sequences. Because of the efficiency of this system, it can be applied to conditional deletions of relatively short coding sequences or regulatory elements but also to more extensive chromosomal rearrangement strategies. Both mechanistic and functional studies of genomic imprinting have benefited from the development of the Cre/loxP technology. Since imprinted genes within large chromosomal regions are regulated by the action of cis-acting sequences known as imprinting centers, chromosomal engineering approaches are particularly well suited to the elucidation of long-range mechanisms controlling the imprinting of autosomal genes. Here we review the applications of the Cre/loxP technology to the study of genomic imprinting, highlight important insights gained from these studies and discuss future directions in the field.

  7. Novel method for the high-throughput production of phosphorylation site-specific monoclonal antibodies

    Science.gov (United States)

    Kurosawa, Nobuyuki; Wakata, Yuka; Inobe, Tomonao; Kitamura, Haruki; Yoshioka, Megumi; Matsuzawa, Shun; Kishi, Yoshihiro; Isobe, Masaharu

    2016-01-01

    Threonine phosphorylation accounts for 10% of all phosphorylation sites compared with 0.05% for tyrosine and 90% for serine. Although monoclonal antibody generation for phospho-serine and -tyrosine proteins is progressing, there has been limited success regarding the production of monoclonal antibodies against phospho-threonine proteins. We developed a novel strategy for generating phosphorylation site-specific monoclonal antibodies by cloning immunoglobulin genes from single plasma cells that were fixed, intracellularly stained with fluorescently labeled peptides and sorted without causing RNA degradation. Our high-throughput fluorescence activated cell sorting-based strategy, which targets abundant intracellular immunoglobulin as a tag for fluorescently labeled antigens, greatly increases the sensitivity and specificity of antigen-specific plasma cell isolation, enabling the high-efficiency production of monoclonal antibodies with desired antigen specificity. This approach yielded yet-undescribed guinea pig monoclonal antibodies against threonine 18-phosphorylated p53 and threonine 68-phosphorylated CHK2 with high affinity and specificity. Our method has the potential to allow the generation of monoclonal antibodies against a variety of phosphorylated proteins. PMID:27125496

  8. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

    Directory of Open Access Journals (Sweden)

    James I. McDonald

    2016-06-01

    Full Text Available Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.

  9. Site-Specific Variability in the Chemical Diversity of the Antarctic Red Alga Plocamium cartilagineum

    Directory of Open Access Journals (Sweden)

    Ryan M. Young

    2013-06-01

    Full Text Available Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like “P. cartilagineum” from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km2 area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation.

  10. Site-specific variability in the chemical diversity of the Antarctic red alga Plocamium cartilagineum.

    Science.gov (United States)

    Young, Ryan M; von Salm, Jacqueline L; Amsler, Margaret O; Lopez-Bautista, Juan; Amsler, Charles D; McClintock, James B; Baker, Bill J

    2013-06-14

    Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like "P. cartilagineum" from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km² area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation.

  11. Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination.

    Science.gov (United States)

    Buchholz, F; Ringrose, L; Angrand, P O; Rossi, F; Stewart, A F

    1996-11-01

    Genomic manipulations using site-specific recombinases rely on their applied characteristics in living systems. To understand their applied properties so that they can be optimally deployed, we compared the recombinases FLP and Cre in two assays. In both Escherichia coli and in vitro, FLP shows a different temperature optimum than Cre. FLP is more thermolabile, having an optimum near 30 degrees C and little detectable activity above 39 degrees C. Cre is optimally efficient at 37 degrees C and above. Consistent with FLP thermolability, recombination in a mammalian cell line mediated by a ligand- regulated FLP-androgen receptor fusion protein is more efficient at 35 degrees C than at higher temperatures. We also document a mutation in a commercially available FLP plasmid (FLP-F70L) which renders this recombinase even more thermolabile. The different temperature optima of FLP, FLP-F70L and Cre influence their strategies of usage. Our results recommend the use of Cre for applications in mice that require efficient recombination. The thermolabilities of FLP and FLP-F70L can be usefully exploited for gain of function and cell culture applications.

  12. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis.

  13. Site-specific muscle hyper-reactivity in musicians with occupational upper limb pain.

    Science.gov (United States)

    Moulton, B; Spence, S H

    1992-07-01

    Fourteen musicians who reported a history of pain in the upper limb associated with the playing of their instruments were compared with a sample of pain-free musicians, matched for age, sex and musical instrument. Four tasks were presented in random order and included neutral, general stressor, personal stressor and pain stressor tasks. Ratings of stressfulness and recordings of skin conductance level confirmed the effectiveness of the experimental manipulations for both subject groups. No differences were found between groups or tasks for frontalis surface electromyograph (EMG) activity. Evidence was found, however, of EMG elevation in flexor and trapezius muscles on the pain side for the pain subjects, in response to the task involving recall of a pain experience. This elevation was not found for the pain-free controls or for other stressor tasks, although some elevation in response to the pain stressor task was found for pain subjects in the trapezius muscles of the non-pain side. The duration of return to baseline of EMG following the pain stressor task was found to be extended in pain subjects for the trapezius, but not for the flexor muscles of the pain side. The findings suggest that site-specific muscle hyper-reactivity may play a role in the development and maintenance of occupational upper limb pain in musicians.

  14. Site-specific identification of heparan and chondroitin sulfate glycosaminoglycans in hybrid proteoglycans

    Science.gov (United States)

    Noborn, Fredrik; Gomez Toledo, Alejandro; Green, Anders; Nasir, Waqas; Sihlbom, Carina; Nilsson, Jonas; Larson, Göran

    2016-01-01

    Heparan sulfate (HS) and chondroitin sulfate (CS) are complex polysaccharides that regulate important biological pathways in virtually all metazoan organisms. The polysaccharides often display opposite effects on cell functions with HS and CS structural motifs presenting unique binding sites for specific ligands. Still, the mechanisms by which glycan biosynthesis generates complex HS and CS polysaccharides required for the regulation of mammalian physiology remain elusive. Here we present a glycoproteomic approach that identifies and differentiates between HS and CS attachment sites and provides identity to the core proteins. Glycopeptides were prepared from perlecan, a complex proteoglycan known to be substituted with both HS and CS chains, further digested with heparinase or chondroitinase ABC to reduce the HS and CS chain lengths respectively, and thereafter analyzed by nLC-MS/MS. This protocol enabled the identification of three consensus HS sites and one hybrid site, carrying either a HS or a CS chain. Inspection of the amino acid sequence at the hybrid attachment locus indicates that certain peptide motifs may encode for the chain type selection process. This analytical approach will become useful when addressing fundamental questions in basic biology specifically in elucidating the functional roles of site-specific glycosylations of proteoglycans. PMID:27694851

  15. Assembled modules technology for site-specific prolonged delivery of norfloxacin.

    Science.gov (United States)

    Oliveira, Paulo Renato; Bernardi, Larissa Sakis; Strusi, Orazio Luca; Mercuri, Salvatore; Segatto Silva, Marcos A; Colombo, Paolo; Sonvico, Fabio

    2011-02-28

    The aim of this research was to design and study norfloxacin (NFX) release in floating conditions from compressed hydrophilic matrices of hydroxypropylmethylcellulose (HPMC) or poly(ethylene oxide) (PEO). Module assembling technology for drug delivery system manufacturing was used. Two differently cylindrical base curved matrix/modules, identified as female and male, were assembled in void configuration by friction interlocking their concave bases obtaining a floating release system. Drug release and floatation behavior of this assembly was investigated. Due to the higher surface area exposed to the release medium, faster release was observed for individual modules compared to their assembled configuration, independently on the polymer used and concentration. The release curves analyzed using the Korsmeyer exponential equation and Peppas & Sahlin binomial equation showed that the drug release was controlled both by drug diffusion and polymer relaxation or erosion mechanisms. However, convective transport was predominant with PEO and at low content of polymers. NFX release from PEO polymeric matrix was more erosion dependent than HPMC. The assembled systems were able to float in vitro for up to 240min, indicating that this drug delivery system of norfloxacin could provide gastro-retentive site-specific release for increasing norfloxacin bioavailability. Copyright © 2010. Published by Elsevier B.V.

  16. Hanford Integrated Planning Process: 1993 Hanford Site-specific science and technology plan

    Energy Technology Data Exchange (ETDEWEB)

    1993-12-01

    This document is the FY 1993 report on Hanford Site-specific science and technology (S&T) needs for cleanup of the Site as developed via the Hanford Integrated Planning Process (HIPP). It identifies cleanup problems that lack demonstrated technology solutions and technologies that require additional development. Recommendations are provided regarding allocation of funding to address Hanford`s highest-priority technology improvement needs, technology development needs, and scientific research needs, all compiled from a Sitewide perspective. In the past, the S&T agenda for Hanford Site cleanup was sometimes driven by scientists and technologists, with minimal input from the ``problem owners`` (i.e., Westinghouse Hanford Company [WHC] staff who are responsible for cleanup activities). At other times, the problem-owners made decisions to proceed with cleanup without adequate scientific and technological inputs. Under both of these scenarios, there was no significant stakeholder involvement in the decision-making process. One of the key objectives of HIPP is to develop an understanding of the integrated S&T requirements to support the cleanup mission, (a) as defined by the needs of the problem owners, the values of the stakeholders, and the technology development expertise that exists at Hanford and elsewhere. This requires a periodic, systematic assessment of these needs and values to appropriately define a comprehensive technology development program and a complementary scientific research program. Basic to our success is a methodology that is defensible from a technical perspective and acceptable to the stakeholders.

  17. Regulation of Transient Site-specific Copy Gain by MicroRNA.

    Science.gov (United States)

    Black, Joshua C; Zhang, Hailei; Kim, Jaegil; Getz, Gad; Whetstine, Johnathan R

    2016-03-04

    Intra-tumor copy number heterogeneity is commonly observed in cancer; however, the molecular mechanisms that contribute to heterogeneity remain poorly understood. Up-regulation of the histone demethylase KDM4A promotes transient site-specific copy gain (TSSG) in cells; therefore, uncovering how KDM4A levels are controlled is important for understanding the regulation of copy number heterogeneity. Here, we demonstrate that KDM4A is regulated by hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137. Altering expression of these microRNAs (miRNAs) regulates KDM4A-dependent TSSG. miRNA inhibition promoted copy gains and increased expression of the drug-resistant oncogene CKS1B, which was further substantiated in primary breast tumors. Consistent with increased CKS1B expression, miRNA inhibition reduced breast cancer cell sensitivity to cisplatin. Our data identify these miRNAs as regulators of TSSG and copy gains of a drug resistance gene.

  18. Regulation of Transient Site-specific Copy Gain by MicroRNA*♦

    Science.gov (United States)

    Black, Joshua C.; Zhang, Hailei; Kim, Jaegil; Getz, Gad; Whetstine, Johnathan R.

    2016-01-01

    Intra-tumor copy number heterogeneity is commonly observed in cancer; however, the molecular mechanisms that contribute to heterogeneity remain poorly understood. Up-regulation of the histone demethylase KDM4A promotes transient site-specific copy gain (TSSG) in cells; therefore, uncovering how KDM4A levels are controlled is important for understanding the regulation of copy number heterogeneity. Here, we demonstrate that KDM4A is regulated by hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137. Altering expression of these microRNAs (miRNAs) regulates KDM4A-dependent TSSG. miRNA inhibition promoted copy gains and increased expression of the drug-resistant oncogene CKS1B, which was further substantiated in primary breast tumors. Consistent with increased CKS1B expression, miRNA inhibition reduced breast cancer cell sensitivity to cisplatin. Our data identify these miRNAs as regulators of TSSG and copy gains of a drug resistance gene. PMID:26755726

  19. TALE homeodomain proteins regulate site-specific terminal differentiation, LCE genes and epidermal barrier.

    Science.gov (United States)

    Jackson, Ben; Brown, Stuart J; Avilion, Ariel A; O'Shaughnessy, Ryan F L; Sully, Katherine; Akinduro, Olufolake; Murphy, Mark; Cleary, Michael L; Byrne, Carolyn

    2011-05-15

    The epidermal barrier varies over the body surface to accommodate regional environmental stresses. Regional skin barrier variation is produced by site-dependent epidermal differentiation from common keratinocyte precursors and often manifests as site-specific skin disease or irritation. There is strong evidence for body-site-dependent dermal programming of epidermal differentiation in which the epidermis responds by altering expression of key barrier proteins, but the underlying mechanisms have not been defined. The LCE multigene cluster encodes barrier proteins that are differentially expressed over the body surface, and perturbation of LCE cluster expression is linked to the common regional skin disease psoriasis. LCE subclusters comprise genes expressed variably in either external barrier-forming epithelia (e.g. skin) or in internal epithelia with less stringent barriers (e.g. tongue). We demonstrate here that a complex of TALE homeobox transcription factors PBX1, PBX2 and Pknox (homologues of Drosophila Extradenticle and Homothorax) preferentially regulate external rather than internal LCE gene expression, competitively binding with SP1 and SP3. Perturbation of TALE protein expression in stratified squamous epithelia in mice produces external but not internal barrier abnormalities. We conclude that epidermal barrier genes, such as the LCE multigene cluster, are regulated by TALE homeodomain transcription factors to produce regional epidermal barriers.

  20. Site-specific assessments of the abundance of three inshore dolphin species to inform conservation and management

    Directory of Open Access Journals (Sweden)

    Alexander Mark Brown

    2016-02-01

    Full Text Available Assessing the abundance of wildlife populations is essential to their effective conservation and management. Concerns have been raised over the vulnerability of tropical inshore dolphins in waters off northern Australia to anthropogenic impacts on local populations, yet a lack of abundance data precludes assessment of their conservation status and the management of threats. Using small vessels as cost-effective research platforms, photo-identification surveys and capture-recapture models were applied to provide the first quantitative abundance data for Australian snubfin (Orcaella heinsohni, Australian humpback (Sousa sahulensis, and Indo-Pacific bottlenose dolphins (Tursiops aduncus at five sites in the Kimberley region of north-western Australia. The abundance of each species was highly variable between different sites, likely reflecting species-specific habitat preferences. Within the c. 130 km2 study sites, the estimated abundance of most species was ≤ 60 individuals (excluding calves, and fewer than 20 humpback dolphins were identified at each site in any one 3-5 week sampling period. However, larger estimates of c. 130 snubfin and c. 160 bottlenose dolphins were obtained at two different sites. Several local populations showed evidence of site fidelity, particularly snubfin dolphins. By implementing a standardized, multi-site approach, data on local populations were provided within a broader, regional context, and indicated that each species is patchily distributed in the region. This highlights the need for site-specific baseline data collection using appropriate survey techniques to quantitatively assess the potential impacts of threatening activities to local populations. These findings further illustrate the need to gain a greater understanding of known and potential threats to inshore dolphin populations, their relative impacts, and to mitigate where necessary. In particular, the level of interactions with inshore gillnet fisheries

  1. Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Brandon L Jutras

    Full Text Available A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.

  2. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.

    Science.gov (United States)

    Anastassiadis, Konstantinos; Fu, Jun; Patsch, Christoph; Hu, Shengbiao; Weidlich, Stefanie; Duerschke, Kristin; Buchholz, Frank; Edenhofer, Frank; Stewart, A Francis

    2009-01-01

    Tyrosine site-specific recombinases (SSRs) including Cre and FLP are essential tools for DNA and genome engineering. Cre has long been recognized as the best SSR for genome engineering, particularly in mice. Obtaining another SSR that is as good as Cre will be a valuable addition to the genomic toolbox. To this end, we have developed and validated reagents for the Dre-rox system. These include an Escherichia coli-inducible expression vector based on the temperature-sensitive pSC101 plasmid, a mammalian expression vector based on the CAGGs promoter, a rox-lacZ reporter embryonic stem (ES) cell line based on targeting at the Rosa26 locus, the accompanying Rosa26-rox reporter mouse line, and a CAGGs-Dre deleter mouse line. We also show that a Dre-progesterone receptor shows good ligand-responsive induction properties. Furthermore, we show that there is no crossover recombination between Cre-rox or Dre-loxP. Hence, we add another set of efficient tools to the genomic toolbox, which will enable the development of more sophisticated mouse models for the analysis of gene function and disease.

  3. The three-dimensional microstructure of trabecular bone: Analysis of site-specific variation in the human jaw bone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jo Eun; Yi, Won Jin; Heo, Min Suk; Lee, Sam Sun; Choi, Soon Chul; Huh, Kyung Hoe [Dept. of Oral and Maxillofacial Radiology and Dental Research Institute, School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Shin, Jae Myung [Dept. of Oral and Maxillofacial Surgery, Ilsan Paik Hospital, Inje University College of Medicine, Goyang (Korea, Republic of); Oh, Sung Ook [A Plus Dental Clinic, Seoul (Korea, Republic of)

    2013-12-15

    This study was performed to analyze human maxillary and mandibular trabecular bone using the data acquired from micro-computed tomography (micro-CT), and to characterize the site-specific microstructures of trabeculae. Sixty-nine cylindrical bone specimens were prepared from the mandible and maxilla. They were divided into 5 groups by region: the anterior maxilla, posterior maxilla, anterior mandible, posterior mandible, and mandibular condyle. After the specimens were scanned using a micro-CT system, three-dimensional microstructural parameters such as the percent bone volume, bone specific surface, trabecular thickness, trabecular separation, trabecular number, structure model index, and degrees of anisotropy were analyzed. Among the regions other than the condylar area, the anterior mandibular region showed the highest trabecular thickness and the lowest value for the bone specific surface. On the other hand, the posterior maxilla region showed the lowest trabecular thickness and the highest value for the bone specific surface. The degree of anisotropy was lowest at the anterior mandible. The condyle showed thinner trabeculae with a more anisotropic arrangement than the other mandibular regions. There were microstructural differences between the regions of the maxilla and mandible. These results suggested that different mechanisms of external force might exist at each site.

  4. Site-specific carbon deposition for hierarchically ordered core/shell-structured graphitic carbon with remarkable electrochemical performance.

    Science.gov (United States)

    Lv, Yingying; Wu, Zhangxiong; Qian, Xufang; Fang, Yin; Feng, Dan; Xia, Yongyao; Tu, Bo; Zhao, Dongyuan

    2013-10-01

    A fascinating core-shell-structured graphitic carbon material composed of ordered microporous core and uniform mesoporous shell is fabricated for the first time through a site-specific chemical vapor deposition process by using a nanozeolite@mesostructured silica composite molecular sieve as the template. The mesostructure-directing agent cetyltrimethylammonium bromide in the shell of the template can be either burned off or carbonized so that it is successfully utilized as a pore switch to turn the shell of the template "on" or "off" to allow selective carbon deposition. The preferred carbon deposition process can be performed only in the inner microporous zeolite cores or just within the outer mesoporous shells, resulting in a zeolite-like ordered microporous carbon or a hollow mesoporous carbon. Full carbon deposition in the template leads to the new core-shell-structured microporous@mesoporous carbon with a nanographene-constructed framework for fast electron transport, a microporous nanocore with large surface area for high-capacity storage of lithium ions, a mesoporous shell with highly opened mesopores as a transport layer for lithium ions and electron channels to access inner cores. The ordered micropores are protected by the mesoporous shell, avoiding pore blockage as the formation of solid electrolyte interphase layers. Such a unique core-shell-structured microporous@mesoporous carbon material represents a newly established lithium ion storage model, demonstrating high reversible energy storage, excellent rate capability, and long cyclic stability.

  5. Wild Marshmallows.

    Science.gov (United States)

    Kallas, John N.

    1984-01-01

    Provides information for teaching a unit on wild plants, including resources to use, plants to learn, safety considerations, list of plants (with scientific name, edible parts, and uses), list of plants that might cause allergic reactions when eaten. Also describes the chickweed, bull thistle, and common mallow. (BC)

  6. An approach to model monitoring and surveillance data of wildlife diseases-exemplified by Classical Swine Fever in wild boar.

    Science.gov (United States)

    Stahnke, N; Liebscher, V; Staubach, C; Ziller, M

    2013-11-01

    The analysis of epidemiological field data from monitoring and surveillance systems (MOSSs) in wild animals is of great importance in order to evaluate the performance of such systems. By parameter estimation from MOSS data, conclusions about disease dynamics in the observed population can be drawn. To strengthen the analysis, the implementation of a maximum likelihood estimation is the main aim of our work. The new approach presented here is based on an underlying simple SIR (susceptible-infected-recovered) model for a disease scenario in a wildlife population. The three corresponding classes are assumed to govern the intensities (number of animals in the classes) of non-homogeneous Poisson processes. A sampling rate was defined which describes the process of data collection (for MOSSs). Further, the performance of the diagnostics was implemented in the model by a diagnostic matrix containing misclassification rates. Both descriptions of these MOSS parts were included in the Poisson process approach. For simulation studies, the combined model demonstrates its ability to validly estimate epidemiological parameters, such as the basic reproduction rate R0. These parameters will help the evaluation of existing disease control systems. They will also enable comparison with other simulation models. The model has been tested with data from a Classical Swine Fever (CSF) outbreak in wild boars (Sus scrofa scrofa L.) from a region of Germany (1999-2002). The results show that the hunting strategy as a sole control tool is insufficient to decrease the threshold for susceptible animals to eradicate the disease, since the estimated R0 confirms an ongoing epidemic of CSF. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Configuration and specifications of an Unmanned Aerial Vehicle (UAV) for early site specific weed management.

    Science.gov (United States)

    Torres-Sánchez, Jorge; López-Granados, Francisca; De Castro, Ana Isabel; Peña-Barragán, José Manuel

    2013-01-01

    A new aerial platform has risen recently for image acquisition, the Unmanned Aerial Vehicle (UAV). This article describes the technical specifications and configuration of a UAV used to capture remote images for early season site- specific weed management (ESSWM). Image spatial and spectral properties required for weed seedling discrimination were also evaluated. Two different sensors, a still visible camera and a six-band multispectral camera, and three flight altitudes (30, 60 and 100 m) were tested over a naturally infested sunflower field. The main phases of the UAV workflow were the following: 1) mission planning, 2) UAV flight and image acquisition, and 3) image pre-processing. Three different aspects were needed to plan the route: flight area, camera specifications and UAV tasks. The pre-processing phase included the correct alignment of the six bands of the multispectral imagery and the orthorectification and mosaicking of the individual images captured in each flight. The image pixel size, area covered by each image and flight timing were very sensitive to flight altitude. At a lower altitude, the UAV captured images of finer spatial resolution, although the number of images needed to cover the whole field may be a limiting factor due to the energy required for a greater flight length and computational requirements for the further mosaicking process. Spectral differences between weeds, crop and bare soil were significant in the vegetation indices studied (Excess Green Index, Normalised Green-Red Difference Index and Normalised Difference Vegetation Index), mainly at a 30 m altitude. However, greater spectral separability was obtained between vegetation and bare soil with the index NDVI. These results suggest that an agreement among spectral and spatial resolutions is needed to optimise the flight mission according to every agronomical objective as affected by the size of the smaller object to be discriminated (weed plants or weed patches).

  8. A site-specific ecological risk assessment for corn-associated insecticides.

    Science.gov (United States)

    Whiting, Sara A; Lydy, Michael J

    2015-07-01

    A site-specific ecological risk assessment (ERA) was conducted to examine the simultaneous use of genetically modified corn (Bt corn) with a neonicotinoid seed coating, clothianidin, and use of a granular insecticide, tefluthrin, to protect crops from pest damage. A field study was conducted on site, and exposure data from the literature were summarized to determine the matrices and exposure concentrations that nontarget species could typically experience within an agricultural ecosystem. To determine ecological effects on nontarget species, acute toxicity bioassays were conducted on earthworms (Eisenia fetida), amphipods (Hyalella azteca), and Elmid riffle beetle larvae (Ancyronyx spp.) in which the test species were exposed to single insecticides as well as the mixture of the 3 insecticides. In the risk characterization section of the ERA, stressor-response profiles for each species tested were compared with field distributions of the insecticides, and a margin of safety at the 10th percentile (MOS10) was calculated to estimate risk. No acute toxicity was observed in any of the 3 nontarget species after exposure to senescent Bt corn leaf tissue. Large MOS10 values were calculated for clothianidin to the nontarget species. When bioassays were compared with tefluthrin field distributions, very low MOS10 values were calculated for earthworms (0.06) and H. azteca (0.08) because the environmental concentrations often exceeded the stressor-response profile. No increased toxicity was observed when nontarget species were exposed to a mixture of the 3 insecticides. In summary, the genetically modified corn insecticidal proteins and clothianidin were not found at environmental concentrations exceeding benchmark values for ecological effects, but tefluthrin was consistently detected in the environment at levels that could be causing toxicity to nontarget species, especially if this pyrethroid is able to travel off site. © 2015 SETAC.

  9. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    Science.gov (United States)

    Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

    2016-07-01

    Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

  10. Current implementation of site-specific technologies in U.S. cotton production

    Science.gov (United States)

    Barnes, Edward M.

    2004-11-01

    The initial adoption of site-specific management for cotton production was slower than by commodities grown in the mid-west. Part of the delayed adoption can be explained by the lack of functional cotton yield monitors. Now that yield monitors are commercially available, cotton producers are beginning to find many applications for geospatial technologies. The emphasis of this paper is on applications that are being implemented at some level on commercial farms. Traditional grid-based soil sampling has found some use for pre-plant application of fertilizer, and soil conductivity mapping has been used to apply variable rate soil amendments in the west. During the growing season, vegetation indices such as the normalized difference vegetation index (NDVI) have been used as a tool to direct scouting for insect infestations. The scouting information and NDVI are combined to create variable rate insecticide application maps. A catalyst to this approach has been the recent development of aerial variable rate application technology. In some production regions, it is necessary to control cotton's vegetative development rate with plant growth regulators (PGRs). Vegetation indices are very useful for defining areas of the field where PGRs are needed. Research is also being conducted on the use of imagery for the development of defoliation application maps before harvest. In the short-term, simple vegetation indices can meet many of management information needs for cotton when combined with directed scouting of the field. However, to be ultimately successful, dependable and frequent sources of imagery will be required. Near-real time delivery is essential, as a majority of the management decisions are often made on a daily basis. Most producer-based applications of these data have been from airborne platforms managed by local image providers where flexible image acquisition schedules are possible.

  11. Synthesis and characterization of carboxymethyl chitosan hydrogel: Application as site specific delivery for lercanidipine hydrochloride

    Indian Academy of Sciences (India)

    Subhash S Vaghani; Madhabhai M Patel; C S Satish; Kandarp M Patel; N P Jivani

    2012-12-01

    In the present study, carboxymethylchitosan (CMCS) was prepared from chitosan, crosslinked with glutaraldehyde and evaluated in vitro as a potential carrier for site specific drug delivery of lercanidipine hydrochloride (LERH). LERH was incorporated at the time of crosslinking of CMCS. The chitosan was evaluated for its degree of deacetylation () and average molecular weight, which were found to be 84.6% and 3.5 × 104 Da, respectively. The degree of substitution on prepared CMCS was found to be 0.68. All hydrogel formulations showed more than 86% and 77% yield and drug loading, respectively. The swelling behaviour of prepared hydrogels were checked in different pH values, 1.2, 6.8 and 7.4, indicated pH responsive swelling characteristic with very less swelling at pH 1.2 and quick swelling at pH 6.8 followed by linear swelling at pH 7.4 with slight increase. In vitro release profile was carried out at the same conditions as in swelling and drug release was found to be dependent on swelling of hydrogels and showed biphasic release pattern with non-fickian diffusion kinetics at higher pH. The carboxymethylation of chitosan, entrapment of drug and its interaction in prepared hydrogels were checked by FTIR, 1H-NMR, DSC and -XRD studies, which confirmed formation of CMCS from chitosan and absence of any significant chemical change in LERH after being entrapped in crosslinked hydrogel formulations. The surface morphology of formulation 6 was checked before and after dissolution, revealed open channel like pores formation after dissolution.

  12. Site-specific DICER and DROSHA RNA products control the DNA-damage response.

    Science.gov (United States)

    Francia, Sofia; Michelini, Flavia; Saxena, Alka; Tang, Dave; de Hoon, Michiel; Anelli, Viviana; Mione, Marina; Carninci, Piero; d'Adda di Fagagna, Fabrizio

    2012-08-09

    Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11–RAD50–NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.

  13. Distinct and site-specific phosphorylation of the retinoblastoma protein at serine 612 in differentiated cells.

    Directory of Open Access Journals (Sweden)

    Takayuki Hattori

    Full Text Available The retinoblastoma susceptibility protein (pRB is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.

  14. Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis.

    Science.gov (United States)

    Yu, Ching-Ching; Kuo, Yu-Ying; Liang, Chien-Fu; Chien, Wei-Ting; Wu, Huan-Ting; Chang, Tsung-Che; Jan, Fan-Dan; Lin, Chun-Cheng

    2012-04-18

    Magnetic nanoparticles (MNPs) are attractive materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field; this could facilitate the recycling of enzymes and broaden their applications in organic synthesis. Herein, we report the methods for the immobilization of water-soluble and membrane-bound enzymes, and the activity difference between free and immobilized enzymes is discussed. Sialyltransferase (PmST1, from Pasteurella multocida ) and cytidine monophosphate (CMP)-sialic acid synthetase (CSS, from Neisseria meningitides ) were chosen as water-soluble enzymes and expressed using an intein expression system. The enzymes were site-specifically and covalently immobilized on PEGylated-N-terminal cysteine MNPs through native chemical ligation (NCL). Increasing the length of the PEG linker between the enzyme and the MNP surface increased the activity of the immobilized enzymes relative to the free parent enzymes. In addition, the use of a fluorescent acceptor tag for PmST1 affected enzyme kinetics. In contrast, sialyltransferase from Neisseria gonorrheae (NgST, a membrane-bound enzyme) was modified with a biotin-labeled cysteine at the C-terminus using NCL, and the enzyme was then assembled on streptavidin-functionalized MNPs. Using a streptavidin-biotin interaction, it was possible to immobilize NgST on a solid support under mild ligation conditions, which prevented the enzyme from high-temperature decomposition and provided an approximately 2-fold increase in activity compared to other immobilization methods on MNPs. Finally, the ganglioside GM3-derivative (sialyl-lactose derivative) was synthesized in a one-pot system by combining the use of immobilized PmST1 and CSS. The enzymes retained 50% activity after being reused ten times. Furthermore, the results obtained using the one-pot two-immobilized-enzyme system demonstrated that it can be applied to large-scale reactions with acceptable yields and

  15. Oral neutrophils display a site-specific phenotype characterized by expression of T-cell receptors.

    Science.gov (United States)

    Lakschevitz, Flavia S; Aboodi, Guy M; Glogauer, Michael

    2013-10-01

    Neutrophils, key cells of the innate immune system, were previously thought to be terminally differentiated cells, incapable of altering their gene expression after differentiation and maturation in the bone marrow. Only recently has it been shown that neutrophils perform rapid and complex changes in gene expression during inflammatory responses. Previous work by the authors has demonstrated differences in reactive oxygen species production between oral and peripheral blood neutrophils isolated from patients with chronic periodontitis, suggesting that oral neutrophils present with a unique oral phenotype. Understanding differences in the neutrophil transcriptome after transit from circulation into the site of inflammation will give new insights into how these innate immune cells function during inflammation. Venous blood and oral rinse samples were obtained from five healthy participants. Blood neutrophils were isolated using a standard gradient method. Oral neutrophils were isolated through nylon mesh filters of different pore sizes (40 to 10 μm). RNA was purified from isolated neutrophils, and gene expression microarray analysis was completed. Results were confirmed by quantitative reverse transcription-polymerase chain reaction and immunofluorescence microscopy. Oral neutrophil isolation, which is critical when analyzing gene expression with samples clear of epithelial cell contamination, was optimized. It was also demonstrated that oral neutrophils present with a significant increase in T-cell receptor expression compared with circulating neutrophils, suggesting a role for oral neutrophils in crosstalk between the innate and adaptive immune system in the mouth. To the best of the authors' knowledge, it is demonstrated for the first time that, compared with circulating neutrophils, oral neutrophils present a site-specific gene expression profile in healthy individuals.

  16. Site-Specific Fragment Identification Guided by Single-Step Free Energy Perturbation Calculations

    Science.gov (United States)

    Raman, E. Prabhu; Vanommeslaeghe, Kenno; MacKerell, Alexander D.

    2012-01-01

    The in-silico Site Identification by Ligand Competitive Saturation (SILCS) approach identifies the binding sites of representative chemical entities on the entire protein surface, information that can be applied for computational fragment-based drug design. In this study, we report an efficient computational protocol that uses sampling of the protein-fragment conformational space obtained from the SILCS simulations and performs single step free energy perturbation (SSFEP) calculations to identify site-specific favorable chemical modifications of benzene involving substitutions of ring hydrogens with individual non-hydrogen atoms. The SSFEP method is able to capture the experimental trends in relative hydration free energies of benzene analogues and for two datasets of experimental relative binding free energies of congeneric series of ligands of the proteins α-thrombin and P38 MAP kinase. The approach includes a protocol in which data obtained from SILCS simulations of the proteins is first analyzed to identify favorable benzene binding sites following which an ensemble of benzene-protein conformations for that site is obtained. The SSFEP protocol applied to that ensemble results in good reproduction of experimental free energies of the α-thrombin ligands, but not for P38 MAP kinase ligands. Comparison with results from a P38 full-ligand simulation and analysis of conformations reveals the reason for the poor agreement being the connectivity with the remainder of the ligand, a limitation inherent in fragment-based methods. Since the SSFEP approach can identify favorable benzene modifications as well as identify the most favorable fragment conformations, the obtained information can be of value for fragment linking or structure-based optimization. PMID:23144598

  17. Site-Specific Fluorescence Depolarization Kinetics Distinguishes the Amyloid Folds Responsible for Distinct Yeast Prion Strains.

    Science.gov (United States)

    Narang, Dominic; Swasthi, Hema M; Mahapatra, Sayanta; Mukhopadhyay, Samrat

    2017-08-25

    The prion determinant of a yeast prion protein, Sup35NM, assembles into β-rich amyloid fibrils that switch the non-prion [psi-] state to the prion [PSI+] state of yeast. Previous studies have shown that two distinct forms of amyloids (Sc4 and Sc37), generated in vitro at two different temperatures (4°C and 37°C), recapitulate the strain phenomenon in Saccharomyces cerevisiae. Sc4 demonstrates a strong [PSI+] phenotype, whereas, Sc37 shows a weak phenotype. In order to discern the residue-specific structural and dynamical attributes associated with the amyloids that display stain-diversity, we took advantage of non-occurrence of tryptophan (Trp) in the NM-domain and created 18 single-Trp variants spanning the entire polypeptide length. The fluorescence readouts from these locations reported the site-specific structural details in Sc4 and Sc37 fibrils. Highly sensitive picosecond fluorescence depolarization measurements at these positions allowed us construct a conformational mobility map. Nearly all the residue positions demonstrated higher local flexibility in Sc4 amyloid that exhibits strong phenotype. The differences in the amplitude of local mobility were more pronounced at the two end segments of the N-domain compared to the central region. The M-domain is partially exposed and exhibits higher amplitude of local mobility indicating a lower degree of chain packing in the amyloid state and exhibited higher mobility in Sc4 state compared to Sc37 state. The altered local conformational dynamics in two distinct amyloid states provide molecular insights into the varied fragility and severing efficiency that govern the inheritance pattern of strong and weak prion strains.

  18. Site-specific genomic (SSG and random domain-localized (RDL mutagenesis in yeast

    Directory of Open Access Journals (Sweden)

    Honigberg Saul M

    2004-04-01

    Full Text Available Abstract Background A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. Results We have modified the existing method for introducing deletions in the yeast (S. cerevisiae genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis or is synthesized by error-prone PCR (RDL mutagenesis. In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. Conclusion SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

  19. Configuration and specifications of an Unmanned Aerial Vehicle (UAV for early site specific weed management.

    Directory of Open Access Journals (Sweden)

    Jorge Torres-Sánchez

    Full Text Available A new aerial platform has risen recently for image acquisition, the Unmanned Aerial Vehicle (UAV. This article describes the technical specifications and configuration of a UAV used to capture remote images for early season site- specific weed management (ESSWM. Image spatial and spectral properties required for weed seedling discrimination were also evaluated. Two different sensors, a still visible camera and a six-band multispectral camera, and three flight altitudes (30, 60 and 100 m were tested over a naturally infested sunflower field. The main phases of the UAV workflow were the following: 1 mission planning, 2 UAV flight and image acquisition, and 3 image pre-processing. Three different aspects were needed to plan the route: flight area, camera specifications and UAV tasks. The pre-processing phase included the correct alignment of the six bands of the multispectral imagery and the orthorectification and mosaicking of the individual images captured in each flight. The image pixel size, area covered by each image and flight timing were very sensitive to flight altitude. At a lower altitude, the UAV captured images of finer spatial resolution, although the number of images needed to cover the whole field may be a limiting factor due to the energy required for a greater flight length and computational requirements for the further mosaicking process. Spectral differences between weeds, crop and bare soil were significant in the vegetation indices studied (Excess Green Index, Normalised Green-Red Difference Index and Normalised Difference Vegetation Index, mainly at a 30 m altitude. However, greater spectral separability was obtained between vegetation and bare soil with the index NDVI. These results suggest that an agreement among spectral and spatial resolutions is needed to optimise the flight mission according to every agronomical objective as affected by the size of the smaller object to be discriminated (weed plants or weed patches.

  20. Site-specific control of N7-metal coordination in DNA by a fluorescent purine derivative.

    Science.gov (United States)

    Dumas, Anaëlle; Luedtke, Nathan W

    2012-01-01

    A synthetic strategy that utilizes O6-protected 8-bromoguanosine gives broad access to C8-guanine derivatives with phenyl, pyridine, thiophene, and furan substituents. The resulting 8-substituted 2'-deoxyguanosines are push-pull fluorophores that can exhibit environmentally sensitive quantum yields (Φ=0.001-0.72) due to excited-state proton-transfer reactions with bulk solvent. Changes in nucleoside fluorescence were used to characterize metal-binding affinity and specificity of 8-substituted 2'-deoxyguanosines. One derivative, 8-(2-pyridyl)-2'-deoxyguanosine (2PyG), exhibits selective binding of Cu(II), Ni(II), Cd(II), and Zn(II) through a bidentate effect provided by the N7 position of guanine and the 2-pyridyl nitrogen atom. Upon incorporation into DNA, 2-pyridine-modified guanine residues selectively bind to Cu(II) and Ni(II) with equilibrium dissociation constants (K(d)) that range from 25 to 850 nM; the affinities depend on the folded state of the oligonucleotide (duplex>G-quadruplex) as well as the identity of the metal ion (Cu>Ni≫Cd). These binding affinities are approximately 10 to 1 000 times higher than for unmodified metal binding sites in DNA, thereby providing site-specific control of metal localization in alternatively folded nucleic acids. Temperature-dependent circular-dichroism studies reveal metal-dependent stabilization of duplexes, but destabilization of G-quadruplex structures upon adding Cu(II) to 2PyG-modified oligonucleotides. These results demonstrate how the addition of a single pyridine group to the C8 position of guanine provides a powerful new tool for studying the effects of N7 metalation on the structure, stability, and electronic properties of nucleic acids.

  1. Human in vivo regional intestinal permeability: quantitation using site-specific drug absorption data.

    Science.gov (United States)

    Sjögren, Erik; Dahlgren, David; Roos, Carl; Lennernäs, Hans

    2015-06-01

    Application of information on regional intestinal permeability has been identified as a key aspect of successful pharmaceutical product development. This study presents the results and evaluation of an approach for the indirect estimation of site-specific in vivo intestinal effective permeability (Peff) in humans. Plasma concentration-time profiles from 15 clinical studies that administered drug solutions to specific intestinal regions were collected and analyzed. The intestinal absorption rate for each drug was acquired by deconvolution, using historical intravenous data as reference, and used with the intestinal surface area and the dose remaining in the lumen to estimate the Peff. Forty-three new Peff values were estimated (15 from the proximal small intestine, 11 from the distal small intestine, and 17 from the large intestine) for 14 active pharmaceutical ingredients representing a wide range of biopharmaceutical properties. A good correlation (r(2) = 0.96, slope = 1.24, intercept = 0.030) was established between these indirect jejunal Peff estimates and jejunal Peff measurements determined directly using the single-pass perfusion double balloon technique. On average, Peff estimates from the distal small intestine and large intestine were 90% and 40%, respectively, of those from the proximal small intestine. These results support the use of the evaluated deconvolution method for indirectly estimating regional intestinal Peff in humans. This study presents the first comprehensive data set of estimated human regional intestinal permeability values for a range of drugs. These biopharmaceutical data can be used to improve the accuracy of gastrointestinal absorption predictions used in drug development decision-making.

  2. Redox-sensitivity and site-specificity of S- and N- denitrosation in proteins.

    Directory of Open Access Journals (Sweden)

    Frances L Jourd'heuil

    Full Text Available BACKGROUND: S-nitrosation--the formation of S-nitrosothiols (RSNOs at cysteine residues in proteins--is a posttranslational modification involved in signal transduction and nitric oxide (NO transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs in proteins in vivo, although their biological significance remains obscure. In this study, we characterized a redox-based mechanism by which N-nitroso-tryptophan residues in proteins may be denitrosated. METHODOLOGY/PRINCIPAL FINDINGS: The denitrosation of N-acetyl-nitroso Trp (NANT by glutathione (GSH required molecular oxygen and was inhibited by superoxide dismutase (SOD. Transnitrosation to form S-nitrosoglutathione (GSNO was observed only in the absence of oxygen or presence of SOD. Protein denitrosation by GSH was studied using a set of mutant recombinant human serum albumin (HSA. Trp-214 and Cys-37 were the only two residues nitrosated by NO under aerobic conditions. Nitroso-Trp-214 in HSA was insensitive to denitrosation by GSH or ascorbate while denitrosation at Cys-37 was evident in the presence of GSH but not ascorbate. GSH-dependent denitrosation of Trp-214 was restored in a peptide fragment of helix II containing Trp-214. Finally, incubation of cell lysates with NANT revealed a pattern of protein nitrosation distinct from that observed with GSNO. CONCLUSIONS: We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ

  3. Site-specific lead exposure from lead pellet ingestion in sentinel mallards

    Science.gov (United States)

    Rocke, T.E.; Brand, C.J.; Mensik, John G.

    1997-01-01

    We monitored lead poisoning from the ingestion of spent lead pellets in sentinel mallards (Anas platyhrynchos) at the Sacramento National Wildlife Refuge (SNWR), Willows, California for 4 years (1986-89) after the conversion to steel shot for waterfowl hunting on refuges in 1986. Sentinel mallards were held in 1.6-ha enclosures in 1 hunted (P8) and 2 non-hunted (T19 and TF) wetlands. We compared site-specific rates of lead exposure, as determined by periodic measurement of blood lead concentrations, and lead poisoning mortality between wetlands with different lead pellet densities, between seasons, and between male and female sentinels. In 1986, the estimated 2-week rate of lead exposure was significantly higher (P 2,000,000 pellets/ha), than in those with lower densities of lead pellets, T19 (18.1%; 173,200 pellets/ha) and TF (0.9%; 15,750 pellets/ha). The probability of mortality from lead poisoning was also significantly higher (P < 0.01) in sentinel mallards enclosed in P8 (0.25) than T19 (0) and TF (0) in 1986 and remained significantly higher (P < 0.001) during the 4-year study. Both lead exposure and the probability of lead poisoning mortality in P8 were significantly higher (P < 0.001) in the fall of 1986 (43.8%; 0.25), before hunting season, than in the spring of 1987 (21.6%; 0.04), after hunting season. We found no significant differences in the rates of lead exposure or lead poisoning mortality between male and female sentinel mallards. The results of this study demonstrate that in some locations, lead exposure and lead poisoning in waterfowl will continue to occur despite the conversion to steel shot for waterfowl hunting.

  4. Environmental restoration and waste management site-specific plan for Richland Operations Office. [Contains glossary

    Energy Technology Data Exchange (ETDEWEB)

    1991-09-01

    This document was prepared to implement and support the US Department of Energy-Headquarters (DOE-HQ) national plan. The national plan, entitled Environmental Restoration and Waste Management Five-Year Plan (DOE 1990b) (hereinafter referred to as the DOE-HQ Five-Year Plan) is the cornerstone of the US Department of Energy's (DOE) long-term strategy in environmental restoration and waste management. The DOE-HQ Five-Year Plan addresses overall philosophy and environmental and waste-related activities under the responsibilities of the DOE Office of Environmental Restoration and Waste Management. The plan also reaffirms DOE-HQ goals to bring its nuclear sites into environmental compliance in cooperation with its regulators and the public, and to clean up and restore the environment by 2019 (the commitment for the Hanford Site is for one year sooner, or 2018). This document is part of the site-specific plan for the US Department of Energy-Richland Operations Office (DOE-RL). It is the first revision of the original plan, which was dated December 1989 (DOE-RL 1989a). This document is a companion document to the Overview of the Hanford Cleanup Five-Year Plan (DOE-RL 1989d) and The Hanford Site Environmental Restoration and Waste Management Five-Year Plan Activity Data Sheets (DOE-RL 1991). Although there are three documents that make up the complete DOE-RL plan, this detailed information volume was prepared so it could be used as a standalone document. 71 refs., 40 figs., 28 tabs.

  5. Site-specific ultrasound reflection properties and superficial collagen content of bovine knee articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Laasanen, Mikko S [Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital and University of Kuopio, POB 1777, FIN-70211 Kuopio (Finland); Department of Surgery, Division of Orthopaedics and Traumatology, Jyvaeskylae Central Hospital, Keskussairaalantie 19, FIN-40620 Jyvaeskylae (Finland); Saarakkala, Simo [Department of Nuclear Medicine, Etelae-Savo Hospital District, Mikkeli Central Hospital, Porrassalmenkatu 35-37, 50100 Mikkeli (Finland); Toeyraes, Juha [Department of Clinical Neurophysiology, Kuopio University Hospital and University of Kuopio, POB 1777, FIN-70211 Kuopio (Finland); Rieppo, Jarno [Department of Anatomy, University of Kuopio, POB 1627, FIN-70211 Kuopio (Finland); Jurvelin, Jukka S [Department of Clinical Physiology and Nuclear Medicine, Kuopio University Hospital and University of Kuopio, POB 1777, FIN-70211 Kuopio (Finland); Department of Applied Physics, University of Kuopio, POB 1627, FIN-70211 Kuopio (Finland)

    2005-07-21

    Previous quantitative 2D-ultrasound imaging studies have demonstrated that the ultrasound reflection measurement of articular cartilage surface sensitively detects degradation of the collagen network, whereas digestion of cartilage proteoglycans has no significant effect on the ultrasound reflection. In this study, the first aim was to characterize the ability of quantitative 2D-ultrasound imaging to detect site-specific differences in ultrasound reflection and backscattering properties of cartilage surface and cartilage-bone interface at visually healthy bovine knee (n = 30). As a second aim, we studied factors controlling ultrasound reflection properties of an intact cartilage surface. The ultrasound reflection coefficient was determined in time (R) and frequency domains (IRC) at medial femoral condyle, lateral patello-femoral groove, medial tibial plateau and patella using a 20 MHz ultrasound imaging instrument. Furthermore, cartilage surface roughness was quantified by calculating the ultrasound roughness index (URI). The superficial collagen content of the cartilage was determined using a FT-IRIS-technique. A significant site-dependent variation was shown in cartilage thickness, ultrasound reflection parameters, URI and superficial collagen content. As compared to R and IRC, URI was a more sensitive parameter in detecting differences between the measurement sites. Ultrasound reflection parameters were not significantly related to superficial collagen content, whereas the correlation between R and URI was high. Ultrasound reflection at the cartilage-bone interface showed insignificant site-dependent variation. The current results suggest that ultrasound reflection from the intact cartilage surface is mainly dependent on the cartilage surface roughness and the collagen content has a less significant role.

  6. Challenges for creating a site-specific groundwater-use record for the Ozark Plateaus aquifer system (central USA) from 1900 to 2010

    Science.gov (United States)

    Knierim, Katherine J.; Nottmeier, Anna M.; Worland, Scott; Westerman, Drew A.; Clark, Brian R.

    2017-09-01

    Hydrologic budgets to determine groundwater availability are important tools for water-resource managers. One challenging component for developing hydrologic budgets is quantifying water use through time because historical and site-specific water-use data can be sparse or poorly documented. This research developed a groundwater-use record for the Ozark Plateaus aquifer system (central USA) from 1900 to 2010 that related county-level aggregated water-use data to site-specific well locations and aquifer units. A simple population-based linear model, constrained to 0 million liters per day in 1900, provided the best means to extrapolate groundwater-withdrawal rates pre-1950s when there was a paucity of water-use data. To disaggregate county-level data to individual wells across a regional aquifer system, a programmatic hierarchical process was developed, based on the level of confidence that a well pumped groundwater for a specific use during a specific year. Statistical models tested on a subset of the best-available site-specific water-use data provided a mechanism to bracket historic groundwater use, such that groundwater-withdrawal rates ranged, on average, plus or minus 38% from modeled values. Groundwater withdrawn for public supply and domestic use accounted for between 48 and 74% of total groundwater use since 1901, highlighting that groundwater provides an important drinking-water resource. The compilation, analysis, and spatial and temporal extrapolation of water-use data remain a challenging task for water scientists, but is of paramount importance to better quantify groundwater use and availability.

  7. Challenges for creating a site-specific groundwater-use record for the Ozark Plateaus aquifer system (central USA) from 1900 to 2010

    Science.gov (United States)

    Knierim, Katherine; Nottmeier, Anna M.; Worland, Scott C.; Westerman, Drew A.; Clark, Brian R.

    2017-01-01

    Hydrologic budgets to determine groundwater availability are important tools for water-resource managers. One challenging component for developing hydrologic budgets is quantifying water use through time because historical and site-specific water-use data can be sparse or poorly documented. This research developed a groundwater-use record for the Ozark Plateaus aquifer system (central USA) from 1900 to 2010 that related county-level aggregated water-use data to site-specific well locations and aquifer units. A simple population-based linear model, constrained to 0 million liters per day in 1900, provided the best means to extrapolate groundwater-withdrawal rates pre-1950s when there was a paucity of water-use data. To disaggregate county-level data to individual wells across a regional aquifer system, a programmatic hierarchical process was developed, based on the level of confidence that a well pumped groundwater for a specific use during a specific year. Statistical models tested on a subset of the best-available site-specific water-use data provided a mechanism to bracket historic groundwater use, such that groundwater-withdrawal rates ranged, on average, plus or minus 38% from modeled values. Groundwater withdrawn for public supply and domestic use accounted for between 48 and 74% of total groundwater use since 1901, highlighting that groundwater provides an important drinking-water resource. The compilation, analysis, and spatial and temporal extrapolation of water-use data remain a challenging task for water scientists, but is of paramount importance to better quantify groundwater use and availability.

  8. Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

    Science.gov (United States)

    Meza, José S; Díaz-Fleischer, Francisco; Sánchez-Velásquez, Lázaro R; Zepeda-Cisneros, Cristina Silvia; Handler, Alfred M; Schetelig, Marc F

    2014-01-01

    Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.

  9. Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy

    Science.gov (United States)

    Broekgaarden, M.; van Vught, R.; Oliveira, S.; Roovers, R. C.; van Bergen En Henegouwen, P. M. P.; Pieters, R. J.; van Gulik, T. M.; Breukink, E.; Heger, M.

    2016-03-01

    Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested.Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested. Electronic supplementary information (ESI) available: Materials and methods. See DOI: 10.1039/c6nr00014b

  10. Site-specific rectocele repair with dermal graft augmentation: comparison of porcine dermal xenograft (Pelvicol) and human dermal allograft.

    Science.gov (United States)

    Biehl, Roger C; Moore, Robert D; Miklos, John R; Kohli, Neeraj; Anand, Indu S; Mattox, T Fleming

    2008-01-01

    This study is a retrospective chart review comparing 195 women who underwent rectocele repair with either a porcine dermal xenograft or human allogenic cadaveric dermal graft augmentation over a two year period. A site-specific defect repair was completed prior to augmentation with the graft. Examinations were performed preoperatively and postoperatively using the pelvic organ prolapse quantification system. Questionnaires were used to assess constipation and dyspareunia. De novo dyspareunia and cure rates for constipation and dyspareunia were not statistically different between the two groups. Site-specific fascial rectocele repairs with xenograft or allograft augmentation were found to have similar complication rates as well as objective and subjective cure rates.

  11. Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG₁.

    Directory of Open Access Journals (Sweden)

    Feifan Yu

    Full Text Available Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG₁ (mIgG₁. Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG₁ monoclonal antibodies (mAbs. The best variant, denoted Z(F5I corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP group as a probe for site-specific photoconjugation to Fc of mIgG₁, The best photocoupling efficiency to mIgG₁ Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG₁ mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG₁ antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG₁ mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP probe. This result indicates that the use of a site-specific and affinity probe

  12. Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery.

    Science.gov (United States)

    Cheng, Ru; Meng, Fenghua; Deng, Chao; Klok, Harm-Anton; Zhong, Zhiyuan

    2013-05-01

    In the past decades, polymeric nanoparticles have emerged as a most promising and viable technology platform for targeted and controlled drug delivery. As vehicles, ideal nanoparticles are obliged to possess high drug loading levels, deliver drug to the specific pathological site and/or target cells without drug leakage on the way, while rapidly unload drug at the site of action. To this end, various "intelligent" polymeric nanoparticles that release drugs in response to an internal or external stimulus such as pH, redox, temperature, magnetic and light have been actively pursued. These stimuli-responsive nanoparticles have demonstrated, though to varying degrees, improved in vitro and/or in vivo drug release profiles. In an effort to further improve drug release performances, novel dual and multi-stimuli responsive polymeric nanoparticles that respond to a combination of two or more signals such as pH/temperature, pH/redox, pH/magnetic field, temperature/reduction, double pH, pH and diols, temperature/magnetic field, temperature/enzyme, temperature/pH/redox, temperature/pH/magnetic, pH/redox/magnetic, temperature/redox/guest molecules, and temperature/pH/guest molecules have recently been developed. Notably, these combined responses take place either simultaneously at the pathological site or in a sequential manner from nanoparticle preparation, nanoparticle transporting pathways, to cellular compartments. These dual and multi-stimuli responsive polymeric nanoparticles have shown unprecedented control over drug delivery and release leading to superior in vitro and/or in vivo anti-cancer efficacy. With programmed site-specific drug delivery feature, dual and multi-stimuli responsive nanoparticulate drug formulations have tremendous potential for targeted cancer therapy. In this review paper, we highlight the recent exciting developments in dual and multi-stimuli responsive polymeric nanoparticles for precision drug delivery applications, with a particular focus

  13. A general method for site specific fluorescent labeling of recombinant chemokines.

    Directory of Open Access Journals (Sweden)

    Tetsuya Kawamura

    Full Text Available Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11-12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4 °C, and co-internalization with their receptors at 37 °C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion by minimally perturbing native interactions. Other advantages of the method

  14. Site-Specific Nutrient Management for Rainfed Maize in Western Mid-Hills of Nepal

    Directory of Open Access Journals (Sweden)

    Prakash Ghimire

    2015-06-01

    Full Text Available On-farm experiment was conducted in eight farmers’ field, of Khasyoli village development committee (952 to 1415 masl, Nepal, from April to September, 2011 to address the major constraint (nutrient management to maize production through site-specific nutrient management (SSNM as this approach is popular among scientists. The experiment comprised three nutrient omission plots (0N, 0P, and 0K, an ample NPK plot, and a farmers’ fertilization practice (FFP plot, arranged in randomized complete block design. Farmers planted open pollinated variety (Manakamana-3 and managed in their way. Field-specific NPK application rates were calculated by considering nutrient demand, indigenous NPK supply and recovery efficiency of fertilizers. Grain yield in FFP (2.32 Mg/ha and 0N (1.79 Mg/ha plots differed significantly from each other and rest of the treatments, but was statistically similar among 0P (3.18 Mg/ha, 0K (3.40 Mg/ha and ample NPK (3.38 Mg/ha plots. Post-harvest grain and stover analysis revealed that indigenous NPK supply (20-71 kg N, 19-68 kg P2O5 and 51-164 kg K2O/ha of soil vary among the farmers’ field. Moreover, soil was poor in indigenous N supply (42 kg/ha, but rich in indigenous P2O5 (35 kg/ha and K2O (90 kg/ha supply, on an average. As per the principles of SSNM, the initial fertilizer recommendation made can vary from 40-222 kg N, 0- 93 kg P2O5, and 0-50 kg K2O/ha. On an average, farmers may apply no or lower dose of P2O5 (18 kg/ha and K2O (3 kg/ha but need to significantly increase dose of N (143 kg/ha fertilizer for enhancing soil and maize productivity.

  15. Evaluation and interpretation of regional and site-specific hydrochemical data bases for water quality assessment

    Directory of Open Access Journals (Sweden)

    M. G. Hutchins

    1999-01-01

    Full Text Available The spatial distribution of stream water composition, as determined by the Geochemical Baseline Survey of the Environment (G-BASE conducted by the British Geological Survey (BGS can be successfully related under baseflow conditions to bedrock geochemistry. Further consideration of results in conjunction with site-specific monitoring data enables factors controlling both spatial and temporal variability in major element composition to be highlighted and allows the value of the survey to be enhanced. Hence, chemical data (i from streams located on Lower Silurian (Llandovery bedrock at 1 km2 resolution collected as part of the G-BASE survey of Wales and the West Midlands and (ii from catchment monitoring studies located in upland mid-Wales (conducted by Institute of Terrestrial Ecology, have been considered together as an example. Classification of the spatial survey data set in terms of potentially controlling factors was carried out so as to illustrate the level of explanation they could give in terms of observed spatial chemical variability. It was therefore hypothesised that on a geological lithostratigraphic series of limited geochemical contrast, altitude and land-use factors provide better explanation of this variability than others such as lithology at sampling site and stream order. At an individual site, temporal variability was also found to be of considerable significance and, at a monthly time-step, is explicable in terms of factors such as antecedent conditions and seasonality. Data suggest that the degree of this variability may show some relationship with stream order and land-use. Monitoring data from the region also reveal that relationships between stream chemistry and land-use may prove to be strong not only at base flow but also in storm flow conditions. In a wider context, predictions of the sensitivity of stream water to acidification based on classifications of soil and geology are successful on a regional scale. However

  16. Site-specific tolerance tables and indexing device to improve patient setup reproducibility.

    Science.gov (United States)

    McCullough, Kiernan T; James, Joshua A; Cetnar, Ashley J; McCullough, Mark A; Wang, Brian

    2015-01-01

    While the implementation of tools such as image-guidance and immobilization devices have helped to prevent geometric misses in radiation therapy, many treatments remain prone to error if these items are not available, not utilized for every fraction, or are misused. The purpose of this project is to design a set of site-specific treatment tolerance tables to be applied to the treatment couch for use in a record and verify (R&V) system that will insure accurate patient setup with minimal workflow interruption. This project also called for the construction of a simple indexing device to help insure reproducible patient setup for patients that could not be indexed with existing equipment. The tolerance tables were created by retrospective analysis on a total of 66 patients and 1,308 treatments, separating them into five categories based on disease site: lung, head and neck (H&N), breast, pelvis, and abdomen. Couch parameter tolerance tables were designed to encompass 95% of treatments, and were generated by calculating the standard deviation of couch vertical, longitudinal, and lateral values using the first day of treatment as a baseline. We also investigated an alternative method for generating the couch tolerances by updating the baseline values when patient position was verified with image guidance. This was done in order to adapt the tolerances to any gradual changes in patient setup that would not correspond with a mistreatment. The tolerance tables and customizable indexing device were then implemented for a trial period in order to determine the feasibility of the system. During this trial period we collected data from 1,054 fractions from 65 patients. We then analyzed the number of treatments that would have been out of tolerance, as well as whether or not the tolerances or setup techniques should be adjusted. When the couch baseline values were updated with every imaging fraction, the average rate of tolerance violations was 10% for the lung, H&N, abdomen

  17. Variable rate application of nematicides on cotton fields: a promising site-specific management strategy.

    Science.gov (United States)

    Ortiz, Brenda V; Perry, Calvin; Sullivan, Dana; Lu, Ping; Kemerait, Robert; Davis, Richard F; Smith, Amanda; Vellidis, George; Nichols, Robert

    2012-03-01

    Field tests were conducted to determine if differences in response to nematicide application (i.e., root-knot nematode (RKN) populations, cotton yield, and profitability) occurred among RKN management zones (MZ). The MZ were delineated using fuzzy clustering of five terrain (TR) and edaphic (ED) field features related to soil texture: apparent soil electrical conductivity shallow (ECa-shallow) and deep (ECa-deep), elevation (EL), slope (SL), and changes in bare soil reflectance. Zones with lowest mean values of ECa- shallow, ECa- deep, NDVI, and SL were designated as at greater risk for high RKN levels. Nematicide-treated plots (4 rows wide and 30 m long) were established in a randomized complete block design within each zone, but the number of replications in each zone varied from four to six depending on the size of the zone.The nematicides aldicarb (Temik 15 G) and 1,3-dichloropropene (1,3-D,Telone II) were applied at two rates (0.51 and 1.0 kg a.i./ha for aldicarb, and 33.1 and 66.2 kg a.i./ha for 1,3-D) to RKN MZ in commercial fields between 2007 and 2009. A consolidated analysis over the entire season showed that regardless of the zone, there were not differences between aldicarb rates and 1,3-D rates. The result across zones showed that 1,3-D provided better RKN control than did aldicarb in zones with low ECa values (high RKN risk zones exhibiting more coarse-textured sandy soils). In contrast, in low risk zones with relatively higher ECa values (heavier textured soil), the effects of 1,3-D and aldicarb were equal and application of any of the treatments provided sufficient control. In low RKN risk zones, a farmer would often have lost money if a high rate of 1,3-D was applied. This study showed that the effect of nematicide type and rate on RKN control and cotton yield varied across management zones (MZ) with the most expensive treatment likely to provide economic benefit only in zones with coarser soil texture. This study demonstrates the value of site

  18. Site-specific thermodynamic stability and unfolding of a de novo designed protein structural motif mapped by 13C isotopically edited IR spectroscopy.

    Science.gov (United States)

    Kubelka, Ginka S; Kubelka, Jan

    2014-04-23

    The mechanism of protein folding remains poorly understood, in part due to limited experimental information available about partially folded states. Isotopically edited infrared (IR) spectroscopy has emerged as a promising method for studying protein structural changes with site-specific resolution, but its full potential to systematically probe folding at multiple protein sites has not yet been realized. We have used (13)C isotopically edited IR spectroscopy to investigate the site-specific thermal unfolding at seven different locations in the de novo designed helix-turn-helix protein αtα. As one of the few stable helix-turn-helix motifs, αtα is an excellent model for studying the roles of secondary and tertiary interactions in folding. Circular dichroism (CD) experiments on the full αtα motif and its two peptide fragments show that interhelical tertiary contacts are critical for stabilization of the secondary structure. The site-specific thermal unfolding probed by (13)C isotopically edited IR is likewise consistent with primarily tertiary stabilization of the local structure. The least thermally stable part of the αtα motif is near the turn where the interhelical contacts are rather loose, while the motif's center with best established core packing has the highest stability. Similar correlation between the local thermal stability and tertiary contacts was found previously for a naturally occurring helix-turn-helix motif. These results underline the importance of native-like tertiary stabilizing interactions in folding, in agreement with recent state-of-the art folding simulations as well as simplified, native-centric models.

  19. Development of an unmanned aerial vehicle-based spray system for highly accurate site-specific application

    Science.gov (United States)

    Application of crop production and protection materials is a crucial component in the high productivity of American agriculture. Agricultural chemical application is frequently needed at a specific time and location for accurate site-specific management of crop pests. Piloted aircrafts that carry ...

  20. 77 FR 12002 - Mount Baker-Snoqualmie National Forest Site-Specific Invasive Plant Treatment Project and Forest...

    Science.gov (United States)

    2012-02-28

    ... Forest Service Mount Baker-Snoqualmie National Forest Site-Specific Invasive Plant Treatment Project and... environmental impact statement. SUMMARY: Invasive plants are currently damaging the ecological integrity of... available with the Pacific Northwest Region Invasive Plant Program, Preventing and Managing Invasive Plants...

  1. Site specific fertilization affects yield, fruit size, quality, and shelf-life of ‘Kent' mango

    Science.gov (United States)

    Site specific fertilization (SSF) defines the type and rate of fertilizer needed for individual orchards. This study presents preliminary results (2010-2011) of a medium term project to quantify the effects of SSF on yield, fruit size, quality, and shelf-life of ‘Kent’ mango. Two orchards are used f...

  2. 76 FR 30027 - Land Disposal Restrictions: Site-Specific Treatment Variance for Hazardous Selenium-Bearing Waste...

    Science.gov (United States)

    2011-05-24

    ... Treatment Variance for Hazardous Selenium-Bearing Waste Treatment Issued to Chemical Waste Management in... Direct Final rule pertains to the treatment of a hazardous waste generated by the Owens-Brockway Glass... AGENCY 40 CFR Part 268 Land Disposal Restrictions: Site-Specific Treatment Variance for Hazardous...

  3. The SSC cycle: a PDCA approach to address site-specific characteristics in a continuous shallow water quality monitoring project.

    Science.gov (United States)

    Miles, Eduardo J

    2008-05-01

    In any water quality-monitoring project there are several critical success factors that must be adequately addressed in order to ensure the implementation and realization of the monitoring objectives. Site selection is one of these critical success factors. The monitoring sites must be selected to comply with the monitoring and data quality objectives. In the real world, ideal monitoring setting conditions are difficult to achieve, and compromises must be made in order to locate the monitoring stations that best represent the environment to be monitored. Site-specific characteristics are all the environmental, logistical and management factors particular to the monitoring site, that could influence the fulfilment of the monitoring and data quality objectives. Therefore, during the site selection process, it is essential to properly consider and evaluate these site-specific characteristics. The SSC cycle was developed with this goal in mind, to assist the monitoring team to systematically address site-specific characteristics. The cycle is a methodology to organize the site-specific characteristics in different categories, and to ensure a comprehensive overview of these characteristics throughout the project life cycle.

  4. Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway

    DEFF Research Database (Denmark)

    Lee, Jae Seong; Beuchert Kallehauge, Thomas; Pedersen, Lasse Ebdrup

    2015-01-01

    gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following...

  5. Probing site-specific 13C/15N-isotope enrichment of spider silk with liquid-state NMR spectroscopy.

    Science.gov (United States)

    Shi, Xiangyan; Yarger, Jeffery L; Holland, Gregory P

    2013-05-01

    Solid-state nuclear magnetic resonance (NMR) has been extensively used to elucidate spider silk protein structure and dynamics. In many of these studies, site-specific isotope enrichment is critical for designing particular NMR methods for silk structure determination. The commonly used isotope analysis techniques, isotope-ratio mass spectroscopy and liquid/gas chromatography-mass spectroscopy, are typically not capable of providing the site-specific isotope information for many systems because an appropriate sample derivatization method is not available. In contrast, NMR does not require any sample derivatization or separation prior to analysis. In this article, conventional liquid-state (1)H NMR was implemented to evaluate incorporation of (13)C/(15)N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific (13)C and (15)N isotope enrichments, an analysis method was developed to fit the (1)H-(13)C and (1)H-(15)N J-splitting (J CH and J NH) (1)H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U-(13)C3,(15)N]-Ala and [1-(13)C,(15)N]-Gly were dissolved in their water supplies. Overall, contents for Ala and Gly isotopomers are extracted for these silk samples. The current methodology can be applied to many fields where site-specific tracking of isotopes is of interest.

  6. Local nutrient regimes determine site-specific environmental triggers of cyanobacterial and microcystin variability in urban lakes

    Directory of Open Access Journals (Sweden)

    S. C. Sinang

    2014-10-01

    Full Text Available Toxic cyanobacterial blooms in urban lakes present serious health hazards to humans and animals and require effective management strategies. In the management of toxic cyanobacteria blooms, understanding the roles of environmental factors is crucial. To date, a range of environmental factors have been proposed as potential triggers for the spatiotemporal variability of cyanobacterial biomass and microcystins in freshwater systems. However, the environmental triggers of cyanobacteria and microcystin variability remain a subject of debate due to contrasting findings. This issue has raised the question if the environmental triggers are site-specific and unique between water bodies. In this study, we investigated the site-specificity of environmental triggers for cyanobacterial bloom and cyanotoxins dynamics. Our study suggests that cyanobacterial dominance and cyanobacterial microcystin content variability were significantly correlated to phosphorus and iron concentrations. However, the correlations between phosphorus and iron with cyanobacterial biomass and microcystin variability were not consistent between lakes, thus suggesting a site specificity of these environmental factors. The discrepancies in the correlations could be explained by differences in local nutrient concentration and the cyanobacterial community in the systems. The findings of this study suggest that identification of site-specific environmental factors under unique local conditions is an important strategy to enhance positive outcomes in cyanobacterial bloom control measures.

  7. Repetitive, Marker-Free, Site-Specific Integration as a Novel Tool for Multiple Chromosomal Integration of DNA

    DEFF Research Database (Denmark)

    Petersen, Kia Vest; Martinussen, Jan; Jensen, Peter Ruhdal

    2013-01-01

    We present a tool for repetitive, marker-free, site-specific integration in Lactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusio...

  8. A Free-Radical-Promoted Site-Specific Cross-Dehydrogenative-Coupling of N-Heterocycles with Fluorinated Alcohols.

    Science.gov (United States)

    Xu, Zhengbao; Hang, Zhaojia; Chai, Li; Liu, Zhong-Quan

    2016-09-16

    A C-C formation of an electron-rich N-heterocycle with fluorinated alcohol is developed. Through this radical-triggered cross-dehydrogenative coupling strategy, a wide range of useful building blocks such as C3 hydroxyfluoroalkylated indoles and pyrroles can be site-specifically synthesized. Mechanistic studies indicate a single-electron-transfer initiated radical cycle would be involved.

  9. Cre/lox-recombinase-mediated cassette exchange for reversible site-specific genomic targeting of the disease vector, Aedes aegypti

    Science.gov (United States)

    Site-specific genome modification is an important tool for mosquito functional genomics studies that help to uncover gene functions, identify gene regulatory elements, and perform comparative gene expression studies, all of which contribute to a better understanding of mosquito biology and are thus ...

  10. Site-specific incorporation of perylene into an N-terminally modified light-harvesting complex II

    NARCIS (Netherlands)

    Peneva, Kalina; Gundlach, Kristina; Herrmann, Andreas; Paulsen, Harald; Muellen, Klaus; Müllen, Klaus

    2010-01-01

    Employing the utility of the native chemical ligation, site-specific attachment of an ultrastable perylene dye to a derivative of the major light-harvesting complex (LHCII) was demonstrated. Biochemical analysis of the conjugate indicated that the structure and function of LHCII remain largely unaff

  11. Assessing, mapping and validating site-specific ecotoxicological risk for pesticide mixtures: a case study for small scale hot spots in aquatic and terrestrial environments.

    Science.gov (United States)

    Vaj, Claudia; Barmaz, Stefania; Sørensen, Peter Borgen; Spurgeon, David; Vighi, Marco

    2011-11-01

    Mixture toxicity is a real world problem and as such requires risk assessment solutions that can be applied within different geographic regions, across different spatial scales and in situations where the quantity of data available for the assessment varies. Moreover, the need for site specific procedures for assessing ecotoxicological risk for non-target species in non-target ecosystems also has to be recognised. The work presented in the paper addresses the real world effects of pesticide mixtures on natural communities. Initially, the location of risk hotspots is theoretically estimated through exposure modelling and the use of available toxicity data to predict potential community effects. The concept of Concentration Addition (CA) is applied to describe responses resulting from exposure of multiple pesticides The developed and refined exposure models are georeferenced (GIS-based) and include environmental and physico-chemical parameters, and site specific information on pesticide usage and land use. As a test of the risk assessment framework, the procedures have been applied on a suitable study areas, notably the River Meolo basin (Northern Italy), a catchment characterised by intensive agriculture, as well as comparative area for some assessments. Within the studied areas, the risks for individual chemicals and complex mixtures have been assessed on aquatic and terrestrial aboveground and belowground communities. Results from ecological surveys have been used to validate these risk assessment model predictions. Value and limitation of the approaches are described and the possibilities for larger scale applications in risk assessment are also discussed.

  12. Site specific prediction equations for peak acceleration of ground motion due to earthquakes induced by underground mining in Legnica-Głogów Copper District in Poland

    Science.gov (United States)

    Lasocki, Stanisław

    2013-10-01

    Ground motion database from the region of Żelazny Most tailings pond, the largest in Europe ore-flotation waste repository, is used to identify ground motion prediction equations (GMPE-s) for peak horizontal and peak vertical acceleration. A GMPE model including both geometrical spreading and anelastic damping terms cannot be correctly identified and the model with only spreading term is accepted. The analysis of variance of this model's residuals with station location as grouping variable indicates that station locations contribute significantly to the observed ground motion variability. Therefore, a site specific GMPE model with relative site amplifications is assessed. Despite short distances among stations, the amplification considerably vary from point to point, up to 1.8 times for the horizontal and 3.5 times for the vertical peak amplitude. The model including site effects enhances GMPE-s fit to observations, explains more than 60% dependent variables variability and correctly accounts for site effects.

  13. Wild Animals

    Institute of Scientific and Technical Information of China (English)

    宁静

    2005-01-01

    Many of us think that all wild animals are dangerous. In fact, very few of them will eat a man if he leaves them alone. If you meet a tiger, I'm sure you will run away, but even a tiger doesn't like meeting a man if it isn't hungry. Tigers only kill and eat man when they are too old to catch their food, such as sheep and other small animals. Some animals get frightened when they only smell a man. Some of themst and and look at a man for a short time before they run away.

  14. Modelling spread of foot-and-mouth disease in wild white-tailed deer and feral pig populations using a geographic-automata model and animal distributions.

    Science.gov (United States)

    Ward, Michael P; Laffan, Shawn W; Highfield, Linda D

    2009-09-01

    We investigated how the size and distribution of wild deer and feral pigs - species that might act as potential foot-and-mouth disease (FMD) virus maintenance hosts - might affect the persistence and spread of FMD. We used a susceptible-latent-infected-recovered geographic-automata model and spatially referenced data from southern Texas, USA. Within this study area, 100 locations were randomly selected and FMD virus spread was simulated (50 simulations each) at each location. As expected, the predicted sizes (km(2)) of the wild deer outbreaks were highly correlated (r(SP)>0.95) with the number of deer at incursion locations, the total number of deer within 2 km of incursion locations, and the minimum and maximum deer herd size within 2 km of incursion locations. However, the predicted sizes of the feral pig outbreaks were only moderately correlated (r(SP) 0.63-0.67) with the total, maximum and variance of the number of feral pigs within 2 km of incursion locations. Lack of continuity within the feral pig herd distribution across the landscape makes predicting disease spread more difficult than for deer, a more homogenously distributed species. When assessing the potential of wild and feral animal species at a locality to act as maintenance hosts of FMD virus, estimates of the population size and distribution might serve as a useful indicator of potential outbreaks in some circumstances.

  15. How wild is your model fire? Constraining WRF-Chem wildfire smoke simulations with satellite observations

    Science.gov (United States)

    Fischer, E. V.; Ford, B.; Lassman, W.; Pierce, J. R.; Pfister, G.; Volckens, J.; Magzamen, S.; Gan, R.

    2015-12-01

    Exposure to high concentrations of particulate matter (PM) present during acute pollution events is associated with adverse health effects. While many anthropogenic pollution sources are regulated in the United States, emissions from wildfires are difficult to characterize and control. With wildfire frequency and intensity in the western U.S. projected to increase, it is important to more precisely determine the effect that wildfire emissions have on human health, and whether improved forecasts of these air pollution events can mitigate the health risks associated with wildfires. One of the challenges associated with determining health risks associated with wildfire emissions is that the low spatial resolution of surface monitors means that surface measurements may not be representative of a population's exposure, due to steep concentration gradients. To obtain better estimates of ambient exposure levels for health studies, a chemical transport model (CTM) can be used to simulate the evolution of a wildfire plume as it travels over populated regions downwind. Improving the performance of a CTM would allow the development of a new forecasting framework that could better help decision makers estimate and potentially mitigate future health impacts. We use the Weather Research and Forecasting model with online chemistry (WRF-Chem) to simulate wildfire plume evolution. By varying the model resolution, meteorology reanalysis initial conditions, and biomass burning inventories, we are able to explore the sensitivity of model simulations to these various parameters. Satellite observations are used first to evaluate model skill, and then to constrain the model results. These data are then used to estimate population-level exposure, with the aim of better characterizing the effects that wildfire emissions have on human health.

  16. Rift Valley Fever Risk Map Model and Seroprevalence in Selected Wild Ungulates and Camels from Kenya

    NARCIS (Netherlands)

    Britch, S.C.; Binepal, Y.S.; Ruder, M.G.; Kariithi, H.M.; Linthicum, K.J.; Anyamba, A.; Small, J.L.; Tucker, C.J.; Ateya, L.O.; Oriko, A.A.; Gacheru, S.; Wilson, W.C.

    2013-01-01

    Since the first isolation of Rift Valley fever virus (RVFV) in the 1930s, there have been multiple epizootics and epidemics in animals and humans in sub-Saharan Africa. Prospective climate-based models have recently been developed that flag areas at risk of RVFV transmission in endemic regions based

  17. Craniofacial Statistical Deformation Models of Wild-type mice and Crouzon mice

    DEFF Research Database (Denmark)

    Ólafsdóttir, Hildur; Darvann, Tron Andre; Ersbøll, Bjarne Kjær

    2007-01-01

    Crouzon syndrome is characterised by the premature fusion of cranial sutures and synchondroses leading to craniofacial growth disturbances. The gene causing the syndrome was discovered approximately a decade ago and recently the first mouse model of the syndrome was generated. In this study, a se...

  18. Wild-type huntingtin ameliorates striatal neuronal atrophy but does not prevent other abnormalities in the YAC128 mouse model of Huntington disease

    Directory of Open Access Journals (Sweden)

    Leavitt Blair R

    2006-12-01

    Full Text Available Abstract Background Huntington disease (HD is an adult onset neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (htt protein. Htt function is essential for embryonic survival as well as normal function during the postnatal period. In addition to having roles in transcription and transport, recent evidence demonstrates that wild-type htt is neuroprotective in vivo. To determine whether treatment with wild-type htt would be beneficial in HD, we crossed the YAC128 mouse model of HD with mice that over-express wild-type htt (YAC18 mice to generate YAC128 mice that over-express wild-type htt (YAC18/128 mice. Results YAC18/128 mice were found to express mutant htt at the same level as YAC128 mice and wild-type htt at the same level as YAC18 mice. YAC18/128 mice show no significant behavioural improvement compared to YAC128 mice in the rotarod test of motor coordination or in an automated open field test. In the brain, YAC18/128 mice show no significant improvement in striatal volume, striatal neuronal numbers or striatal DARPP-32 expression compared to YAC128 mice. In contrast, striatal neuronal cross-sectional area showed significant improvement in YAC18/128 mice compared to YAC128 mice. Conclusion While the over-expression of wild-type htt results in a mild improvement in striatal neuropathology in YAC128 mice, our findings suggest that treatment with wild-type htt may not be sufficient to ameliorate the symptoms of HD in this model.

  19. A journey into the wild of the cnidarian model system Aiptasia and its symbionts

    KAUST Repository

    Voolstra, Christian R.

    2013-08-27

    The existence of coral reef ecosystems relies critically on the mutualistic relationship between calcifying cnidarians and photosynthetic, dinoflagellate endosymbionts in the genus Symbiodinium. Reef-corals have declined globally due to anthropogenic stressors, for example, rising sea-surface temperatures and pollution that often disrupt these symbiotic relationships (known as coral bleaching), exacerbating mass mortality and the spread of disease. This threatens one of the most biodiverse marine ecosystems providing habitats to millions of species and supporting an estimated 500 million people globally (Hoegh-Guldberg et al. 2007). Our understanding of cnidarian-dinoflagellate symbioses has improved notably with the recent application of genomic and transcriptomic tools (e.g. Voolstra et al. 2009; Bayer et al. 2012; Davy et al. 2012), but a model system that allows for easy manipulation in a laboratory environment is needed to decipher underlying cellular mechanisms important to the functioning of these symbioses. To this end, the sea anemone Aiptasia, otherwise known as a \\'pest\\' to aquarium hobbyists, is emerging as such a model system (Schoenberg & Trench 1980; Sunagawa et al. 2009; Lehnert et al. 2012). Aiptasia is easy to grow in culture and, in contrast to its stony relatives, can be maintained aposymbiotically (i.e. dinoflagellate free) with regular feeding. However, we lack basic information on the natural distribution and genetic diversity of these anemones and their endosymbiotic dinoflagellates. These data are essential for placing the significance of this model system into an ecological context. In this issue of Molecular Ecology, Thornhill et al. (2013) are the first to present genetic evidence on the global distribution, diversity and population structure of Aiptasia and its associated Symbiodinium spp. By integrating analyses of the host and symbiont, this research concludes that the current Aitpasia taxonomy probably needs revision and that two

  20. Site-specific mutations of FtsZ - effects on GTPase and in vitro assembly

    Directory of Open Access Journals (Sweden)

    Stricker Jesse

    2001-05-01

    Full Text Available Abstract Background FtsZ, the major cytoskeletal protein in bacterial cytokinesis, assembles in vitro into protofilaments, which can further associate into sheets, bundles or tubes. We have constructed 16 site-directed mutants of E. coli ftsZ, and tested them for GTP hydrolysis and assembly in vitro, and for their ability to complement the temperature sensitive ftsZ84 mutation in E. coli. Results The mutants were grouped into three classes. Benign mutants, which mapped mostly to the front and back surface of the protofilament, were able to complement ftsZ84 in vivo and showed normal assembly in vitro. GTP contact mutations had less than 10% of wild type GTPase activity. They could all assemble in vitro, and several of these mutants could complement ftsZ84. A third, and newly discovered, class of mutations mapped to the sides of the protofilaments. These lateral mutants had mostly normal GTPase and assembly in vitro, but none of them complemented ftsZ84. The non-complementing mutants showed greatly reduced expression from the pBS58 vector, suggesting possible dominant negative effects. Conclusions Several mutants with greatly reduced GTPase could still complement ftsZ84, suggesting that the high level of GTPase observed in vitro is not essential for in vivo function. All of the lateral mutants failed to complement ftsZ84, which suggests that these surfaces of the protofilaments are important for function in cell division. These lateral surfaces may mediate association of FtsZ protofilaments into pairs or small sheets, although their structure is apparently different from the sheets assembled in DEAE dextran or calcium.

  1. Apolipoprotein C-III Nanodiscs Studied by Site-Specific Tryptophan Fluorescence.

    Science.gov (United States)

    Brisbois, Chase A; Lee, Jennifer C

    2016-09-06

    Apolipoprotein C-III (ApoC-III) is found on high-density lipoproteins (HDLs) and remodels 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles into HDL-like particles known as nanodiscs. Using single-Trp-containing ApoC-III mutants, we have studied local side chain environments and interactions in nanodiscs at positions W42, W54, and W65. Using transmission electron microscopy and circular dichroism spectroscopy, nanodiscs were characterized at the ultrastructural and secondary conformational levels, respectively. Nearly identical particles (15 ± 2 nm) were produced from all proteins containing approximately 25 ± 4 proteins per particle with an average helicity of 45-51% per protein. Distinct residue-specific fluorescence properties were observed with W54 residing in the most hydrophobic environment followed by W42 and W65. Interestingly, time-resolved anisotropy measurements revealed that Trp side chain mobility is uncorrelated to the polarity of its surroundings. W54 is the most mobile compared to W65 and W42, which are more immobile in a nanodisc-bound state. On the basis of Trp spectral comparisons of ApoC-III in micellar and vesicle environments, ApoC-III binding within nanodiscs more closely resembles a bilayer-bound state. Despite the nanodiscs being structurally similar, we found marked differences during nanodisc formation by the Trp variants as a function of temperature, with W42 behaving the most like the wild-type protein. Our data suggest that despite the modest mutations of Trp to Phe at two of the three native sites, the interfacial location of W42 is important for lipid binding and nanodisc assembly, which may be biologically meaningful as of the three Trp residues, only W42 is invariant among mammals.

  2. Transcriptome discovery in non-model wild fish species for the development of quantitative transcript abundance assays

    Science.gov (United States)

    Hahn, Cassidy M.; Iwanowicz, Luke R.; Cornman, Robert S.; Mazik, Patricia M.; Blazer, Vicki S.

    2016-01-01

    Environmental studies increasingly identify the presence of both contaminants of emerging concern (CECs) and legacy contaminants in aquatic environments; however, the biological effects of these compounds on resident fishes remain largely unknown. High throughput methodologies were employed to establish partial transcriptomes for three wild-caught, non-model fish species; smallmouth bass (Micropterus dolomieu), white sucker (Catostomus commersonii) and brown bullhead (Ameiurus nebulosus). Sequences from these transcriptome databases were utilized in the development of a custom nCounter CodeSet that allowed for direct multiplexed measurement of 50 transcript abundance endpoints in liver tissue. Sequence information was also utilized in the development of quantitative real-time PCR (qPCR) primers. Cross-species hybridization allowed the smallmouth bass nCounter CodeSet to be used for quantitative transcript abundance analysis of an additional non-model species, largemouth bass (Micropterus salmoides). We validated the nCounter analysis data system with qPCR for a subset of genes and confirmed concordant results. Changes in transcript abundance biomarkers between sexes and seasons were evaluated to provide baseline data on transcript modulation for each species of interest.

  3. Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity.

    Science.gov (United States)

    Flint, David J; Binart, Nadine; Boumard, Stephanie; Kopchick, John J; Kelly, Paul

    2006-10-01

    Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40-50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.

  4. The scaling of wild events in stochastic models: The Fisher limit, the Mandelbrot limit, and FARIMA as a model of the intermediate cases

    Science.gov (United States)

    Watkins, Nicholas

    2013-04-01

    Stochastic modelling is of increasing importance, both specifically in climate science and more broadly across the whole of nonlinear geophysics. Traditionally, the noise components of such models would be spectrally white (delta-correlated) and Gaussian in amplitude, and their variance (first named by Fisher in 1918) would well characterise the likely size of fluctuations. Integration, for example in autoregressive models like AR(1), would redden a noise spectrum, while multiplication in turbulent cascades could greatly increase the range of fluctuation amplitudes, but such processes would still inherit aspects of their finite variance building blocks. In the 60s and 70s, however, Mandelbrot and others [see e.g. Watkins, GRL Frontiers, 2013] began to present evidence in nature for much stronger departures from Gaussianity (via very heavy tailed, infinite variance, distributions) and from white noise (through long range dependence (LRD) in time). He also observed intermittency, defined here as correlations between absolute magnitudes in some time series, in, for example, finance and turbulence. He proposed various models, including self-similar ones for heavy tails and LRD, and multifractal cascades for intermittency. In this presentation we compare contrasting types of model by looking at the "wild" events that they produce. The notion of a "wild" event can be made more precise in many ways, including by its duration in time, peak amplitude, and spatial extent. Our chosen measure will be the "burst", defined as the area of a time series above a fixed threshold. We will compare burst scaling in a self-similar, LRD, heavy tailed model (LFSM, e.g. Watkins et al, PRE, 2009] with our newer results for multifractal random walks [with M. Rypdal and O. Lovsletten], and for the heavy tailed extended version of the FARIMA (1,d,0) process, which combines long range dependence with the high frequency structure familiar from AR(1). We will also discuss the physical meaning of

  5. Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS.

    Science.gov (United States)

    Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Gun Wook; Hwang, Heeyoun; Jeong, Hoi Keun; Yun, Ki Na; Ji, Eun Sun; Kim, Kwang Hoe; Kim, Jun Seok; Kim, Jong Won; Yun, Sung Ho; Choi, Chi-Won; Kim, Seung Il; Lim, Jong-Sun; Jeong, Seul-Ki; Paik, Young-Ki; Lee, Soo-Youn; Park, Jisook; Kim, Su Yeon; Choi, Young-Jin; Kim, Yong-In; Seo, Jawon; Cho, Je-Yoel; Oh, Myoung Jin; Seo, Nari; An, Hyun Joo; Kim, Jin Young; Yoo, Jong Shin

    2016-12-02

    Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was N-glycopeptide isoforms of AGP from control and disease plasma sample.

  6. Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv.

    Science.gov (United States)

    Tobiason, D M; Buchner, J M; Thiel, W H; Gernert, K M; Karls, A C

    2001-02-01

    Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.

  7. Late-Quaternary biogeographic scenarios for the brown bear ( Ursus arctos), a wild mammal model species

    Science.gov (United States)

    Davison, John; Ho, Simon Y. W.; Bray, Sarah C.; Korsten, Marju; Tammeleht, Egle; Hindrikson, Maris; Østbye, Kjartan; Østbye, Eivind; Lauritzen, Stein-Erik; Austin, Jeremy; Cooper, Alan; Saarma, Urmas

    2011-02-01

    This review provides an up-to-date synthesis of the matrilineal phylogeography of a uniquely well-studied Holarctic mammal, the brown bear. We extend current knowledge by presenting a DNA sequence derived from one of the earliest known fossils of a polar bear (dated to 115 000 years before present), a species that shares a paraphyletic mitochondrial association with brown bears. A molecular clock analysis of 140 mitochondrial DNA sequences, including our new polar bear sequence, provides novel insights into the times of origin for different brown bear clades. We propose a number of regional biogeographic scenarios based on genetic data, divergence time estimates and paleontological records. The case of the brown bear provides an example for researchers working with less well-studied taxa: it shows clearly that phylogeographic models based on patterns of modern genetic variation alone can be substantially improved by including data on historical patterns of genetic diversity in the form of ancient DNA sequences derived from accurately dated samples and by using an approach to divergence-time estimation that suits the data under analysis. Using such approaches it has been possible to (i) establish that the processes shaping modern genetic diversity in brown bears acted recently, within the last three glacial cycles; (ii) distinguish among hypotheses concerning species' responses to climatic oscillations in accordance with the lack of phylogeographic structure that existed in brown bears prior to the last glacial maximum (LGM); (iii) reassess theories linking monophyletic brown bear populations to particular LGM refuge areas; and (iv) identify vicariance events and track analogous patterns of migration by brown bears out of Eurasia to North America and Japan.

  8. A noncanonical function of sortase enables site-specific conjugation of small molecules to lysine residues in proteins.

    Science.gov (United States)

    Bellucci, Joseph J; Bhattacharyya, Jayanta; Chilkoti, Ashutosh

    2015-01-07

    We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full-length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε-amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site-specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site-specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.

  9. Preliminary evaluation of AERMOD using site specific stack and ambient sampling data

    Science.gov (United States)

    A cotton ginning industry-supported project was initiated in 2008 to develop a robust particulate matter (PM) dispersion modeling dataset that could be used for evaluating current and future PM dispersion models. This paper compares total PM data collected by the industry-supported study at one gin ...

  10. Site-specific human risk assessment of soil contamination with volatile compounds

    NARCIS (Netherlands)

    Bakker J; Lijzen JPA; van Wijnen HJ; SEC; LER

    2009-01-01

    Het RIVM heeft het zogeheten VOLASOIL-model verbeterd. Het model schat voor woningen en andere gebouwen de binnenluchtconcentraties die ontstaan als gevolg van bodemverontreiniging met vluchtige verbindingen. Deze verontreiniging van bodem en grondwater kan zich voordoen in de omgeving van bijvoor

  11. Clearing the waters: Evaluating the need for site-specific field fluorescence corrections based on turbidity measurements

    Science.gov (United States)

    Saraceno, John F.; Shanley, James B.; Downing, Bryan D.; Pellerin, Brian A.

    2017-01-01

    In situ fluorescent dissolved organic matter (fDOM) measurements have gained increasing popularity as a proxy for dissolved organic carbon (DOC) concentrations in streams. One challenge to accurate fDOM measurements in many streams is light attenuation due to suspended particles. Downing et al. (2012) evaluated the need for corrections to compensate for particle interference on fDOM measurements using a single sediment standard in a laboratory study. The application of those results to a large river improved unfiltered field fDOM accuracy. We tested the same correction equation in a headwater tropical stream and found that it overcompensated fDOM when turbidity exceeded ∼300 formazin nephelometric units (FNU). Therefore, we developed a site-specific, field-based fDOM correction equation through paired in situ fDOM measurements of filtered and unfiltered streamwater. The site-specific correction increased fDOM accuracy up to a turbidity as high as 700 FNU, the maximum observed in this study. The difference in performance between the laboratory-based correction equation of Downing et al. (2012) and our site-specific, field-based correction equation likely arises from differences in particle size distribution between the sediment standard used in the lab (silt) and that observed in our study (fine to medium sand), particularly during high flows. Therefore, a particle interference correction equation based on a single sediment type may not be ideal when field sediment size is significantly different. Given that field fDOM corrections for particle interference under turbid conditions are a critical component in generating accurate DOC estimates, we describe a way to develop site-specific corrections.

  12. Superhydrophobic Surface Enhanced Raman Scattering Sensing using Janus Particle Arrays Realized by Site-Specific Electrochemical Growth

    OpenAIRE

    Yang, Shikuan; Hricko, Patrick John; Huang, Po-Hsun; Li, Sixing; Zhao, Yanhui; Xie, Yuliang; Guo, Feng; Wang, Lin; Huang, Tony Jun

    2013-01-01

    Site-specific electrochemical deposition is used to prepare polystyrene (PS)-Ag Janus particle arrays with superhydrophobic properties. The analyte molecules can be significantly enriched using the superhydrophobic property of the PS-Ag Janus particle array before SERS detections, enabling an extremely sensitive detection of molecules in a highly diluted solution (e.g., femtomolar level). This superhydrophobic surface enhanced Raman scattering sensing concept described here is of critical sig...

  13. Measurement of spatial proximity and accessibility of chromosomal loci in yeast using Cre/loxP site-specific recombination

    OpenAIRE

    Lui, Doris; Burgess, Sean M.

    2009-01-01

    Several methods have been developed to measure interactions between homologous chromosomes during meiosis in budding yeast. These include cytological analysis of fixed, spread nuclei using fluorescence in situ Hybridization (FISH) (1, 2), visualization of GFP-labeled chromosomal loci in living cells (3), and Chromosome-Conformation Capture (3C) (4). Here we describe a quantitative genetic assay that uses exogenous site-specific recombination to monitor the level of homolog associations betwee...

  14. Local nutrient regimes determine site-specific environmental triggers of cyanobacterial and microcystin variability in urban lakes

    Science.gov (United States)

    Sinang, S. C.; Reichwaldt, E. S.; Ghadouani, A.

    2015-05-01

    Toxic cyanobacterial blooms in urban lakes present serious health hazards to humans and animals and require effective management strategies. Managing such blooms requires a sufficient understanding of the controlling environmental factors. A range of them has been proposed in the literature as potential triggers for cyanobacterial biomass development and cyanotoxin (e.g. microcystin) production in freshwater systems. However, the environmental triggers of cyanobacteria and microcystin variability remain a subject of debate due to contrasting findings. This issue has raised the question of whether the relevance of environmental triggers may depend on site-specific combinations of environmental factors. In this study, we investigated the site-specificity of environmental triggers for cyanobacterial bloom and microcystin dynamics in three urban lakes in Western Australia. Our study suggests that cyanobacterial biomass, cyanobacterial dominance and cyanobacterial microcystin content variability were significantly correlated to phosphorus and iron concentrations. However, the correlations were different between lakes, thus suggesting a site-specific effect of these environmental factors. The discrepancies in the correlations could be explained by differences in local nutrient concentration. For instance, we found no correlation between cyanobacterial fraction and total phosphorous (TP) in the lake with the highest TP concentration, while correlations were significant and negative in the other two lakes. In addition, our study indicates that the difference of the correlation between total iron (TFe) and the cyanobacterial fraction between lakes might have been a consequence of differences in the cyanobacterial community structure, specifically the presence or absence of nitrogen-fixing species. In conclusion, our study suggests that identification of significant environmental factors under site-specific conditions is an important strategy to enhance successful outcomes

  15. In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates.

    Directory of Open Access Journals (Sweden)

    Dowdy Jackson

    Full Text Available Antibody drug conjugates (ADCs are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR, can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

  16. Site-Specific Management of Meloidogyne chitwoodi in Idaho Potatoes Using 1,3-Dichloropropene; Approach, Experiences, and Economics.

    Science.gov (United States)

    King, Bradley A; Taberna, John P

    2013-09-01

    Fumigation for nematode management in irrigated potato production systems of Idaho is widely practiced. Soil injection is the only labeled application method for 1,3-dichloropropene that is conventionally applied on a whole-field basis. Plant-parasitic nematode species exhibit spatially variable population densities that provide an opportunity to practice site-specific fumigation to reduce chemical usage and production costs. During 2002 to 2008, 62 fields intended for commercial potato production in eastern Idaho were sampled using a geo-referenced grid sampling system for plant-parasitic nematode population densities. In total, 4,030 grid samples were collected representing nearly 3,200 ha of commercial potato production. Collectively, 73% of the grid samples had Columbia root knot (CRN) (Meloidogyne chitwoodi) population densities below detectable levels. Site-specific fumigation is the practice of varying application rate of a fumigant based on nematode population density. In 2007, 640 ha of potato production were site-specific fumigated for CRN nematode control in eastern Idaho. On average, this practice resulted in a 30% reduction in chemical usage and production cost savings of $209/ha when 1,3-dichloropropene was used as the sole source of nematode suppression. Reductions in usage of 1,3-dichloropropene can exceed 50% if used in combination with a nonfumigant nematicide such as oxamyl. This combination approach can have production cost savings exceeding $200/ha. Based on farm-gate receipts and USDA inspections provided by potato producers from 2001 to 2011, potato tuber yield and quality have not been adversely affected using site-specific fumigation.

  17. Site-specific probabilistic seismic hazard analyses for the Idaho National Engineering Laboratory. Volume 1: Final report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-05-01

    This report describes and summarizes a probabilistic evaluation of ground motions for the Idaho National Engineering Laboratory (INEL). The purpose of this evaluation is to provide a basis for updating the seismic design criteria for the INEL. In this study, site-specific seismic hazard curves were developed for seven facility sites as prescribed by DOE Standards 1022-93 and 1023-96. These sites include the: Advanced Test Reactor (ATR); Argonne National Laboratory West (ANL); Idaho Chemical Processing Plant (ICPP or CPP); Power Burst Facility (PBF); Radioactive Waste Management Complex (RWMC); Naval Reactor Facility (NRF); and Test Area North (TAN). The results, probabilistic peak ground accelerations and uniform hazard spectra, contained in this report are not to be used for purposes of seismic design at INEL. A subsequent study will be performed to translate the results of this probabilistic seismic hazard analysis to site-specific seismic design values for the INEL as per the requirements of DOE Standard 1020-94. These site-specific seismic design values will be incorporated into the INEL Architectural and Engineering Standards.

  18. Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry.

    Science.gov (United States)

    Someya, Tatsuhiko; Ando, Ami; Kimoto, Michiko; Hirao, Ichiro

    2015-08-18

    Site-specific labeling of long-chain RNAs with desired molecular probes is an imperative technique to facilitate studies of functional RNA molecules. By genetic alphabet expansion using an artificial third base pair, called an unnatural base pair, we present a post-transcriptional modification method for RNA transcripts containing an incorporated azide-linked unnatural base at specific positions, using a copper-free click reaction. The unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions in transcription. Thus, we chemically synthesized a triphosphate substrate of 4-(4-azidopentyl)-pyrrole-2-carbaldehyde (N3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N3-Pa incorporated in the transcripts was modified with dibenzocyclooctyne (DIBO) derivatives. We demonstrated the transcription of 17-, 76- and 260-mer RNA molecules and their site-specific labeling with Alexa 488, Alexa 594 and biotin. This method will be useful for preparing RNA molecules labeled with any functional groups of interest, toward in vivo experiments.

  19. Analysis of the site-specific integration system of the Streptomyces aureofaciens phage μ1/6.

    Science.gov (United States)

    Farkašovská, Jarmila; Godány, Andrej

    2012-03-01

    The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3' end of a putative tRNA(Thr) gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.

  20. Symptomatic and quality of life outcomes after site-specific fascial reattachment for pelvic organ prolapse repair.

    Science.gov (United States)

    Fayyad, Abdalla M; Redhead, Emma; Awan, Noveen; Kyrgiou, Maria; Prashar, Sanjeev; Hill, Simon R

    2008-02-01

    The aim of this study was to assess symptomatic and quality of life outcome scores following site specific fascial reattachment surgery for pelvic organ prolapse using the validated Prolapse Quality of Life (P-QOL) questionnaires. One hundred and ninety two women underwent surgery for pelvic organ prolapse; ninety four underwent anterior repair (thirty four of them had vaginal hysterectomy), and ninety eight had posterior repair. Patients filled P-QOL questionnaires 24 hours prior to surgery and a postal P-QOL questionnaire six months post operatively. Pre and post operative questionnaires were paired. Quality of life and symptoms scores were calculated using Wilcoxon signed rank test. One hundred and one women returned their questionnaires and were suitable to include in the study. Forty nine underwent anterior repair (fifteen had vaginal hysterectomy) and 52 underwent posterior repair. Quality of life scores showed significant improvement in the anterior and posterior repair groups with the exception of general health in the anterior repair group and general health and prolapse impact in the posterior repair group. Anterior repair significantly improved urinary voiding and storage symptoms. Posterior repair group showed significant improvement in defecatory symptoms. Both groups showed improvement in sexual function and general prolapse symptoms. Prolapse repair with site specific fascial reattachment results in significant improvement in quality of life scores six months after surgery. Anterior repair improves urinary voiding and storage symptoms and posterior repair improves defecatory dysfunction and urinary voiding. Sexual function improves following prolapse repair with site specific fascial reattachment.

  1. Refining health risk assessment by incorporating site-specific background concentration and bioaccessibility data of Nickel in soil.

    Science.gov (United States)

    Zhong, Mao-Sheng; Jiang, Lin

    2017-03-01

    Risk assessment of Nickel (Ni) in a brownfield site contaminated was refined by incorporating the site-specific background level (SSBL) derived using multiple lines of evidence technology and bioaccessibility measured by the Unified Bioaccessibility Method (UBM) and Inhalation Bioaccessibility Method (IBM). The results revealed that the SSBL was 80mg/kg, which was much higher than the general soil screening value (GSSL) (50mg/kg) and more than two times regional background level reported for Tianjing (33mg/kg). The average bioaccessibility for the gastric and intestinal phases was 24.2% (8.6%-54.4%) and 12.6% (7.7%-17.1%), respectively. In simulated lung fluid, only 2.1% (1.0%-4.6%) of Ni in the soil particle was bioaccessible. The amount of samples exceeding the acceptable level was reduced by 50% when SSBL, other than GSSL, was used as the screening value. Moreover, the site-specific target level (SSTL) under acceptable risk level was increased from 94mg/kg to 283mg/kg when bioaccessibility was considered, and the amount of samples above SSTL was reduced to 10%. The study indicated that incorporating site-specific background levels and bioaccessibility data of contaminants in a given site has the potential to support health risk assessment decisions and can reduce the remediation cost greatly. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Cardiac myosin binding protein C phosphorylation affects cross-bridge cycle's elementary steps in a site-specific manner.

    Directory of Open Access Journals (Sweden)

    Li Wang

    Full Text Available Based on our recent finding that cardiac myosin binding protein C (cMyBP-C phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302, DAD (Asp273-Ala282-Asp302, SAS (Ser273-Ala282-Ser302, and t/t (cMyBP-C null genotypes, and the results were compared to transgenic mice expressing wide-type (WT cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi, and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc, and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.

  3. A statistical model of the international spread of wild poliovirus in Africa used to predict and prevent outbreaks.

    Directory of Open Access Journals (Sweden)

    Kathleen M O'Reilly

    2011-10-01

    Full Text Available Outbreaks of poliomyelitis in African countries that were previously free of wild-type poliovirus cost the Global Polio Eradication Initiative US$850 million during 2003-2009, and have limited the ability of the program to focus on endemic countries. A quantitative understanding of the factors that predict the distribution and timing of outbreaks will enable their prevention and facilitate the completion of global eradication.Children with poliomyelitis in Africa from 1 January 2003 to 31 December 2010 were identified through routine surveillance of cases of acute flaccid paralysis, and separate outbreaks associated with importation of wild-type poliovirus were defined using the genetic relatedness of these viruses in the VP1/2A region. Potential explanatory variables were examined for their association with the number, size, and duration of poliomyelitis outbreaks in 6-mo periods using multivariable regression analysis. The predictive ability of 6-mo-ahead forecasts of poliomyelitis outbreaks in each country based on the regression model was assessed. A total of 142 genetically distinct outbreaks of poliomyelitis were recorded in 25 African countries, resulting in 1-228 cases (median of two cases. The estimated number of people arriving from infected countries and <5-y childhood mortality were independently associated with the number of outbreaks. Immunisation coverage based on the reported vaccination history of children with non-polio acute flaccid paralysis was associated with the duration and size of each outbreak, as well as the number of outbreaks. Six-month-ahead forecasts of the number of outbreaks in a country or region changed over time and had a predictive ability of 82%.Outbreaks of poliomyelitis resulted primarily from continued transmission in Nigeria and the poor immunisation status of populations in neighbouring countries. From 1 January 2010 to 30 June 2011, reduced transmission in Nigeria and increased incidence in reinfected

  4. Volume-based solvation models out-perform area-based models in combined studies of wild-type and mutated protein-protein interfaces

    Directory of Open Access Journals (Sweden)

    Warwicker Jim

    2008-10-01

    Full Text Available Abstract Background Empirical binding models have previously been investigated for the energetics of protein complexation (ΔG models and for the influence of mutations on complexation (i.e. differences between wild-type and mutant complexes, ΔΔG models. We construct binding models to directly compare these processes, which have generally been studied separately. Results Although reasonable fit models were found for both ΔG and ΔΔG cases, they differ substantially. In a dataset curated for the absence of mainchain rearrangement upon binding, non-polar area burial is a major determinant of ΔG models. However this ΔG model does not fit well to the data for binding differences upon mutation. Burial of non-polar area is weighted down in fitting of ΔΔG models. These calculations were made with no repacking of sidechains upon complexation, and only minimal packing upon mutation. We investigated the consequences of more extensive packing changes with a modified mean-field packing scheme. Rather than emphasising solvent exposure with relatively extended sidechains, rotamers are selected that exhibit maximal packing with protein. This provides solvent accessible areas for proteins that are much closer to those of experimental structures than the more extended sidechain regime. The new packing scheme increases changes in non-polar burial for mutants compared to wild-type proteins, but does not substantially improve agreement between ΔG and ΔΔG binding models. Conclusion We conclude that solvent accessible area, based on modelled mutant structures, is a poor correlate for ΔΔG upon mutation. A simple volume-based, rather than solvent accessibility-based, model is constructed for ΔG and ΔΔG systems. This shows a more consistent behaviour. We discuss the efficacy of volume, as opposed to area, approaches to describe the energetic consequences of mutations at interfaces. This knowledge can be used to develop simple computational screens for

  5. Site-specific critical acid load estimates for forest soils in the Osborn Creek watershed, Michigan

    Science.gov (United States)

    Trevor Hobbs; Jason Lynch; Randy. Kolka

    2017-01-01

    Anthropogenic acid deposition has the potential to accelerate leaching of soil cations, and in turn, deplete nutrients essential to forest vegetation. The critical load concept, employing a simple mass balance (SMB) approach, is often used to model this process. In an evaluation under the U.S. Forest Service Watershed Condition Framework program, soils in all 6th level...

  6. Site-Specific Study of In-Building Wireless Solutions with Poisson Traffic

    DEFF Research Database (Denmark)

    Liu, Zhen; Sørensen, Troels Bundgaard; Mogensen, Preben

    2011-01-01

    traffic model with fixed buffer size and Poisson arrival. Our new results show better performance for Femto cells with frequency reuse 1 at light to medium load, although the intelligent distributed system still obtains considerable better cell edge user throughput for the same number of access points....

  7. Predicting functional divergence in protein evolution by site-specific rate shifts

    Science.gov (United States)

    Gaucher, Eric A.; Gu, Xun; Miyamoto, Michael M.; Benner, Steven A.

    2002-01-01

    Most modern tools that analyze protein evolution allow individual sites to mutate at constant rates over the history of the protein family. However, Walter Fitch observed in the 1970s that, if a protein changes its function, the mutability of individual sites might also change. This observation is captured in the "non-homogeneous gamma model", which extracts functional information from gene families by examining the different rates at which individual sites evolve. This model has recently been coupled with structural and molecular biology to identify sites that are likely to be involved in changing function within the gene family. Applying this to multiple gene families highlights the widespread divergence of functional behavior among proteins to generate paralogs and orthologs.

  8. LCA of contaminated site remediation - integration of site-specific impact assessment of local toxic impacts

    DEFF Research Database (Denmark)

    Lemming, Gitte; Hauschild, Michael Zwicky; Chambon, Julie Claire Claudia

    2011-01-01

    The environmental impacts from remediation can be divided into primary and secondary impacts. Primary impacts cover the local impacts associated with the on-site contamination, whereas the secondary impacts are impacts on the local, regional and global scale generated by the remediation activities...... impacts have typically been assessed using site-generic characterization models representing a continental scale and excluding the groundwater compartment. Soil contaminants have therefore generally been assigned as emissions to surface soil or surface water compartments. However, such site...

  9. Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish

    OpenAIRE

    Atsuo Kawahara; Yu Hisano; Satoshi Ota; Kiyohito Taimatsu

    2016-01-01

    The zebrafish (Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and i...

  10. Site-specific blockade of RAGE-Vd prevents amyloid-beta oligomer neurotoxicity

    OpenAIRE

    Sturchler, E; Galichet, A; Weibel, M; Leclerc, E; Heizmann, C W

    2008-01-01

    In the genesis of Alzheimer's disease (AD), converging lines of evidence suggest that amyloid-beta peptide (Abeta) triggers a pathogenic cascade leading to neuronal loss. It was long assumed that Abeta had to be assembled into extracellular amyloid fibrils or aggregates to exert its cytotoxic effects. Over the past decade, characterization of soluble oligomeric Abeta species in the brains of AD patients and in transgenic models has raised the possibility that different conformations of Abeta ...

  11. A simple method for assessing available weather data quality for site specific nutrient management

    Science.gov (United States)

    Fuka, D. R.; Collick, A.; Kleinman, P. J. A.; Sommerlot, A.; Easton, Z. M.

    2014-12-01

    Phosphorous (P) Indices are an assessment tool used to identifying agricultural fields most vulnerable to P losses. In the two decades since its introduction, the P Indexing concept has evolved, and there are now P Indices that serve as Best Management Practice (BMP) selection and targeting. While the use of observed P loss data under various management scenarios is the ideal way to assess the accuracy of the P Indices, water quality data, particularly at the field scale, are not widely available and can require years of costly field research to generate. In place of in-situ water quality measurements, the use of locally relevant and corroborated water quality models is a more expedient option to conduct index assessments in the short time required for new standards. The input forcing data required by these models include precipitation and temperature. Unfortunately obtaining representative meteorological data for watershed-scale hydrological modelling can be difficult and time consuming. Land-based weather stations do not always adequately represent the weather occurring over a watershed because they can be far from the watershed of interest and can have gaps in their data series, or recent data are not available. Fuka et al. (2013) has shown that readily available short term forecasting data can match the accuracy of using traditional weather gauging stations, especially when the closest stations are more than 10km from the watershed. In this study we demonstrate a methodology to ascertain the most representative openly accessible weather forcing data for running water quality models in any location in the US. For this P-Index assessment, nine representative project watersheds from the three regional consortiums--Heartland, Chesapeake Bay, and Southern Regions--are used to demonstrate this method of determining what is the most representative weather forcing data for any location within the regions.

  12. Spatiotemporal modelling of hormonal crosstalk explains the level and patterning of hormones and gene expression in Arabidopsis thaliana wild-type and mutant roots

    Science.gov (United States)

    Moore, Simon; Zhang, Xiaoxian; Mudge, Anna; Rowe, James H; Topping, Jennifer F; Liu, Junli; Lindsey, Keith

    2015-01-01

    • Patterning in Arabidopsis root development is coordinated via a localized auxin concentration maximum in the root tip, requiring the regulated expression of specific genes. However, little is known about how hormone and gene expression patterning is generated. • Using a variety of experimental data, we develop a spatiotemporal hormonal crosstalk model that describes the integrated action of auxin, ethylene and cytokinin signalling, the POLARIS protein, and the functions of PIN and AUX1 auxin transporters. We also conduct novel experiments to confirm our modelling predictions. • The model reproduces auxin patterning and trends in wild-type and mutants; reveals that coordinated PIN and AUX1 activities are required to generate correct auxin patterning; correctly predicts shoot to root auxin flux, auxin patterning in the aux1 mutant, the amounts of cytokinin, ethylene and PIN protein, and PIN protein patterning in wild-type and mutant roots. Modelling analysis further reveals how PIN protein patterning is related to the POLARIS protein through ethylene signalling. Modelling prediction of the patterning of POLARIS expression is confirmed experimentally. • Our combined modelling and experimental analysis reveals that a hormonal crosstalk network regulates the emergence of patterns and levels of hormones and gene expression in wild-type and mutants. PMID:25906686

  13. Comparison of unmanned aircraft systems (UAS) to LiDAR for streambank erosion measurement at the site-specific and river network scales

    Science.gov (United States)

    Hamshaw, S. D.; Dewoolkar, M. M.; Rizzo, D.; ONeil-Dunne, J.; Frolik, J.

    2016-12-01

    Measurement of rates and extent of streambank erosion along river corridors is an important component of many catchment studies and necessary for engineering projects such as river restoration, hazard assessment, and total maximum daily load (TMDL) development. A variety of methods have been developed to quantify streambank erosion, including bank pins, ground surveys, photogrammetry, LiDAR, and analytical models. However, these methods are not only resource intensive, but many are feasible and appropriate only for site-specific studies and not practical for erosion estimates at larger scales. Recent advancements in unmanned aircraft systems (UAS) and photogrammetry software provide capabilities for more rapid and economical quantification of streambank erosion and deposition at multiple scales (from site-specific to river network). At the site-specific scale, the capability of UAS to quantify streambank erosion was compared to terrestrial laser scanning (TLS) and RTK-GPS ground survey and assessed at seven streambank monitoring sites in central Vermont. Across all sites, the UAS-derived bank topography had mean errors of 0.21 m compared to TLS and GPS data. Highest accuracies were achieved in early spring conditions where mean errors approached 10 cm. The cross sectional area of bank erosion at a typical, vegetated streambank site was found to be reliably calculated within 10% of actual for erosion areas greater than 3.5 m2. At the river network-level scale, 20 km of river corridor along the New Haven, Winooski, and Mad Rivers was flown on multiple dates with UAS and used to generate digital elevation models (DEMs) that were then compared for change detection analysis. Airborne LiDAR data collected prior to UAS surveys was also compared to UAS data to determine multi-year rates of bank erosion. UAS-based photogrammetry for generation of fine scale topographic data shows promise for the monitoring of streambank erosion both at the individual site scale and river

  14. Development and Characterization of Novel Site Specific Hollow Floating Microspheres Bearing 5-Fu for Stomach Targeting

    Directory of Open Access Journals (Sweden)

    Peeyush Bhardwaj

    2014-01-01

    Full Text Available Multiple-unit-type oral floating hollow microspheres of 5-fluorouracil (5-Fu were developed using modified solvent evaporation technique to prolong gastric residence time, to target stomach cancer, and to increase drug bioavailability. The prepared microspheres were characterized for micromeritic properties, floating behavior, entrapment efficiency, and scanning electron microscopy (SEM. The in vitro drug release and floating behavior were studied in simulated gastric fluid (SGF at pH 1.2. The yield of microspheres was obtained up to 84.46±6.47%. Microspheres showed passable flow properties. Based on optical microscopy, particle size was found to be ranging from 158.65±12.02 to 198.67±17.45 μm. SEM confirmed spherical size, perforated smooth surface, and a hollow cavity inside the microspheres. Different kinetic models for drug release were also applied on selected batches.

  15. Gene Regulatory Network Analysis Reveals Differences in Site-specific Cell Fate Determination in Mammalian Brain

    Directory of Open Access Journals (Sweden)

    Gokhan eErtaylan

    2014-12-01

    Full Text Available Neurogenesis - the generation of new neurons - is an ongoing process that persists in the adult mammalian brain of several species, including humans. In this work we analyze two discrete brain regions: the subventricular zone (SVZ lining the walls of the lateral ventricles; and the subgranular zone (SGZ of the dentate gyrus of the hippocampus in mice and shed light on the SVZ and SGZ specific neurogenesis. We propose a computational model that relies on the construction and analysis of region specific gene regulatory networks from the publicly available data on these two regions. Using this model a number of putative factors involved in neuronal stem cell (NSC identity and maintenance were identified. We also demonstrate potential gender and niche-derived differences based on cell surface and nuclear receptors via Ar, Hif1a and Nr3c1.We have also conducted cell fate determinant analysis for SVZ NSC populations to Olfactory Bulb interneurons and SGZ NSC populations to the granule cells of the Granular Cell Layer. We report thirty-one candidate cell fate determinant gene pairs, ready to be validated. We focus on Ar - Pax6 in SVZ and Sox2 - Ncor1 in SGZ. Both pairs are expressed and localized in the suggested anatomical structures as shown by in situ hybridization and found to physically interact.Finally, we conclude that there are fundamental differences between SGZ and SVZ neurogenesis. We argue that these regulatory mechanisms are linked to the observed differential neurogenic potential of these regions. The presence of nuclear and cell surface receptors in the region specific regulatory circuits indicate the significance of niche derived extracellular factors, hormones and region specific factors such as the oxygen sensitivity, dictating SGZ and SVZ specific neurogenesis.

  16. Light-stimulated cargo release from a core–shell structured nanocomposite for site-specific delivery

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Yun; Ling, Li; Li, Xiaofang [Department of Neurology, Affiliated Hospital of Hebei University, Baoding 071000 (China); Chen, Meng [Department of Rheumatology, Affiliated Hospital of Hebei University, Baoding 071000 (China); Su, Likai, E-mail: zhangdong19992003@163.com [Department of Neurology, Affiliated Hospital of Hebei University, Baoding 071000 (China)

    2015-03-15

    This paper reported a core–shell structured site-specific delivery system with a light switch triggered by low energy light (λ=510 nm). Its core was composed of supermagnetic Fe{sub 3}O{sub 4} nanoparticles for magnetic guiding and targeting. Its outer shell consisted of mesoporous silica molecular sieve MCM-41 which offered highly ordered hexagonal tunnels for cargo capacity. A light switch N1-(4aH-cyclopenta[1,2-b:5,4-b′]dipyridin-5(5aH)-ylidene)benzene-1, 4-diamine (CBD) was covalently grafted into these hexagonal tunnels, serving as light stimuli acceptor with loading content of 1.1 μM/g. This composite was fully characterized and confirmed by SEM, TEM, XRD patterns, N{sub 2} adsorption/desorption, thermogravimetric analysis, IR, UV–vis absorption and emission spectra. Experimental data suggested that this composite had a core as wide as 150 nm and could be magnetically guided to specific sites. Its hexagonal tunnels were as long as 180 nm. Upon light stimuli of “on” and “off” states, controllable release was observed with short release time of ~900 s (90% capacity). - Graphical abstract: A core–shell structured site-specific delivery system with a light switch triggered by yellow light was constructed. Controllable release was observed with short release time of ~900 s (90% capacity). - Highlights: • A core–shell structured site-specific delivery system was constructed. • It consisted of Fe{sub 3}O{sub 4} core and MCM-41 shell grafted with light switch. • This delivery system was triggered by low energy light. • Controllable release was observed with short release time of ~900 s.

  17. Molecular cytogenetic characterization of chromosome site-specific repetitive sequences in the Arctic lamprey (Lethenteron camtschaticum, Petromyzontidae)

    Science.gov (United States)

    Ishijima, Junko; Uno, Yoshinobu; Nunome, Mitsuo; Nishida, Chizuko; Kuraku, Shigehiro

    2017-01-01

    Abstract All extant lamprey karyotypes are characterized by almost all dot-shaped microchromosomes. To understand the molecular basis of chromosome structure in lampreys, we performed chromosome C-banding and silver staining and chromosome mapping of the 18S–28S and 5S ribosomal RNA (rRNA) genes and telomeric TTAGGG repeats in the Arctic lamprey (Lethenteron camtschaticum). In addition, we cloned chromosome site-specific repetitive DNA sequences and characterized them by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. Three types of repetitive sequences were detected; a 200-bp AT-rich repetitive sequence, LCA-EcoRIa that co-localized with the 18S–28S rRNA gene clusters of 3 chromosomal pairs; a 364-bp AT-rich LCA-EcoRIb sequence that showed homology to the EcoRI sequence family from the sea lamprey (Petromyzon marinus), which contains short repeats as centromeric motifs; and a GC-rich 702-bp LCA-ApaI sequence that was distributed on nearly all chromosomes and showed significant homology with the integrase-coding region of a Ty3/Gypsy family long terminal repeat (LTR) retrotransposon. All three repetitive sequences are highly conserved within the Petromyzontidae or within Petromyzontidae and Mordaciidae. Molecular cytogenetic characterization of these site-specific repeats showed that they may be correlated with programed genome rearrangement (LCA-EcoRIa), centromere structure and function (LCA-EcoRIb), and site-specific amplification of LTR retroelements through homogenization between non-homologous chromosomes (LCA-ApaI). PMID:28025319

  18. Accounting for both local aquatic community composition and bioavailability in setting site-specific quality standards for zinc.

    Science.gov (United States)

    Peters, Adam; Simpson, Peter; Moccia, Alessandra

    2014-01-01

    Recent years have seen considerable improvement in water quality standards (QS) for metals by taking account of the effect of local water chemistry conditions on their bioavailability. We describe preliminary efforts to further refine water quality standards, by taking account of the composition of the local ecological community (the ultimate protection objective) in addition to bioavailability. Relevance of QS to the local ecological community is critical as it is important to minimise instances where quality classification using QS does not reconcile with a quality classification based on an assessment of the composition of the local ecology (e.g. using benthic macroinvertebrate quality assessment metrics such as River InVertebrate Prediction and Classification System (RIVPACS)), particularly where ecology is assessed to be at good or better status, whilst chemical quality is determined to be failing relevant standards. The alternative approach outlined here describes a method to derive a site-specific species sensitivity distribution (SSD) based on the ecological community which is expected to be present at the site in the absence of anthropogenic pressures (reference conditions). The method combines a conventional laboratory ecotoxicity dataset normalised for bioavailability with field measurements of the response of benthic macroinvertebrate abundance to chemical exposure. Site-specific QSref are then derived from the 5%ile of this SSD. Using this method, site QSref have been derived for zinc in an area impacted by historic mining activities. Application of QSref can result in greater agreement between chemical and ecological metrics of environmental quality compared with the use of either conventional (QScon) or bioavailability-based QS (QSbio). In addition to zinc, the approach is likely to be applicable to other metals and possibly other types of chemical stressors (e.g. pesticides). However, the methodology for deriving site-specific targets requires

  19. A novel bench-scale column assay to investigate site-specific nitrification biokinetics in biological rapid sand filters

    DEFF Research Database (Denmark)

    Tatari, Karolina; Smets, Barth F.; Albrechtsen, Hans-Jørgen

    2013-01-01

    A bench-scale assay was developed to obtain site-specific nitrification biokinetic information from biological rapid sand filters employed in groundwater treatment. The experimental set-up uses granular material subsampled from a full-scale filter, packed in a column, and operated with controlled....../h could easily be determined at 7.5 g NH4+–N/m3 packed sand/h. This assay, with conditions reflecting full-scale observations, and where the biological activity is subject to minimal physical disturbance, provides a simple and fast, yet powerful tool to gain insight in nitrification kinetics in rapid sand...

  20. Molecular recognition: monomer of the yeast transcriptional activator GCN4 recognizes its dimer DNA binding target sites specifically

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    It is widely believed that dimerization is a requirement for the yeast transcriptional activator GCN4 to recognize its specific DNA target sites. We used the basic region (226-252) of the yeast transcriptional activator GCN4, as both a monomeric peptide and a disulfide-linked dimer to investigate the interaction of the peptides with the DNA target sites AP-1 and CRE. CD and ITC experiments indicate that although the monomeric peptide GCN4-M has a weaker affinity with the DNA relative to the disulfide-linked dimer peptide GCN4-D, it recognizes AP-1 and CRE target sites specifically.

  1. A procedure for estimating site specific derived limits for the discharge of radioactive material to the atmosphere

    CERN Document Server

    Hallam, J; Jones, J A

    1983-01-01

    Generalised Derived Limits (GDLs) for the discharge of radioactive material to the atmosphere are evaluated using parameter values to ensure that the exposure of the critical group is unlikely to be underestimated significantly. Where the discharge is greater than about 5% of the GDL, a more rigorous estimate of the derived limit may be warranted. This report describes a procedure for estimating site specific derived limits for discharges of radioactivity to the atmosphere taking into account the conditions of the release and the location and habits of the exposed population. A worksheet is provided to assist in carrying out the required calculations.

  2. The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Sass, Ehud; Suski, Catherine

    2014-01-01

    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus......, to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it is neither...

  3. Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo.

    Science.gov (United States)

    Schuch, Ronaldo; Agelopoulos, Konstantin; Neumann, Anna; Brandt, Burkhard; Bürger, Horst; Korsching, Eberhard

    2012-02-20

    Transcription factors (TFs) and their binding sites (TFBSs) play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP). However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs. The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase), through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue. The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially in cancer biology. The established site-specific enzyme

  4. Site-specific chromatin immunoprecipitation: a selective method to individually analyze neighboring transcription factor binding sites in vivo

    Directory of Open Access Journals (Sweden)

    Schuch Ronaldo

    2012-02-01

    Full Text Available Abstract Background Transcription factors (TFs and their binding sites (TFBSs play a central role in the regulation of gene expression. It is therefore vital to know how the allocation pattern of TFBSs affects the functioning of any particular gene in vivo. A widely used method to analyze TFBSs in vivo is the chromatin immunoprecipitation (ChIP. However, this method in its present state does not enable the individual investigation of densely arranged TFBSs due to the underlying unspecific DNA fragmentation technique. This study describes a site-specific ChIP which aggregates the benefits of both EMSA and in vivo footprinting in only one assay, thereby allowing the individual detection and analysis of single binding motifs. Findings The standard ChIP protocol was modified by replacing the conventional DNA fragmentation, i. e. via sonication or undirected enzymatic digestion (by MNase, through a sequence specific enzymatic digestion step. This alteration enables the specific immunoprecipitation and individual examination of occupied sites, even in a complex system of adjacent binding motifs in vivo. Immunoprecipitated chromatin was analyzed by PCR using two primer sets - one for the specific detection of precipitated TFBSs and one for the validation of completeness of the enzyme digestion step. The method was established exemplary for Sp1 TFBSs within the egfr promoter region. Using this site-specific ChIP, we were able to confirm four previously described Sp1 binding sites within egfr promoter region to be occupied by Sp1 in vivo. Despite the dense arrangement of the Sp1 TFBSs the improved ChIP method was able to individually examine the allocation of all adjacent Sp1 TFBS at once. The broad applicability of this site-specific ChIP could be demonstrated by analyzing these SP1 motifs in both osteosarcoma cells and kidney carcinoma tissue. Conclusions The ChIP technology is a powerful tool for investigating transcription factors in vivo, especially

  5. Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    For the temporally and spatially regulated expression of the barnase gene in plant,two kinds of plasmids with cre gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure.The transgenic plants were conformed by PCR analysis.The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome.Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.

  6. MONITORING OF IN-FIELD VARIABILITY FOR SITE SPECIFIC CROP MANAGEMENT THROUGH OPEN GEOSPATIAL INFORMATION

    Directory of Open Access Journals (Sweden)

    T. Řezník

    2016-06-01

    Full Text Available The agricultural sector is in a unique position due to its strategic importance around the world. It is crucial for both citizens (consumers and the economy (both regional and global, which, ideally, should ensure that the whole sector is a network of interacting organisations. It is important to develop new tools, management methods, and applications to improve the management and logistic operations of agricultural producers (farms and agricultural service providers. From a geospatial perspective, this involves identifying cost optimization pathways, reducing transport, reducing environmental loads, and improving the energy balance, while maintaining production levels, etc. This paper describes the benefits of, and open issues arising from, the development of the Open Farm Management Information System. Emphasis is placed on descriptions of available remote sensing and other geospatial data, and their harmonization, processing, and presentation to users. At the same time, the FOODIE platform also offers a novel approach of yield potential estimations. Validation for one farm demonstrated 70% successful rate when comparing yield results at a farm counting 1’284 hectares on one hand and results of a theoretical model of yield potential on the other hand. The presented Open Farm Management Information System has already been successfully registered under Phase 8 of the Global Earth Observation System of Systems (GEOSS Architecture Implementation Pilot in order to support the wide variety of demands that are primarily aimed at agriculture and water pollution monitoring by means of remote sensing.

  7. Monitoring of In-Field Variability for Site Specific Crop Management Through Open Geospatial Information

    Science.gov (United States)

    Řezník, T.; Lukas, V.; Charvát, K.; Charvát, K., Jr.; Horáková, Š.; Křivánek, Z.; Herman, L.

    2016-06-01

    The agricultural sector is in a unique position due to its strategic importance around the world. It is crucial for both citizens (consumers) and the economy (both regional and global), which, ideally, should ensure that the whole sector is a network of interacting organisations. It is important to develop new tools, management methods, and applications to improve the management and logistic operations of agricultural producers (farms) and agricultural service providers. From a geospatial perspective, this involves identifying cost optimization pathways, reducing transport, reducing environmental loads, and improving the energy balance, while maintaining production levels, etc. This paper describes the benefits of, and open issues arising from, the development of the Open Farm Management Information System. Emphasis is placed on descriptions of available remote sensing and other geospatial data, and their harmonization, processing, and presentation to users. At the same time, the FOODIE platform also offers a novel approach of yield potential estimations. Validation for one farm demonstrated 70% successful rate when comparing yield results at a farm counting 1'284 hectares on one hand and results of a theoretical model of yield potential on the other hand. The presented Open Farm Management Information System has already been successfully registered under Phase 8 of the Global Earth Observation System of Systems (GEOSS) Architecture Implementation Pilot in order to support the wide variety of demands that are primarily aimed at agriculture and water pollution monitoring by means of remote sensing.

  8. Alcohol consumption and site-specific cancer risk: a comprehensive dose–response meta-analysis

    Science.gov (United States)

    Bagnardi, V; Rota, M; Botteri, E; Tramacere, I; Islami, F; Fedirko, V; Scotti, L; Jenab, M; Turati, F; Pasquali, E; Pelucchi, C; Galeone, C; Bellocco, R; Negri, E; Corrao, G; Boffetta, P; La Vecchia, C

    2015-01-01

    Background: Alcohol is a risk factor for cancer of the oral cavity, pharynx, oesophagus, colorectum, liver, larynx and female breast, whereas its impact on other cancers remains controversial. Methods: We investigated the effect of alcohol on 23 cancer types through a meta-analytic approach. We used dose–response meta-regression models and investigated potential sources of heterogeneity. Results: A total of 572 studies, including 486 538 cancer cases, were identified. Relative risks (RRs) for heavy drinkers compared with nondrinkers and occasional drinkers were 5.13 for oral and pharyngeal cancer, 4.95 for oesophageal squamous cell carcinoma, 1.44 for colorectal, 2.65 for laryngeal and 1.61 for breast cancer; for those neoplasms there was a clear dose–risk relationship. Heavy drinkers also had a significantly higher risk of cancer of the stomach (RR 1.21), liver (2.07), gallbladder (2.64), pancreas (1.19) and lung (1.15). There was indication of a positive association between alcohol consumption and risk of melanoma and prostate cancer. Alcohol consumption and risk of Hodgkin's and Non-Hodgkin's lymphomas were inversely associated. Conclusions: Alcohol increases risk of cancer of oral cavity and pharynx, oesophagus, colorectum, liver, larynx and female breast. There is accumulating evidence that alcohol drinking is associated with some other cancers such as pancreas and prostate cancer and melanoma. PMID:25422909

  9. Effects of salt stress on wild type and vte4 mutant Arabidopsis thaliana: Model plant to engineer tolerance towards salinity

    Directory of Open Access Journals (Sweden)

    Khalatbari Amir Ali

    2013-01-01

    Full Text Available One of the major environmental constraints impairing plant distribution and yield is believed to be salt stress. Additionally, engineered abiotic stress resistance or/and tolerance is considered as an indispensable target in order to enhance plant productivity. In this study, the effects of salinity on physiological and morphological of wild type (Columbia-0 and vte4 mutant Arabidopsis thaliana were investigated under different NaCl concentrations. These salt treatments, including control condition, 50mM and 100mM NaCl were imposed on the plants. Each salt treatment was replicated three times in a complete randomized design with factorial arrangement. Wild type and mutant A.thaliana plants were subjected to the abiotic stress (salinity for up to 11 days to evaluate the parameters of growth, development and water relations. As a result, the performance of wild type plants was stronger than vte4 mutant under different salt treatments. Under control condition, rosette dry weight, maximum quantum efficiency (PSII and specific leaf area obtained the highest values of 13.85 mg, considered, wild type A.thaliana recorded higher value of 0.82 gW/gFW for relative water content (RWC under 50mM NaCl whereas mutant plants gained the value of 0.78 gW/gFW under the same condition. However, root mass fraction indicated an increase for both wild type and vte4 mutant plants after 11 days of salt stress onset. The reduction of water potential was observed for wild type and mutant A.thaliana where it scored -1.3 MPa and -1.4, respectively. As a conclusion, these findings implied that under different salt treatments morphological and physiological responses of wild type and vte4 mutant were affected in which wild type plants showed more tolerance. Lack of γ-tocopherol methyltransferase (γ -TMT gene in vte4 seemed to impair defence mechanism of this mutant against salinity.

  10. Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Samuel Cordey

    Full Text Available Enterovirus 71 (EV71 is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R, a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V; and a silent mutation in protein 3D (L175. Infectious clones were constructed using both BrCr (lineage A and the clinical strain (lineage C backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP1₉₇ Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.

  11. Vascular nanomedicine: Site specific delivery of elastin stabilizing therapeutics to damaged arteries

    Science.gov (United States)

    Sinha, Aditi

    improved resistance to elastolytic digestion. We further show that the same polyphenols interact with monomeric tropoelastin released by the vascular cells and dramatically increasing their self-assembly in-vitro. In addition, we demonstrate the elastogenic ability of these polyphenols in aiding the crosslinking of tropoelastin released by aneurysmal cells converting it into mature elastin. Finally, we developed a nanoparticle system functionalized with elastin antibody on the surface that, upon systemic delivery, can recognize and bind to sites of damaged elastin in the aorta. We are able to show that this nanoparticle system works in representative animal models for MAC and AAA. These nanoparticles demonstrated spatial and functional specificity for degraded elastin. In conclusion, our work is focused on understanding the role of elastin degradation in vascular calcification and aortic aneurysms. We tested approaches to halt elastin degradation and to regenerate elastin in arteries so that homeostasis can be achieved.

  12. Virus Capsids as Targeted Nanoscale Delivery Vessels of Photoactive Compounds for Site-Specific Photodynamic Therapy

    Science.gov (United States)

    Cohen, Brian A.

    The research presented in this work details the use of a viral capsid as an addressable delivery vessel of photoactive compounds for use in photodynamic therapy. Photodynamic therapy is a treatment that involves the interaction of light with a photosensitizing molecule to create singlet oxygen, a reactive oxygen species. Overproduction of singlet oxygen in cells can cause oxidative damage leading to cytotoxicity and eventually cell death. Challenges with the current generation of FDA-approved photosensitizers for photodynamic therapy primarily stem from their lack of tissue specificity. This work describes the packaging of photoactive cationic porphyrins inside the MS2 bacteriophage capsid, followed by external modification of the capsid with cancer cell-targeting G-quadruplex DNA aptamers to generate a tumor-specific photosensitizing agent. First, a cationic porphyrin is loaded into the capsids via nucleotide-driven packaging, a process that involves charge interaction between the porphyrin and the RNA inside the capsid. Results show that over 250 porphyrin molecules associate with the RNA within each MS2 capsid. Removal of RNA from the capsid severely inhibits the packaging of the cationic porphyrins. Porphyrin-virus constructs were then shown to photogenerate singlet oxygen, and cytotoxicity in non-targeted photodynamic treatment experiments. Next, each porphyrin-loaded capsid is externally modified with approximately 60 targeting DNA aptamers by employing a heterobifunctional crosslinking agent. The targeting aptamer is known to bind the protein nucleolin, a ubiquitous protein that is overexpressed on the cell surface by many cancer cell types. MCF-7 human breast carcinoma cells and MCF-10A human mammary epithelial cells were selected as an in vitro model for breast cancer and normal tissue, respectively. Fluorescently tagged virus-aptamer constructs are shown to selectively target MCF-7 cells versus MCF-10A cells. Finally, results are shown in which porphyrin

  13. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    Science.gov (United States)

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

  14. Site-specific conformational determination in thermal unfolding studies of helical peptides using vibrational circular dichroism with isotopic substitution

    Science.gov (United States)

    Silva, R. A. G. D.; Kubelka, Jan; Bour, Petr; Decatur, Sean M.; Keiderling, Timothy A.

    2000-01-01

    Understanding the detailed mechanism of protein folding requires dynamic, site-specific stereochemical information. The short time response of vibrational spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unfolding. For a series of alanine-rich peptides that form α-helices in aqueous solution, we used isotopic labeling and VCD to demonstrate that the α-helix noncooperatively unwinds from the ends with increasing temperature. For these blocked peptides, the C-terminal is frayed at 5°C. Ab initio level theoretical simulations of the IR and VCD band shapes are used to analyze the spectra and to confirm the conformation of the labeled components. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled segments are detectable and stereochemically defined in moderately large peptides in this report of site-specific peptide VCD conformational analysis. PMID:10880566

  15. Site-specific branching ratios for H-atom production from primary haloalkanes photolyzed at 193, 222, and 248 nm

    Science.gov (United States)

    Brum, Jeffrey L.; Deshmukh, Subhash; Wang, Zhongrui; Koplitz, Brent

    1993-01-01

    Selectively deuterated compounds are used to investigate the ``site-specific'' nature of H-atom production resulting from the photolysis of primary haloalkanes. The molecules investigated are 1-iodopropane, 1-bromopropane, iodoethane, bromoethane, and chloroethane, with photolysis being initiated at 193, 222, and 248 nm. Hydrogen and deuterium isotopes are systematically used to label chemically distinct carbon atoms within a given molecule. H- and D-atom Doppler profiles are generated via two-photon (121.6+364.7 nm) ionization resonant with Lyman-α, and the relative H/D ratios are used to quantify the probability for hydrogen production from each carbon site. In general, photolysis of an intermediate, presumably the alkyl radical, is implicated as being a key step in the overall process. When using 248 nm radiation, the photolysis process is dominated by C-H (or C-D) bond cleavage at the β carbon position regardless of the system investigated. In contrast, results using 193 nm excitation display no obvious preference for bond cleavage at a particular carbon site, again regardless of the system. The data produced by 222 nm photolysis show a qualitative tendency for C-H (or C-D) bond cleavage at the β carbon, but the quantitative results are not as self-consistent as either the 248 nm or the 193 nm data. Also, the results of ab initio calculations on the ground and excited states of the ethyl radical are used to help interpret the observed site specificity.

  16. Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.

    Science.gov (United States)

    Park, Gun Wook; Kim, Jin Young; Hwang, Heeyoun; Lee, Ju Yeon; Ahn, Young Hee; Lee, Hyun Kyoung; Ji, Eun Sun; Kim, Kwang Hoe; Jeong, Hoi Keun; Yun, Ki Na; Kim, Yong-Sam; Ko, Jeong-Heon; An, Hyun Joo; Kim, Jae Han; Paik, Young-Ki; Yoo, Jong Shin

    2016-02-17

    Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project.

  17. Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells

    Science.gov (United States)

    Yu, Yuan; Tong, Qi; Li, Zhongxia; Tian, Jinhai; Wang, Yizhi; Su, Feng; Wang, Yongsheng; Liu, Jun; Zhang, Yong

    2014-02-01

    PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.

  18. Targeting of Magnetic Nanoparticle-coated Microbubbles to the Vascular Wall Empowers Site-specific Lentiviral Gene Delivery in vivo

    Science.gov (United States)

    Heun, Yvonn; Hildebrand, Staffan; Heidsieck, Alexandra; Gleich, Bernhard; Anton, Martina; Pircher, Joachim; Ribeiro, Andrea; Mykhaylyk, Olga; Eberbeck, Dietmar; Wenzel, Daniela; Pfeifer, Alexander; Woernle, Markus; Krötz, Florian; Pohl, Ulrich; Mannell, Hanna

    2017-01-01

    In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy. PMID:28042335

  19. Concise site-specific synthesis of DTPA-peptide conjugates: application to imaging probes for the chemokine receptor CXCR4.

    Science.gov (United States)

    Masuda, Ryo; Oishi, Shinya; Ohno, Hiroaki; Kimura, Hiroyuki; Saji, Hideo; Fujii, Nobutaka

    2011-05-15

    Diethylenetriaminepentaacetic acid (DTPA) is a useful chelating agent for radionuclides such as (68)Ga, (99m)Tc and (111)In, which are applicable to nuclear medicine imaging. In this study, we established a facile synthetic protocol for the production of mono-DTPA-conjugated peptide probes. A novel monoreactive DTPA precursor reagent was synthesized in two steps using the chemistry of the o-nitrobenzenesulfonyl (Ns) protecting group, and under mild conditions this DTPA precursor was incorporated onto an N(ε)-bromoacetylated Lys of a protected peptide resin. The site-specific DTPA conjugation was facilitated by using a highly acid-labile 4-methyltrityl (Mtt) protecting group for the target site of the bioactive peptide during the solid-phase synthesis. A combination of both techniques yielded peptides with disulfide bonds, such as octreotide and polyphemusin II-derived CXCR4 antagonists. DTPA-peptide conjugates were purified in a single step following cleavage from the resin and disulfide bond formation. This site-specific on-resin construction strategy was used for the design and synthesis of a novel In-DTPA-labeled CXCR4 antagonist, which exhibited highly potent inhibitory activity against SDF-1-CXCR4 binding. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates.

    Science.gov (United States)

    Siegmund, Vanessa; Piater, Birgit; Zakeri, Bijan; Eichhorn, Thomas; Fischer, Frank; Deutsch, Carl; Becker, Stefan; Toleikis, Lars; Hock, Björn; Betz, Ulrich A K; Kolmar, Harald

    2016-12-16

    Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.