Is semantic priming (ir)rational? Insights from the speeded word fragment completion task.

PubMed

Heyman, Tom; Hutchison, Keith A; Storms, Gert

2016-10-01

Semantic priming, the phenomenon that a target is recognized faster if it is preceded by a semantically related prime, is a well-established effect. However, the mechanisms producing semantic priming are subject of debate. Several theories assume that the underlying processes are controllable and tuned to prime utility. In contrast, purely automatic processes, like automatic spreading activation, should be independent of the prime's usefulness. The present study sought to disentangle both accounts by creating a situation where prime processing is actually detrimental. Specifically, participants were asked to quickly complete word fragments with either the letter a or e (e.g., sh_ve to be completed as shave). Critical fragments were preceded by a prime that was either related (e.g., push) or unrelated (write) to a prohibited completion of the target (e.g., shove). In 2 experiments, we found a significant inhibitory priming effect, which is inconsistent with purely "rational" explanations of semantic priming. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Breccia Formation at a Complex Impact Crater: Slate Islands, Lake Superior, Ontario, Canada

NASA Technical Reports Server (NTRS)

Dressler, B. O.; Sharpton, V. L.

1997-01-01

The Slate Islands impact structure is the eroded remnant of a approximately 30-32 km-diameter complex impact structure located in northern Lake Superior, Ontario, Canada. Target rocks are Archean supracrustal and igneous rocks and Proterozoic metavolcanics, metasediments, and diabase. A wide variety of breccias occurs on the islands, many of which contain fragments exhibiting shock metamorphic features. Aphanitic, narrow and inclusion-poor pseudotachylite veins, commonly with more or less parallel boundaries and apophyses branching off them, represent the earliest breccias formed during the compression stage of the impact process. Coarse-grained, polymictic elastic matrix breccias form small to very large, inclusion-rich dikes and irregularly shaped bodies that may contain altered glass fragments. These breccias have sharp contacts with their host rocks and include a wide range of fragment types some of which were transported over minimum distances of approximately 2 km away from the center of the structure. They cut across pseudotachylite veins and contain inclusions of them. Field and petrographic evidence indicate that these polymictic breccias formed predominantly during the excavation and central uplift stages of the impact process. Monomictic breccias, characterized by angular fragments and transitional contacts with their host rocks, occur in parautochthonous target rocks, mainly on the outlying islands of the Slate Islands archipelago. A few contain fragmented and disrupted, coarse-grained, polymictic clastic matrix breccia dikes. This is an indication that at least some of these monomictic breccias formed late in the impact process and that they are probably related to a late crater modification stage. A small number of relatively large occurrences of glass-poor, suevitic breccias occur at the flanks of the central uplift and along the inner flank of the outer ring of the Slate Islands complex crater. A coarse, glass-free, allogenic breccia, containing shatter-coned fragments derived from Proterozoic target rocks (upper target strata), observed at two locations may be analogous to the 'Bunt Breccia' of the Ries crater in Germany. At one of these locations this breccia lies close to a crater suevite deposit. At the other, it overlies parautochthonous, monomictic breccia. The State Islands impact breccias are superbly exposed, much better than breccias in most other terrestrial impact structures. Observations, including those indicative of multiple and and sequential processes, provide insight on how impact breccias form and how they relate to the various phases of the impact process. Eventually they will lead to an improved understanding of planetary impact processes.

Effects of target shape and impact speed on the outcome of catastrophic disruptions

NASA Astrophysics Data System (ADS)

Campo~Bagatin, A.; Durda, D.; Alemañ, R.; Flynn, G.; Strait, M.; Clayton, A.; Patmore, E.

2014-07-01

Because of the propensity of previous laboratory investigations to focus on idealized spherical targets, there is a bit of ambiguity in decoupling the relative importance/influence of low speed or spherical shape in producing the 'onion shell' fragment shape outcomes found in impacts into spherical targets [1,2]. If due primarily to impact speed/energy density as suggested by [3], this could play an important role in main-belt impacts due to the presence of non-spherical targets and non-negligible probability of low-speed (i.e., below about 3-4 km/s, subsonic in rock) impacts [4]. Also, [5] and [6] suggested that the shape of targets may affect the outcome of shattering processes, both in terms of fragment shape and mass distribution. To examine explicitly the effects of target shape in impact outcomes, we chose to conduct impact experiments on both spherical and naturally-occurring irregularly-shaped basalt targets. We impacted a total of six targets (two spheres and four irregular targets). We focused on shots with impact speeds in the ˜4 to 6 km/s range by 3/16th-inch diameter Al-sphere projectiles fired at the NASA AVGR. Following each shot, the debris were recovered (>95 %) and large fragments (>0.20 g) were individually weighed, allowing us to carefully measure the mass-frequency distribution from each impact experiment. The 36 largest fragments of each shot were photographed and their largest axes accurately measured by the program ''ImageJ''. Their shortest axes were measured by means of a digital caliber. High-speed video of each impact was obtained to aid interpretation of the fragmentation mode of the targets. Images clearly show that shell-like fragments can be produced in shattering events not in the target's surface. Instead, those fragments may form around the core, well inside the target structure, independently on the target shape itself. This is a feature not reported to date. In order to understand what the bulk macro-porosity of a non-coherent set of fragments is, we gathered randomly together the fragments with weighed mass mimicking the post-shattering gravitational re-accumulation of fragments into an asteroid rubble-pile. For each set, we wrapped the fragments in a thin plastic film and measured the bulk volume by hanging and plunging the assemblage into distilled water. The volume is calculated straightforward from the density of water at the given temperature. Cumulative mass distributions are derived and exponents 0.75<β <1.2 are found for the relationship N(>m)=A m^{-β} (m is the fragment mass, A is the corresponding constant) in the stationary part of the distribution. The exponent of each distribution and the mass of each largest fragment are found to be related to the corresponding specific energy of each impact as expected [3]. The mass distributions seem to show slightly larger values of β in the case of spherical targets when comparing two sets of close specific energy impacts. However, this feature needs further sets of impact experiments to be properly investigated. As for the shapes of fragments, b/a and c/a ratios were calculated along with the shape metrics Ψ=[ c^2/(ab)]^{1/3}, F=(a-b)/(a-c) for deviation from the spherical shape and relative flatness, respectively [7,8]. The average relationship between a, b, and c axes is 1:0.7:0.4, slightly different (flatter) than reported by former investigations (1:0.7:0.5) carried on in the 70s and 80s [7]. This result is quite stable and no differences are found in average shapes among spherical and irregular targets nor for different specific energy up to a factor of ˜3. This does not mean that fragments look like triaxial ellipsoids, instead they are quite irregular but their average relative sizes are distributed very nicely as described. Finally, the study of the macro-porosities of randomly aggregated fragments shows values in the 45 to 50 % range. This result may be useful in the interpretation of small asteroids' bulk densities and in the calibration of numerical modelling of internal structures.

Kinematics of current region fragmentation in semi-inclusive deeply inelastic scattering

DOE PAGES

Boglione, M.; Collins, J.; Gamberg, L.; ...

2017-01-16

Different kinematical regions of semi-inclusive deeply inelastic scattering (SIDIS) processes correspond to different underlying partonic pictures, and it is important to understand the transition between them. We find criteria in semi-inclusive deeply inelastic scattering (SIDIS) for identifying the current fragmentation region — the kinematical region where a factorization picture with fragmentation functions is appropriate, especially for studies of transverse-momentum-dependent (TMD) functions. This region is distinguished from the central (soft) and target fragmentation regions. The basis of our argument is in the errors in approximations used in deriving factorization. As compared with previous work, we show that it is essential tomore » take account of the transverse momentum of the detected hadron, and we find a much more restricted range for genuine current fragmentation. As a result, we show that it is important to develop an extended factorization formulation to treat hadronization in the central region, as well as the current and target fragmentation regions, and to obtain a unified formalism spanning all rapidities for the detected hadron.« less

FIRST experiment: Fragmentation of Ions Relevant for Space and Therapy

NASA Astrophysics Data System (ADS)

Agodi, C.; Abou-Haidar, Z.; Alvarez, M. A. G.; Aumann, T.; Balestra, F.; Battistoni, G.; Bocci, A.; Bohlen, T. T.; Bondì, M.; Boudard, A.; Brunetti, A.; Carpinelli, M.; Cappuzzello, F.; Cavallaro, M.; Carbone, D.; Cirrone, G. A. P.; Cortes-Giraldo, M. A.; Cuttone, G.; De Napoli, M.; Durante, M.; Fernandez-Garcia, J. P.; Finck, C.; Foti, A.; Gallardo, M. I.; Golosio, B.; Iarocci, E.; Iazzi, F.; Ickert, G.; Introzzi, R.; Juliani, D.; Krimmer, J.; Kurz, N.; Labalme, M.; Lavagno, A.; Leifels, Y.; Le Fevre, A.; Leray, S.; Marchetto, F.; Monaco, V.; Morone, M. C.; Nicolosi, D.; Oliva, P.; Paoloni, A.; Patera, V.; Piersanti, L.; Pleskac, R.; Quesada, J. M.; Randazzo, N.; Romano, F.; Rossi, D.; Rosso, V.; Rousseau, M.; Sacchi, R.; Sala, P.; Sarti, A.; Scheidenberger, C.; Schuy, C.; Sciubba, A.; Sfienti, C.; Simon, H.; Sipala, V.; Spiriti, E.; Stuttge, L.; Tropea, S.; Younis, H.

2013-03-01

Nuclear fragmentation processes are relevant in different fields of basic research and applied physics and are of particular interest for tumor therapy and for space radiation protection applications. The FIRST (Fragmentation of Ions Relevant for Space and Therapy) experiment at SIS accelerator of GSI laboratory in Darmstadt, has been designed for the measurement of different ions fragmentation cross sections at different energies between 100 and 1000 MeV/nucleon. The experiment is performed by an international collaboration made of institutions from Germany, France, Italy and Spain. The experimental apparatus is partly based on an already existing setup made of the ALADIN magnet, the MUSIC IV TPC, the LAND2 neutron detector and the TOFWALL scintillator TOF system, integrated with newly designed detectors in the interaction Region (IR) around the carbon removable target: a scintillator Start Counter, a Beam Monitor drift chamber, a silicon Vertex Detector and a Proton Tagger for detection of light fragments emitted at large angles (KENTROS). The scientific program of the FIRST experiment started on summer 2011 with the study of the 400 MeV/nucleon 12C beam fragmentation on thin (8mm) carbon target.

Evaluating differences in forest fragmentation and restoration between western natural forests and southeastern plantation forests in the United States.

PubMed

Ren, Xinyu; Lv, Yingying; Li, Mingshi

2017-03-01

Changes in forest ecosystem structure and functions are considered some of the research issues in landscape ecology. In this study, advancing Forman's theory, we considered five spatially explicit processes associated with fragmentation, including perforation, dissection, subdivision, shrinkage, and attrition, and two processes associated with restoration, i.e., increment and expansion processes. Following this theory, a forest fragmentation and restoration process model that can detect the spatially explicit processes and ecological consequences of forest landscape change was developed and tested in the current analysis. Using the National Land Cover Databases (2001, 2006 and 2011), the forest fragmentation and restoration process model was applied to US western natural forests and southeastern plantation forests to quantify and classify forest patch losses into one of the four fragmentation processes (the dissection process was merged into the subdivision process) and to classify the newly gained forest patches based on the two restoration processes. At the same time, the spatio-temporal differences in fragmentation and restoration patterns and trends between natural forests and plantations were further compared. Then, through overlaying the forest fragmentation/restoration processes maps with targeting year land cover data and land ownership vectors, the results from forest fragmentation and the contributors to forest restoration in federal and nonfederal lands were identified. Results showed that, in natural forests, the forest change patches concentrated around the urban/forest, cultivated/forest, and shrubland/forest interfaces, while the patterns of plantation change patches were scattered sparsely and irregularly. The shrinkage process was the most common type in forest fragmentation, and the average size was the smallest. Expansion, the most common restoration process, was observed in both natural forests and plantations and often occurred around the previous expansion or covered the previous subdivision or shrinkage processes. The overall temporal fragmentation pattern of natural forests had a "perforation-subdivision/shrinkage-attrition" pathway, which corresponded to Forman's landscape fragmentation rule, while the plantation forests did not follow the rule strictly. The main land cover types resulted from forest fragmentation in natural forests and plantation forests were shrubland and herbaceous, mainly through subdivision and shrinkages process. The processes and effects of restoration of plantation forests were more diverse and efficient, compared to the natural forest, which were simpler with a lower regrowth rate. The fragmentation mostly occurred in nonfederal lands. In natural forests, forest fragmentation pattern differed in different land tenures, yet plantations remained the same in federal and nonfederal lands. Copyright © 2016 Elsevier Ltd. All rights reserved.

EUV emission spectra in collisions of highly charged tantalum ions with nitrogen and oxygen molecules

NASA Astrophysics Data System (ADS)

Tanuma, Hajime; Numadate, Naoki; Uchikura, Yoshiyuki; Shimada, Kento; Akutsu, Takuto; Long, Elaine; O'Sullivan, Gerry

2017-10-01

We have performed ion beam collision experiments using multiply charged tantalum ions and observed EUV (extreme ultra-violet) emission spectra in collisions of ions with molecular targets, N2 and O2. Broad UTAs (un-resolved transition arrays) from multiply charged Ta ions were observed, and the mean wavelengths of the UTAs shifted and became shorter at higher charge statea of Ta ions. These UTAs may be attributed to the 4f-5d and 4f-5g transitions. Not only the UTA emission from incident ions, but also the sharp emission lines from multiply charged fragment atomic ions were observed. Production of temporary highly charged molecular ions, their kinetic energy and fragmentation processes have been investigated with coincident detection technique. However, the observation of emission from the fragments might be for the first time. The formation mechanisms of the multiply charged fragment atomic ions from target molecules are discussed.

BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR

PubMed Central

Aguirre, Clémentine; ten Brink, Tim; Walker, Olivier; Guillière, Florence; Davesne, Dany; Krimm, Isabelle

2013-01-01

Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices 2, 3 and the very beginning of 5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach. PMID:23717610

Target immobilization as a strategy for NMR-based fragment screening: comparison of TINS, STD, and SPR for fragment hit identification.

PubMed

Kobayashi, Masakazu; Retra, Kim; Figaroa, Francis; Hollander, Johan G; Ab, Eiso; Heetebrij, Robert J; Irth, Hubertus; Siegal, Gregg

2010-09-01

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM K(D) is limited. Similarly, they find that SPR detection is most suited to ligands that bind with K(D) better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.

Fragmentation mechanisms for methane induced by 55 eV, 75 eV, and 100 eV electron impact.

PubMed

Wei, B; Zhang, Y; Wang, X; Lu, D; Lu, G C; Zhang, B H; Tang, Y J; Hutton, R; Zou, Y

2014-03-28

The fragmentation of CH4 (2+) dications following 55 eV, 75 eV, and 100 eV electron impact double ionization of methane was studied using a cold target recoil-ion momentum spectroscopy. From the measured momentum of each recoil ion, the momentum of the neutral particles has been deduced and the kinetic energy release distribution for the different fragmentation channels has been obtained. The doubly charged molecular ions break up into three or more fragments in one or two-step processes, resulting in different signatures in the data. We observed the fragmentation of CH4 (2+) dications through different mechanisms according to the momentum of the neutral particles. For example, our result shows that there are three reaction channels to form CH2 (+), H(+), and H, one synchronous concerted reaction channel and two two-step reaction channels. For even more complicated fragmentation processes of CH4 (2+) dications, the fragmentation mechanism can still be identified in the present measurements. The slopes of the peak in the ion-ion coincidence spectra were also estimated here, as they are also related to the fragmentation mechanism.

Generation of Polar Semi-Saturated Bicyclic Pyrazoles for Fragment-Based Drug Discovery Campaigns.

PubMed

Luise, Nicola; Wyatt, Paul

2018-05-07

Synthesising polar semi-saturated bicyclic heterocycles can lead to better starting points for fragment-based drug discovery (FBDD) programs. This communication highlights the application of diverse chemistry to construct bicyclic systems from a common intermediate, where pyrazole, a privileged heteroaromatic able to bind effectively to biological targets, is fused to diverse saturated counterparts. The generated fragments can be further developed either after confirmation of their binding pose or early in the process, as their synthetic intermediates. Essential quality control (QC) for selection of small molecules to add to a fragment library is discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Selectable fragmentation warhead

DOEpatents

Bryan, Courtney S.; Paisley, Dennis L.; Montoya, Nelson I.; Stahl, David B.

1993-01-01

A selectable fragmentation warhead capable of producing a predetermined number of fragments from a metal plate, and accelerating the fragments toward a target. A first explosive located adjacent to the plate is detonated at selected number of points by laser-driven slapper detonators. In one embodiment, a smoother-disk and a second explosive, located adjacent to the first explosive, serve to increase acceleration of the fragments toward a target. The ability to produce a selected number of fragments allows for effective destruction of a chosen target.

Fission-Fusion: A new reaction mechanism for nuclear astrophysics based on laser-ion acceleration

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

Thirolf, P. G.; Gross, M.; Allinger, K.

We propose to produce neutron-rich nuclei in the range of the astrophysical r-process around the waiting point N = 126 by fissioning a dense laser-accelerated thorium ion bunch in a thorium target (covered by a CH{sub 2} layer), where the light fission fragments of the beam fuse with the light fission fragments of the target. Via the 'hole-boring' mode of laser Radiation Pressure Acceleration using a high-intensity, short pulse laser, very efficiently bunches of {sup 232}Th with solid-state density can be generated from a Th target and a deuterated CD{sub 2} foil, both forming the production target assembly. Laser-accelerated Thmore » ions with about 7 MeV/u will pass through a thin CH{sub 2} layer placed in front of a thicker second Th foil (both forming the reaction target) closely behind the production target and disintegrate into light and heavy fission fragments. In addition, light ions (d,C) from the CD{sub 2} layer of the production target will be accelerated as well, inducing the fission process of {sup 232}Th also in the second Th layer. The laser-accelerated ion bunches with solid-state density, which are about 10{sup 14} times more dense than classically accelerated ion bunches, allow for a high probability that generated fission products can fuse again. The high ion beam density may lead to a strong collective modification of the stopping power, leading to significant range and thus yield enhancement. Using a high-intensity laser as envisaged for the ELI-Nuclear Physics project in Bucharest (ELI-NP), order-of-magnitude estimates promise a fusion yield of about 10{sup 3} ions per laser pulse in the mass range of A = 180-190, thus enabling to approach the r-process waiting point at N = 126.« less

Effect of isospin diffusion on the production of neutron-rich nuclei in multinucleon transfer reactions

NASA Astrophysics Data System (ADS)

Niu, Fei; Chen, Peng-Hui; Guo, Ya-Fei; Ma, Chun-Wang; Feng, Zhao-Qing

2018-03-01

The isospin dissipation dynamics in multinucleon transfer reactions has been investigated within the dinuclear system model. Production cross sections of neutron-rich isotopes around projectile-like and target-like fragments are estimated in collisions of Ni,6458+208Pb and 78.86,91Kr +198Pt near Coulomb barrier energies. The isospin diffusion in the nucleon transfer process is coupled to the dissipation of relative motion energy and angular momentum of colliding system. The available data of projectile-like fragments via multinucleon transfer reactions are nicely reproduced. It is found that the light projectile-like fragments are produced in the neutron-rich region because of the isospin equilibrium in two colliding nuclei. However, the heavy target-like fragments tend to be formed on the neutron-poor side above the β -stability line. The neutron-rich projectiles move the maximal yields of heavy nuclei to the neutron-rich domain and are available for producing the heavy exotic isotopes, in particular around the neutron shell closure of N =126 .

Biosensor-based small molecule fragment screening with biolayer interferometry

NASA Astrophysics Data System (ADS)

Wartchow, Charles A.; Podlaski, Frank; Li, Shirley; Rowan, Karen; Zhang, Xiaolei; Mark, David; Huang, Kuo-Sen

2011-07-01

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions. The BLI format is based on a disposable sensor that is immersed in 96-well or 384-well plates. BLI has been validated for small molecule detection and fragment screening with model systems and well-characterized targets where affinity constants and binding profiles are generally similar to those obtained with surface plasmon resonsance (SPR). Screens with challenging targets involved in protein-protein interactions including BCL-2, JNK1, and eIF4E were performed with a fragment library of 6,500 compounds, and hit rates were compared for these targets. For eIF4E, a protein containing a PPI site and a nucleotide binding site, results from a BLI fragment screen were compared to results obtained in biochemical HTS screens. Overlapping hits were observed for the PPI site, and hits unique to the BLI screen were identified. Hit assessments with SPR and BLI are described.

Fragment-based lead generation: identification of seed fragments by a highly efficient fragment screening technology

NASA Astrophysics Data System (ADS)

Neumann, Lars; Ritscher, Allegra; Müller, Gerhard; Hafenbradl, Doris

2009-08-01

For the detection of the precise and unambiguous binding of fragments to a specific binding site on the target protein, we have developed a novel reporter displacement binding assay technology. The application of this technology for the fragment screening as well as the fragment evolution process with a specific modelling based design strategy is demonstrated for inhibitors of the protein kinase p38alpha. In a fragment screening approach seed fragments were identified which were then used to build compounds from the deep-pocket towards the hinge binding area of the protein kinase p38alpha based on a modelling approach. BIRB796 was used as a blueprint for the alignment of the fragments. The fragment evolution of these deep-pocket binding fragments towards the fully optimized inhibitor BIRB796 included the modulation of the residence time as well as the affinity. The goal of our study was to evaluate the robustness and efficiency of our novel fragment screening technology at high fragment concentrations, compare the screening data with biochemical activity data and to demonstrate the evolution of the hit fragments with fast kinetics, into slow kinetic inhibitors in an in silico approach.

First Observation of Three-Neutron Sequential Emission from 25O

NASA Astrophysics Data System (ADS)

Sword, C.; Brett, J.; Deyoung, P. A.; Frank, N.; Karrick, H.; Kuchera, A. N.; MoNA Collaboration

2017-09-01

An active area of nuclear physics research is to evaluate models of the nuclear force by studying the structure of neutron-rich isotopes. In this experiment, a 101.3 MeV/u 27Ne beam from the National Superconducting Cyclotron Laboratory collided with a liquid deuterium target. The collision resulted in two-proton removal from the 27Ne beam which created excited 25O that decayed into three neutrons and an 22O fragment. The neutrons were detected by arrays of scintillating plastic bars, while a 4-Tesla dipole magnet placed directly after the target redirected charged fragments to a series of charged-particle detectors. From measured velocities of the neutrons and 22O fragments, the decay energy of 25O was calculated on an event-by-event basis with invariant mass spectroscopy. Using GEANT4, we simulated the decay of all nuclei that could have been created by the beam collision. By successfully fitting simulated decay processes to experimental data, we determined the decay processes present in the experiment. This work is supported by the National Science Foundation under Grants No. PHY-1306074 and No. PHY-1613188.

Quantum control of molecular fragmentation in strong laser fields

NASA Astrophysics Data System (ADS)

Zohrabi, Mohammad

Present advances in laser technology allow the production of ultrashort (<˜5 fs, approaching single cycle at 800 nm), intense tabletop laser pulses. At these high intensities laser-matter interactions cannot be described with perturbation theory since multiphoton processes are involved. This is in contrast to photodissociation by the absorption of a single photon, which is well described by perturbation theory. For example, at high intensities (<˜5x1013 W/cm2) the fragmentation of molecular hydrogen ions has been observed via the absorption of three or more photons. In another example, an intriguing dissociation mechanism has been observed where molecular hydrogen ions seem to fragment by apparently absorbing no photons. This is actually a two photon process, photoabsorption followed by stimulated emission, resulting in low energy fragments. We are interested in exploring these kinds of multiphoton processes. Our research group has studied the dynamics and control of fragmentation induced by strong laser fields in a variety of molecular targets. The main goal is to provide a basic understanding of fragmentation mechanisms and possible control schemes of benchmark systems such as H2+. This knowledge is further extended to more complex systems like the benchmark H3+ polyatomic and other molecules. In this dissertation, we report research based on two types of experiments. In the first part, we describe laser-induced fragmentation of molecular ion-beam targets. In the latter part, we discuss the formation of highly-excited neutral fragments from hydrogen molecules using ultrashort laser pulses. In carrying out these experiments, we have also extended experimental techniques beyond their previous capabilities. We have performed a few experiments to advance our understanding of laser-induced fragmentation of molecular-ion beams. For instance, we explored vibrationally resolved spectra of O2+ dissociation using various wavelengths. We observed a vibrational suppression effect in the dissociation spectra due to the small magnitude of the dipole transition moment, which depends on the photon energy --- a phenomenon known as Cooper minima. By changing the laser wavelength, the Cooper minima shift, a fact that was used to identify the dissociation pathways. In another project, we studied the carrier-envelope phase (CEP) dependences of highly-excited fragments from hydrogen molecules. General CEP theory predicts a CEP dependence in the total dissociation yield due to the interference of dissociation pathways differing by an even net number of photons, and our measurements are consistent with this prediction. Moreover, we were able to extract the difference in the net number of photons involved in the interfering pathways by using a Fourier analysis. In terms of our experimental method, we have implemented a pump-probe style technique on a thin molecular ion-beam target and explored the feasibility of such experiments. The results presented in this work should lead to a better understanding of the dynamics and control in molecular fragmentation induced by intense laser fields.

Fragmentation mechanisms for methane induced by 55 eV, 75 eV, and 100 eV electron impact

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

Wei, B.; Zhang, Y.; Wang, X., E-mail: xinchengwang@fudan.edu.cn

2014-03-28

The fragmentation of CH{sub 4}{sup 2+} dications following 55 eV, 75 eV, and 100 eV electron impact double ionization of methane was studied using a cold target recoil-ion momentum spectroscopy. From the measured momentum of each recoil ion, the momentum of the neutral particles has been deduced and the kinetic energy release distribution for the different fragmentation channels has been obtained. The doubly charged molecular ions break up into three or more fragments in one or two-step processes, resulting in different signatures in the data. We observed the fragmentation of CH{sub 4}{sup 2+} dications through different mechanisms according to themore » momentum of the neutral particles. For example, our result shows that there are three reaction channels to form CH{sub 2}{sup +}, H{sup +}, and H, one synchronous concerted reaction channel and two two-step reaction channels. For even more complicated fragmentation processes of CH{sub 4}{sup 2+} dications, the fragmentation mechanism can still be identified in the present measurements. The slopes of the peak in the ion-ion coincidence spectra were also estimated here, as they are also related to the fragmentation mechanism.« less

Comparing forest fragmentation and its drivers in China and the USA with Globcover v2.2

USGS Publications Warehouse

Chen, Mingshi; Mao, Lijun; Zhou, Chunguo; Vogelmann, James E.; Zhu, Zhiliang

2010-01-01

Forest loss and fragmentation are of major concern to the international community, in large part because they impact so many important environmental processes. The main objective of this study was to assess the differences in forest fragmentation patterns and drivers between China and the conterminous United States (USA). Using the latest 300-m resolution global land cover product, Globcover v2.2, a comparative analysis of forest fragmentation patterns and drivers was made. The fragmentation patterns were characterized by using a forest fragmentation model built on the sliding window analysis technique in association with landscape indices. Results showed that China’s forests were substantially more fragmented than those of the USA. This was evidenced by a large difference in the amount of interior forest area share, with China having 48% interior forest versus the 66% for the USA. China’s forest fragmentation was primarily attributed to anthropogenic disturbances, driven particularly by agricultural expansion from an increasing and large population, as well as poor forest management practices. In contrast, USA forests were principally fragmented by natural land cover types. However, USA urban sprawl contributed more to forest fragmentation than in China. This is closely tied to the USA’s economy, lifestyle and institutional processes. Fragmentation maps were generated from this study, which provide valuable insights and implications regarding habitat planning for rare and endangered species. Such maps enable development of strategic plans for sustainable forest management by identifying areas with high amounts of human-induced fragmentation, which improve risk assessments and enable better targeting for protection and remediation efforts. Because forest fragmentation is a long-term, complex process that is highly related to political, institutional, economic and philosophical arenas, both nations need to take effective and comprehensive measures to mitigate the negative effects of forest loss and fragmentation on the existing forest ecosystems.

Comparing forest fragmentation and its drivers in China and the USA with Globcover v2.2.

PubMed

Li, Mingshi; Mao, Lijun; Zhou, Chunguo; Vogelmann, James E; Zhu, Zhiliang

2010-12-01

Forest loss and fragmentation are of major concern to the international community, in large part because they impact so many important environmental processes. The main objective of this study was to assess the differences in forest fragmentation patterns and drivers between China and the conterminous United States (USA). Using the latest 300-m resolution global land cover product, Globcover v2.2, a comparative analysis of forest fragmentation patterns and drivers was made. The fragmentation patterns were characterized by using a forest fragmentation model built on the sliding window analysis technique in association with landscape indices. Results showed that China's forests were substantially more fragmented than those of the USA. This was evidenced by a large difference in the amount of interior forest area share, with China having 48% interior forest versus the 66% for the USA. China's forest fragmentation was primarily attributed to anthropogenic disturbances, driven particularly by agricultural expansion from an increasing and large population, as well as poor forest management practices. In contrast, USA forests were principally fragmented by natural land cover types. However, USA urban sprawl contributed more to forest fragmentation than in China. This is closely tied to the USA's economy, lifestyle and institutional processes. Fragmentation maps were generated from this study, which provide valuable insights and implications regarding habitat planning for rare and endangered species. Such maps enable development of strategic plans for sustainable forest management by identifying areas with high amounts of human-induced fragmentation, which improve risk assessments and enable better targeting for protection and remediation efforts. Because forest fragmentation is a long-term, complex process that is highly related to political, institutional, economic and philosophical arenas, both nations need to take effective and comprehensive measures to mitigate the negative effects of forest loss and fragmentation on the existing forest ecosystems. Copyright © 2010 Elsevier Ltd. All rights reserved.

Knowledge-based fragment binding prediction.

PubMed

Tang, Grace W; Altman, Russ B

2014-04-01

Target-based drug discovery must assess many drug-like compounds for potential activity. Focusing on low-molecular-weight compounds (fragments) can dramatically reduce the chemical search space. However, approaches for determining protein-fragment interactions have limitations. Experimental assays are time-consuming, expensive, and not always applicable. At the same time, computational approaches using physics-based methods have limited accuracy. With increasing high-resolution structural data for protein-ligand complexes, there is now an opportunity for data-driven approaches to fragment binding prediction. We present FragFEATURE, a machine learning approach to predict small molecule fragments preferred by a target protein structure. We first create a knowledge base of protein structural environments annotated with the small molecule substructures they bind. These substructures have low-molecular weight and serve as a proxy for fragments. FragFEATURE then compares the structural environments within a target protein to those in the knowledge base to retrieve statistically preferred fragments. It merges information across diverse ligands with shared substructures to generate predictions. Our results demonstrate FragFEATURE's ability to rediscover fragments corresponding to the ligand bound with 74% precision and 82% recall on average. For many protein targets, it identifies high scoring fragments that are substructures of known inhibitors. FragFEATURE thus predicts fragments that can serve as inputs to fragment-based drug design or serve as refinement criteria for creating target-specific compound libraries for experimental or computational screening.

Knowledge-based Fragment Binding Prediction

PubMed Central

Tang, Grace W.; Altman, Russ B.

2014-01-01

Target-based drug discovery must assess many drug-like compounds for potential activity. Focusing on low-molecular-weight compounds (fragments) can dramatically reduce the chemical search space. However, approaches for determining protein-fragment interactions have limitations. Experimental assays are time-consuming, expensive, and not always applicable. At the same time, computational approaches using physics-based methods have limited accuracy. With increasing high-resolution structural data for protein-ligand complexes, there is now an opportunity for data-driven approaches to fragment binding prediction. We present FragFEATURE, a machine learning approach to predict small molecule fragments preferred by a target protein structure. We first create a knowledge base of protein structural environments annotated with the small molecule substructures they bind. These substructures have low-molecular weight and serve as a proxy for fragments. FragFEATURE then compares the structural environments within a target protein to those in the knowledge base to retrieve statistically preferred fragments. It merges information across diverse ligands with shared substructures to generate predictions. Our results demonstrate FragFEATURE's ability to rediscover fragments corresponding to the ligand bound with 74% precision and 82% recall on average. For many protein targets, it identifies high scoring fragments that are substructures of known inhibitors. FragFEATURE thus predicts fragments that can serve as inputs to fragment-based drug design or serve as refinement criteria for creating target-specific compound libraries for experimental or computational screening. PMID:24762971

An optimized OPC and MDP flow for reducing mask write time and mask cost

NASA Astrophysics Data System (ADS)

Yang, Ellyn; Li, Cheng He; Park, Se Jin; Zhu, Yu; Guo, Eric

2010-09-01

In the process of optical proximity correction, layout edge or fragment is migrating to proper position in order to minimize edge placement error (EPE). During this fragment migration, several factors other than EPE can be also taken into account as a part of cost function for optimal fragment displacement. Several factors are devised in favor of OPC stability, which can accommodate room for high mask error enhancement factor (MEEF), lack of process window, catastrophic pattern failure such as pinch/bridge and improper fragmentation. As technology node becomes finer, there happens conflict between OPC accuracy and stability. Especially for metal layers, OPC has focused on the stability by loss of accurate OPC results. On this purpose, several techniques have been introduced, which are target smoothing, process window aware OPC, model-based retargeting and adaptive OPC. By utilizing those techniques, OPC enables more stabilized patterning, instead of realizing design target exactly on wafer. Inevitably, post-OPC layouts become more complicated because those techniques invoke additional edge, or fragments prior to correction or during OPC iteration. As a result, jogs of post OPC layer can be dramatically increased, which results in huge number of shot count after data fracturing. In other words, there is trade-off relationship between data complexity and various methods for OPC stability. In this paper, those relationships have been investigated with respect to several technology nodes. The mask shot count reduction is achieved by reducing the number of jogs with which EPE difference are within pre-specified value. The effect of jog smoothing on OPC output - in view of OPC performance and mask data preparation - was studied quantitatively for respective technology nodes.

Scleroderma Autoantigens Are Uniquely Fragmented by Metal-catalyzed Oxidation Reactions: Implications for Pathogenesis

PubMed Central

Casciola-Rosen, Livia; Wigley, Fredrick; Rosen, Antony

1997-01-01

The observation that revelation of immunocryptic epitopes in self antigens may initiate the autoimmune response has prompted the search for processes which induce novel fragmentation of autoantigens as potential initiators of autoimmunity. The reversible ischemia reperfusion which characterizes scleroderma has focused attention on reactive oxygen species as molecules which might induce autoantigen fragmentation. We demonstrate that several of the autoantigens targeted in diffuse scleroderma are uniquely susceptible to cleavage by reactive oxygen species, in a metal-dependent manner. Multiple features of the fragmentation reaction and its inhibition indicate that these autoantigens possess metal-binding sites, which focus metal-catalyzed oxidation reactions (and consequent fragmentation) to specific regions of the antigens. These data suggest that the autoantibody response in scleroderma is the immune marker of unique protein fragmentation, induced by ischemia reperfusion in the presence of appropriate metals, and focus attention on abnormal metal status as a potential pathogenic principle in this disease. PMID:8996243

Activation of accelerator construction materials by heavy ions

NASA Astrophysics Data System (ADS)

Katrík, P.; Mustafin, E.; Hoffmann, D. H. H.; Pavlovič, M.; Strašík, I.

2015-12-01

Activation data for an aluminum target irradiated by 200 MeV/u 238U ion beam are presented in the paper. The target was irradiated in the stacked-foil geometry and analyzed using gamma-ray spectroscopy. The purpose of the experiment was to study the role of primary particles, projectile fragments, and target fragments in the activation process using the depth profiling of residual activity. The study brought information on which particles contribute dominantly to the target activation. The experimental data were compared with the Monte Carlo simulations by the FLUKA 2011.2c.0 code. This study is a part of a research program devoted to activation of accelerator construction materials by high-energy (⩾200 MeV/u) heavy ions at GSI Darmstadt. The experimental data are needed to validate the computer codes used for simulation of interaction of swift heavy ions with matter.

Fragment properties at the catastrophic disruption threshold: The effect of the parent body’s internal structure

NASA Astrophysics Data System (ADS)

Jutzi, Martin; Michel, Patrick; Benz, Willy; Richardson, Derek C.

2010-05-01

Numerical simulations of asteroid breakups, including both the fragmentation of the parent body and the gravitational interactions between the fragments, have allowed us to reproduce successfully the main properties of asteroid families formed in different regimes of impact energy, starting from a non-porous parent body. In this paper, using the same approach, we concentrate on a single regime of impact energy, the so-called catastrophic threshold usually designated by QD*, which results in the escape of half of the target's mass. Thanks to our recent implementation of a model of fragmentation of porous materials, we can characterize QD* for both porous and non-porous targets with a wide range of diameters. We can then analyze the potential influence of porosity on the value of QD*, and by computing the gravitational phase of the collision in the gravity regime, we can characterize the collisional outcome in terms of the fragment size and ejection speed distributions, which are the main outcome properties used by collisional models to study the evolutions of the different populations of small bodies. We also check the dependency of QD* on the impact speed of the projectile. In the strength regime, which corresponds to target sizes below a few hundreds of meters, we find that porous targets are more difficult to disrupt than non-porous ones. In the gravity regime, the outcome is controlled purely by gravity and porosity in the case of porous targets. In the case of non-porous targets, the outcome also depends on strength. Indeed, decreasing the strength of non-porous targets make them easier to disrupt in this regime, while increasing the strength of porous targets has much less influence on the value of QD*. Therefore, one cannot say that non-porous targets are systematically easier or more difficult to disrupt than porous ones, as the outcome highly depends on the assumed strength values. In the gravity regime, we also confirm that the process of gravitational reaccumulation is at the origin of the largest remnant's mass in both cases. We then propose some power-law relationships between QD* and both target's size and impact speed that can be used in collisional evolution models. The resulting fragment size distributions can also be reasonably fitted by a power-law whose exponent ranges between -2.2 and -2.7 for all target diameters in both cases and independently on the impact velocity (at least in the small range investigated between 3 and 5 km/s). Then, although ejection velocities in the gravity regime tend to be higher from porous targets, they remain on the same order as the ones from non-porous targets.

Impacts into Coarse-Grained Spheres at Moderate Impact Velocities: Implications for Cratering on Asteroids and Planets

NASA Technical Reports Server (NTRS)

Barnouin, Olivier S.; Daly, R. Terik; Cintala, Mark J.; Crawford, David A.

2018-01-01

The surfaces of many planets and asteroids contain coarsely fragmental material generated by impacts or other geologic processes. The presence of such pre-existing structures may affect subsequent impacts, particularly when the width of the shock is comparable to or smaller than the size of pre-existing structures. Reasonable theoretical predictions and low speed (<300m/s) impact experiments suggest that in such targets the cratering process should be highly dissipative, which would reduce cratering efficiencies and cause a rapid decay in ejection velocity as a function of distance from the impact point. In this study, we assess whether these results apply at higher impact speeds between 0.5 and 2.5 km s-1. This study shows little change in cratering efficiency when 3.18 mm diameter glass beads are launched into targets composed of these same beads. These impacts are very efficient, and ejection velocity decays slowly as function of distance from the impact point. This slow decay in ejection velocity probably indicates a correspondingly slow decay of the shock stresses. However, these experiments reveal that initial interactions between projectile and target strongly influence the cratering process and lead to asymmetries in crater shape and ejection angles, as well as significant variations in ejection velocity at a given launch position. Such effects of asymmetric coupling could be further enhanced by heterogeneity in the initial distribution of grains in the target and by mechanical collisions between grains. These experiments help to explain why so few craters are seen on the rubble-pile asteroid Itokawa: impacts into its coarsely fragmental surface by projectiles comparable to or smaller than the size of these fragments likely yield craters that are not easily recognizable.

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry

PubMed Central

2018-01-01

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain. PMID:29436819

Fragment-Based Screening of a Natural Product Library against 62 Potential Malaria Drug Targets Employing Native Mass Spectrometry.

PubMed

Vu, Hoan; Pedro, Liliana; Mak, Tin; McCormick, Brendan; Rowley, Jessica; Liu, Miaomiao; Di Capua, Angela; Williams-Noonan, Billy; Pham, Ngoc B; Pouwer, Rebecca; Nguyen, Bao; Andrews, Katherine T; Skinner-Adams, Tina; Kim, Jessica; Hol, Wim G J; Hui, Raymond; Crowther, Gregory J; Van Voorhis, Wesley C; Quinn, Ronald J

2018-04-13

Natural products are well known for their biological relevance, high degree of three-dimensionality, and access to areas of largely unexplored chemical space. To shape our understanding of the interaction between natural products and protein targets in the postgenomic era, we have used native mass spectrometry to investigate 62 potential protein targets for malaria using a natural-product-based fragment library. We reveal here 96 low-molecular-weight natural products identified as binding partners of 32 of the putative malarial targets. Seventy-nine (79) fragments have direct growth inhibition on Plasmodium falciparum at concentrations that are promising for the development of fragment hits against these protein targets. This adds a fragment library to the published HTS active libraries in the public domain.

Lead generation and examples opinion regarding how to follow up hits.

PubMed

Orita, Masaya; Ohno, Kazuki; Warizaya, Masaichi; Amano, Yasushi; Niimi, Tatsuya

2011-01-01

In fragment-based drug discovery (FBDD), not only identifying the starting fragment hit to be developed but also generating a drug lead from that starting fragment hit is important. Converting fragment hits to leads is generally similar to a high-throughput screening (HTS) hits-to-leads approach in that properties associated with activity for a target protein, such as selectivity against other targets and absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox), and physicochemical properties should be taken into account. However, enhancing the potency of the fragment hit is a key requirement in FBDD, unlike HTS, because initial fragment hits are generally weak. This enhancement is presently achieved by adding additional chemical groups which bind to additional parts of the target protein or by joining or combining two or more hit fragments; however, strategies for effecting greater improvements in effective activity are needed. X-ray analysis is a key technology attractive for converting fragments to drug leads. This method makes it clear whether a fragment hit can act as an anchor and provides insight regarding introduction of functional groups to improve fragment activity. Data on follow-up chemical synthesis of fragment hits has allowed for the differentiation of four different strategies: fragment optimization, fragment linking, fragment self-assembly, and fragment evolution. Here, we discuss our opinion regarding how to follow up on fragment hits, with a focus on the importance of fragment hits as an anchor moiety to so-called hot spots in the target protein using crystallographic data. Copyright © 2011 Elsevier Inc. All rights reserved.

Large scale meta-analysis of fragment-based screening campaigns: privileged fragments and complementary technologies.

PubMed

Kutchukian, Peter S; Wassermann, Anne Mai; Lindvall, Mika K; Wright, S Kirk; Ottl, Johannes; Jacob, Jaison; Scheufler, Clemens; Marzinzik, Andreas; Brooijmans, Natasja; Glick, Meir

2015-06-01

A first step in fragment-based drug discovery (FBDD) often entails a fragment-based screen (FBS) to identify fragment "hits." However, the integration of conflicting results from orthogonal screens remains a challenge. Here we present a meta-analysis of 35 fragment-based campaigns at Novartis, which employed a generic 1400-fragment library against diverse target families using various biophysical and biochemical techniques. By statistically interrogating the multidimensional FBS data, we sought to investigate three questions: (1) What makes a fragment amenable for FBS? (2) How do hits from different fragment screening technologies and target classes compare with each other? (3) What is the best way to pair FBS assay technologies? In doing so, we identified substructures that were privileged for specific target classes, as well as fragments that were privileged for authentic activity against many targets. We also revealed some of the discrepancies between technologies. Finally, we uncovered a simple rule of thumb in screening strategy: when choosing two technologies for a campaign, pairing a biochemical and biophysical screen tends to yield the greatest coverage of authentic hits. © 2014 Society for Laboratory Automation and Screening.

Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

PubMed

Khashan, Raed S

2015-01-01

As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

The Behavior of Lipid Debris Left on Cell Surfaces from Microbubble Based Ultrasound Molecular Imaging

PubMed Central

Ibsen, Stuart; Shi, Guixin; Schutt, Carolyn; Shi, Linda; Suico, Kyle-David; Benchimol, Michael; Serra, Viviana; Simberg, Dmitri; Berns, Michael; Esener, Sadik

2014-01-01

Lipid monolayer coated microbubbles are currently being developed to identify vascular regions that express certain surface proteins as part of the new technique of ultrasound molecular imaging. The microbubbles are functionalized with targeting ligands which bind to the desired cells holding the microbubbles in place as the remaining unbound microbubbles are eliminated from circulation. Subsequent scanning with ultrasound can detect the highly reflectant microbubbles that are left behind. The ultrasound scanning and detection process results in the destruction of the microbubble, creating lipid fragments from the monolayer. Here we demonstrate that microbubbles targeted to 4T1 murine breast cancer cells and human umbilical cord endothelial cells leave behind adhered fragments of the lipid monolayer after exposure to ultrasound with peak negative pressures of 0.18 and 0.8 MPa. Most of the observed fragments were large enough to be resistant to receptor mediated endocytosis. The fragments were not observed to incorporate into the lipid membrane of the cell over a period of 96 min. They were not observed to break into smaller pieces or significantly change shape but they were observed to undergo translation and rotation across the cell surface as the cells migrated over the substrate. These large fragments will apparently remain on the surface of the targeted cells for significant periods of time and need to be considered for their potential effects on blood flow through the microcapillaries and potential for immune system recognition. PMID:25059435

Fragment-based approaches to the discovery of kinase inhibitors.

PubMed

Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

2014-01-01

Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

PubMed

Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

2012-11-01

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

An ab initio study of ion induced charge transfer dynamics in collision of carbon ions with thymine.

PubMed

Bacchus-Montabonel, Marie-Christine; Tergiman, Yvette Suzanne

2011-05-28

Charge transfer in collisions of carbon ions on a thymine target has been studied theoretically in a wide collision range by means of ab initio quantum chemistry molecular methods. The process appears markedly anisotropic in the whole energy domain, significantly favoured in the perpendicular orientation. A specific decrease of the charge transfer cross sections at low collision energies may be pointed out and could induce an enhancement of the complementary fragmentation processes for collision energies down to about 10 eV, as observed for the low-electron fragmentation process. Such feature may be of important interest in ion-induced biomolecular radiation damage. This journal is © the Owner Societies 2011

Evaluating Fragment Construction Policies for SDT Systems

DTIC Science & Technology

2006-01-01

allocates a fragment and begins translation. Once a termination condition is met, Strata emits any trampolines that are necessary. Trampolines are pieces... trampolines (unless its target previously exists in the fragment cache). Once a CTI’s target instruction becomes available in the fragment cache, the CTI is...linked directly to the destination, avoiding future uses of the trampoline . This mechanism is called Fragment Linking and avoids significant overhead

Integration of fragment screening and library design.

PubMed

Siegal, Gregg; Ab, Eiso; Schultz, Jan

2007-12-01

With more than 10 years of practical experience and theoretical analysis, fragment-based drug discovery (FBDD) has entered the mainstream of the pharmaceutical and biotech industries. An array of biophysical techniques has been used to detect the weak interaction between a fragment and the target. Each technique presents its own requirements regarding the fragment collection and the target; therefore, in order to optimize the potential of FBDD, the nature of the target should be a driving factor for simultaneous development of both the library and the screening technology. A roadmap is now available to guide fragment-to-lead evolution when structural information is available. The next challenge is to apply FBDD to targets for which high-resolution structural information is not available.

Electromagnetic dissociation of U-238 in heavy-ion collisions at 120 MeV/A

NASA Astrophysics Data System (ADS)

Justice, M. L.

1991-04-01

This thesis describes a measurement of the heavy-ion induced electromagnetic dissociation of a 120 MeV/A U-238 beam incident on five targets: Be-9, Al-27, Cu, Ag, and U. Electromagnetic dissociation at this beam energy is essentially a two step process involving the excitation of a giant resonance followed by particle decay. At 120 MeV/A there is predicted to be a significant contribution of the giant quadrupole resonance to the EMD cross sections. The specific exit channel which was looked at was projectile fission. The two fission fragments were detected in coincidence by an array of solid-state (Delta)E-E detectors, allowing the changes of the fragments to be determined to within (+/-) .5 units. The events were sorted on the basis of the sums of the fragments' charges, acceptance corrections were applied, and total cross sections for the most peripheral events were determined. Electromagnetic fission at the beam energy of this experiment always leads to a true charge sum of 92. Due to the imperfect resolution of the detectors, charge sums of 91 and 93 were included in order to account for all of the electromagnetic fission events. The experimentally observed cross sections are due to nuclear interaction processes as well as electromagnetic processes. Under the conditions of this experiment, the cross sections for the beryllium target are almost entirely due to nuclear processes. The nuclear cross sections for the other four targets were determined by extrapolation from the beryllium data using a geometrical scaling model. After subtraction of the nuclear cross sections, the resulting electromagnetic cross sections are compared to theoretical calculations based on the equivalent photon approximation. Systematic uncertainties are discussed and suggestions for improving the experiment are given.

Multinucleon transfer dynamics in heavy-ion collisions near Coulomb-barrier energies

NASA Astrophysics Data System (ADS)

Niu, Fei; Chen, Peng-Hui; Guo, Ya-Fei; Ma, Chun-Wang; Feng, Zhao-Qing

2017-12-01

Multinucleon transfer reactions near barrier energies have been investigated with a multistep model based on the dinuclear system (DNS) concept, in which the capture of two colliding nuclei, the transfer dynamics, and the deexcitation process of primary fragments are described by an analytical formula, diffusion theory, and a statistical model, respectively. The nucleon transfer takes place after forming the DNS and is coupled to the dissipation of relative motion energy and angular momentum by solving a set of microscopically derived master equations within the potential energy surface. Specific reactions of Ca,4840+124Sn , 40Ca(40Ar,58Ni)+232Th , 40Ca(58Ni)+238U , and Ca,4840(58Ni)+248Cm near barrier energies are investigated. It is found that fragments are produced by multinucleon transfer reactions with maximal yields along the β -stability line. The isospin relaxation is particularly significant in the process of fragment formation. The incident energy dependence of heavy target-like fragments in the reaction of 58Ni+248Cm is analyzed thoroughly.

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

PubMed

Chimenti, Michael S; Bulfer, Stacie L; Neitz, R Jeffrey; Renslo, Adam R; Jacobson, Matthew P; James, Thomas L; Arkin, Michelle R; Kelly, Mark J S

2015-07-01

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding. © 2015 Society for Laboratory Automation and Screening.

The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

PubMed Central

Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

2015-01-01

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

AlphaSpace: Fragment-Centric Topographical Mapping To Target Protein–Protein Interaction Interfaces

PubMed Central

2016-01-01

Inhibition of protein–protein interactions (PPIs) is emerging as a promising therapeutic strategy despite the difficulty in targeting such interfaces with drug-like small molecules. PPIs generally feature large and flat binding surfaces as compared to typical drug targets. These features pose a challenge for structural characterization of the surface using geometry-based pocket-detection methods. An attractive mapping strategy—that builds on the principles of fragment-based drug discovery (FBDD)—is to detect the fragment-centric modularity at the protein surface and then characterize the large PPI interface as a set of localized, fragment-targetable interaction regions. Here, we introduce AlphaSpace, a computational analysis tool designed for fragment-centric topographical mapping (FCTM) of PPI interfaces. Our approach uses the alpha sphere construct, a geometric feature of a protein’s Voronoi diagram, to map out concave interaction space at the protein surface. We introduce two new features—alpha-atom and alpha-space—and the concept of the alpha-atom/alpha-space pair to rank pockets for fragment-targetability and to facilitate the evaluation of pocket/fragment complementarity. The resulting high-resolution interfacial map of targetable pocket space can be used to guide the rational design and optimization of small molecule or biomimetic PPI inhibitors. PMID:26225450

Momentum distributions of isotopes produced by fragmentation of relativistic C-12 and O-16 projectiles

NASA Technical Reports Server (NTRS)

Greiner, D. E.; Lindstrom, P. J.; Heckman, H. H.; Cork, B.; Bieser, F. S.

1975-01-01

The fragment momentum distributions in the projectile rest frame are, typically, Gaussian shaped, narrow, consistent with isotropy, depend on fragment and projectile, and have no significant correlation with target mass or beam energy. The nuclear temperature is inferred from the momentum distributions of the fragments and is approximately equal to the projectile nuclear binding energy, indicative of small energy transfer between target and fragment.

T-cell-restricted intracellular antigen 1 facilitates mitochondrial fragmentation by enhancing the expression of mitochondrial fission factor

PubMed Central

Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung

2017-01-01

Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012

The FIRST experiment at GSI

NASA Astrophysics Data System (ADS)

Pleskac, R.; Abou-Haidar, Z.; Agodi, C.; Alvarez, M. A. G.; Aumann, T.; Battistoni, G.; Bocci, A.; Böhlen, T. T.; Boudard, A.; Brunetti, A.; Carpinelli, M.; Cirrone, G. A. P.; Cortes-Giraldo, M. A.; Cuttone, G.; De Napoli, M.; Durante, M.; Fernández-García, J. P.; Finck, C.; Golosio, B.; Gallardo, M. I.; Iarocci, E.; Iazzi, F.; Ickert, G.; Introzzi, R.; Juliani, D.; Krimmer, J.; Kurz, N.; Labalme, M.; Leifels, Y.; Le Fevre, A.; Leray, S.; Marchetto, F.; Monaco, V.; Morone, M. C.; Oliva, P.; Paoloni, A.; Piersanti, L.; Quesada, J. M.; Raciti, G.; Randazzo, N.; Romano, F.; Rossi, D.; Rousseau, M.; Sacchi, R.; Sala, P.; Sarti, A.; Scheidenberger, C.; Schuy, C.; Sciubba, A.; Sfienti, C.; Simon, H.; Sipala, V.; Spiriti, E.; Stuttge, L.; Tropea, S.; Younis, H.; Patera, V.

2012-06-01

The FIRST (Fragmentation of Ions Relevant for Space and Therapy) experiment at the SIS accelerator of GSI laboratory in Darmstadt has been designed for the measurement of ion fragmentation cross-sections at different angles and energies between 100 and 1000 MeV/nucleon. Nuclear fragmentation processes are relevant in several fields of basic research and applied physics and are of particular interest for tumor therapy and for space radiation protection applications. The start of the scientific program of the FIRST experiment was on summer 2011 and was focused on the measurement of 400 MeV/nucleon 12C beam fragmentation on thin (8 mm) graphite target. The detector is partly based on an already existing setup made of a dipole magnet (ALADiN), a time projection chamber (TP-MUSIC IV), a neutron detector (LAND) and a time of flight scintillator system (TOFWALL). This pre-existing setup has been integrated with newly designed detectors in the Interaction Region, around the carbon target placed in a sample changer. The new detectors are a scintillator Start Counter, a Beam Monitor drift chamber, a silicon Vertex Detector and a Proton Tagger scintillator system optimized for the detection of light fragments emitted at large angles. In this paper we review the experimental setup, then we present the simulation software, the data acquisition system and finally the trigger strategy of the experiment.

[Development of pseudoviral competitive internal controls for RT-PCR detection of dengue virus].

PubMed

Hang, Xiao-Tong; Li, Jian-Dong; Zhang, Quan-Fu; Li, Chuan; Zhang, Shuo; Liang, Mi-Fang; Li, De-Xin

2010-02-01

Development of pseudoviral competitive internal controls for RT-PCR laboratory detection of dengue virus. The internal controls target gene were obtained by insertion of a 180 bp non-related DNA fragment into RT-PCR detection target of dengue virus between the forward and reverse PCR primer binding regions. A yellow florescence protein reporter gene was induced at downstream of internal controls target gene via internal ribosome entry site gene. HEK 293T cells were transfected with plasmid containing this whole cassette and lentiviral packaging support plasmid. Pseudoviral particle was recovered from the supernatant and analyzed quantitatively and qualitatively in simulated samples at the same tube under different experimental conditions. The established pseudoviral competitive internal controls can be used in the RT-PCR detection of different serotype dengue virus and the whole detection process can be monitored. The obtained fragment is easy to be differentiated in agarose electrophoresis. The pseudoviral competitive internal controls could be used for the quality control of the laboratory diagnosis process, simple to prepare, stable for storage, easy to be transformed into internal controls for other RNA virus.

TagFinder for the quantitative analysis of gas chromatography--mass spectrometry (GC-MS)-based metabolite profiling experiments.

PubMed

Luedemann, Alexander; Strassburg, Katrin; Erban, Alexander; Kopka, Joachim

2008-03-01

Typical GC-MS-based metabolite profiling experiments may comprise hundreds of chromatogram files, which each contain up to 1000 mass spectral tags (MSTs). MSTs are the characteristic patterns of approximately 25-250 fragment ions and respective isotopomers, which are generated after gas chromatography (GC) by electron impact ionization (EI) of the separated chemical molecules. These fragment ions are subsequently detected by time-of-flight (TOF) mass spectrometry (MS). MSTs of profiling experiments are typically reported as a list of ions, which are characterized by mass, chromatographic retention index (RI) or retention time (RT), and arbitrary abundance. The first two parameters allow the identification, the later the quantification of the represented chemical compounds. Many software tools have been reported for the pre-processing, the so-called curve resolution and deconvolution, of GC-(EI-TOF)-MS files. Pre-processing tools generate numerical data matrices, which contain all aligned MSTs and samples of an experiment. This process, however, is error prone mainly due to (i) the imprecise RI or RT alignment of MSTs and (ii) the high complexity of biological samples. This complexity causes co-elution of compounds and as a consequence non-selective, in other words impure MSTs. The selection and validation of optimal fragment ions for the specific and selective quantification of simultaneously eluting compounds is, therefore, mandatory. Currently validation is performed in most laboratories under human supervision. So far no software tool supports the non-targeted and user-independent quality assessment of the data matrices prior to statistical analysis. TagFinder may fill this gap. TagFinder facilitates the analysis of all fragment ions, which are observed in GC-(EI-TOF)-MS profiling experiments. The non-targeted approach allows the discovery of novel and unexpected compounds. In addition, mass isotopomer resolution is maintained by TagFinder processing. This feature is essential for metabolic flux analyses and highly useful, but not required for metabolite profiling. Whenever possible, TagFinder gives precedence to chemical means of standardization, for example, the use of internal reference compounds for retention time calibration or quantitative standardization. In addition, external standardization is supported for both compound identification and calibration. The workflow of TagFinder comprises, (i) the import of fragment ion data, namely mass, time and arbitrary abundance (intensity), from a chromatography file interchange format or from peak lists provided by other chromatogram pre-processing software, (ii) the annotation of sample information and grouping of samples into classes, (iii) the RI calculation, (iv) the binning of observed fragment ions of equal mass from different chromatograms into RI windows, (v) the combination of these bins, so-called mass tags, into time groups of co-eluting fragment ions, (vi) the test of time groups for intensity correlated mass tags, (vii) the data matrix generation and (viii) the extraction of selective mass tags supported by compound identification. Thus, TagFinder supports both non-targeted fingerprinting analyses and metabolite targeted profiling. Exemplary TagFinder workspaces and test data sets are made available upon request to the contact authors. TagFinder is made freely available for academic use from http://www-en.mpimp-golm.mpg.de/03-research/researchGroups/01-dept1/Root_Metabolism/smp/TagFinder/index.html.

Multiplicity distributions of shower particles and target fragments in 84 Kr 36-emulsion interactions at 1 GeV per nucleon

NASA Astrophysics Data System (ADS)

Singh, M. K.; Soma, A. K.; Pathak, Ramji; Singh, V.

2014-03-01

This article focuses on multiplicity distributions of shower particles and target fragments for interaction of 84 Kr 36 with NIKFI BR-2 nuclear emulsion target at kinetic energy of 1 GeV per nucleon. Experimental multiplicity distributions of shower particles, grey particles, black particles and heavily ionization particles are well described by multi-component Erlang distribution of multi-source thermal model. We have observed a linear correlation in multiplicities for the above mentioned particles or fragments. Further experimental studies have shown a saturation phenomenon in shower particle multiplicity with the increase of target fragment multiplicity.

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

PubMed

Sangiuolo, Federica; Scaldaferri, Maria Lucia; Filareto, Antonio; Spitalieri, Paola; Guerra, Lorenzo; Favia, Maria; Caroppo, Rosa; Mango, Ruggiero; Bruscia, Emanuela; Gruenert, Dieter C; Casavola, Valeria; De Felici, Massimo; Novelli, Giuseppe

2008-01-01

Different gene targeting approaches have been developed to modify endogenous genomic DNA in both human and mouse cells. Briefly, the process involves the targeting of a specific mutation in situ leading to the gene correction and the restoration of a normal gene function. Most of these protocols with therapeutic potential are oligonucleotide based, and rely on endogenous enzymatic pathways. One gene targeting approach, "Small Fragment Homologous Replacement (SFHR)", has been found to be effective in modifying genomic DNA. This approach uses small DNA fragments (SDF) to target specific genomic loci and induce sequence and subsequent phenotypic alterations. This study shows that SFHR can stably introduce a 3-bp deletion (deltaF508, the most frequent cystic fibrosis (CF) mutation) into the Cftr (CF Transmembrane Conductance Regulator) locus in the mouse embryonic stem (ES) cell genome. After transfection of deltaF508-SDF into murine ES cells, SFHR-mediated modification was evaluated at the molecular levels on DNA and mRNA obtained from transfected ES cells. About 12% of transcript corresponding to deleted allele was detected, while 60% of the electroporated cells completely lost any measurable CFTR-dependent chloride efflux. The data indicate that the SFHR technique can be used to effectively target and modify genomic sequences in ES cells. Once the SFHR-modified ES cells differentiate into different cell lineages they can be useful for elucidating tissue-specific gene function and for the development of transplantation-based cellular and therapeutic protocols.

Novel in vitro protein fragment complementation assay applicable to high-throughput screening in a 1536-well format.

PubMed

Hashimoto, Junko; Watanabe, Taku; Seki, Tatsuya; Karasawa, Satoshi; Izumikawa, Miho; Seki, Tomoe; Iemura, Shun-Ichiro; Natsume, Tohru; Nomura, Nobuo; Goshima, Naoki; Miyawaki, Atsushi; Takagi, Motoki; Shin-Ya, Kazuo

2009-09-01

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 microM and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 microM. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library.

What iconic gesture fragments reveal about gesture-speech integration: when synchrony is lost, memory can help.

PubMed

Obermeier, Christian; Holle, Henning; Gunter, Thomas C

2011-07-01

The present series of experiments explores several issues related to gesture-speech integration and synchrony during sentence processing. To be able to more precisely manipulate gesture-speech synchrony, we used gesture fragments instead of complete gestures, thereby avoiding the usual long temporal overlap of gestures with their coexpressive speech. In a pretest, the minimal duration of an iconic gesture fragment needed to disambiguate a homonym (i.e., disambiguation point) was therefore identified. In three subsequent ERP experiments, we then investigated whether the gesture information available at the disambiguation point has immediate as well as delayed consequences on the processing of a temporarily ambiguous spoken sentence, and whether these gesture-speech integration processes are susceptible to temporal synchrony. Experiment 1, which used asynchronous stimuli as well as an explicit task, showed clear N400 effects at the homonym as well as at the target word presented further downstream, suggesting that asynchrony does not prevent integration under explicit task conditions. No such effects were found when asynchronous stimuli were presented using a more shallow task (Experiment 2). Finally, when gesture fragment and homonym were synchronous, similar results as in Experiment 1 were found, even under shallow task conditions (Experiment 3). We conclude that when iconic gesture fragments and speech are in synchrony, their interaction is more or less automatic. When they are not, more controlled, active memory processes are necessary to be able to combine the gesture fragment and speech context in such a way that the homonym is disambiguated correctly.

The shapes of fragments in hypervelocity impact experiments ranging from cratering to catastrophic disruption

NASA Astrophysics Data System (ADS)

Michikami, T.; Hagermann, A.; Kadokawa, T.; Yoshida, A.; Shimada, A.; Hasegawa, S.; Tsuchiyama, A.

2015-12-01

Laboratory impact experiments have found that the shapes of impact fragments as defined by axes a, b and c, these being the maximum dimensions of the fragment in three mutually orthogonal planes (a ≥ b ≥ c) are distributed around mean values of the axial ratios b/a ~0.7 and c/a ~0.5, i.e., corresponding to a : b: c in the simple proportion 2: √2: 1. The shape distributions of some boulders on asteroid Eros, the small- and fast-rotating asteroids (diameter < 200 m and rotation period < 1 h), and asteroids in young families, are similar to those of laboratory fragments in catastrophic disruption. However, the shapes of laboratory fragments were obtained from the experiments that　resulted in catastrophic disruption, a process that is different from impact cratering. In order to systematically investigate the shapes of fragments in the range from impact cratering to catastrophic disruption, impact experiments for basalt targets 5 to 15 cm in size were performed. A total of 28 impact experiments were carried out by a spherical nylon projectile (diameter 7.14 mm) perpendicularly into the target surface at velocities of 1.6 to 7.0 km/s. More than 13,000 fragments with b ≥ 4 mm generated in the impact experiments were measured. In the experiments, the mean value of c/a in each impact decreases with decreasing impact energy per unit target mass. For instance, the mean value of c/a in an impact cratering event is nearly 0.2, which is less than that c/a in a catastrophic disruption (~0.5). To apply the experimental results to real collisions on asteroids, we investigated the shapes of 21 arbitrarily selected boulders (> 8 m) on asteroid Itokawa. The mean value of c/a of these boulders is 0.46, which is similar to the value for catastrophic disruption. This implies that the parent body of Itokawa could have experienced a catastrophic disruption.

XRN2 is required for the degradation of target RNAs by RNase H1-dependent antisense oligonucleotides

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

Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Obika, Satoshi, E-mail: obika@phs.osaka-u.ac.jp

Antisense oligonucleotides (ASOs) can suppress the expression of a target gene by cleaving pre-mRNA and/or mature mRNA via RNase H1. Following the initial endonucleolytic cleavage by RNase H1, the target RNAs are degraded by a mechanism that is poorly understood. To better understand this degradation pathway, we depleted the expression of two major 5′ to 3′ exoribonucleases (XRNs), named XRN1 and XRN2, and analyzed the levels of 3′ fragments of the target RNAs in vitro. We found that the 3′ fragments of target pre-mRNA generated by ASO were almost completely degraded from their 5′ ends by nuclear XRN2 after RNase H1-mediatedmore » cleavage, whereas the 3′ fragments of mature mRNA were partially degraded by XRN2. In contrast to ASO, small interference RNA (siRNA) could reduce the expression level of only mature mRNA, and the 3′ fragment was degraded by cytoplasmic XRN1. Our findings indicate that the RNAs targeted by RNase H1-dependent ASO are rapidly degraded in the nucleus, contrary to the cytoplasmic degradation pathway mediated by siRNA. - Highlights: • We compared the degradation mechanism of the transcript targeted by ASO and siRNA. • We focused on two 5′ to 3′ exoribonucleases, cytoplasmic XRN1, and nuclear XRN2. • The 3′ fragment of target pre-mRNA generated by ASO was degraded by XRN2. • The 3′ fragment of target mRNA generated by ASO was partially degraded by XRN2. • XRN1 depletion promoted accumulation of the 3′ fragment of mRNA generated by siRNA.« less

Assessment of Dengue virus helicase and methyltransferase as targets for fragment-based drug discovery.

PubMed

Coutard, Bruno; Decroly, Etienne; Li, Changqing; Sharff, Andrew; Lescar, Julien; Bricogne, Gérard; Barral, Karine

2014-06-01

Seasonal and pandemic flaviviruses continue to be leading global health concerns. With the view to help drug discovery against Dengue virus (DENV), a fragment-based experimental approach was applied to identify small molecule ligands targeting two main components of the flavivirus replication complex: the NS3 helicase (Hel) and the NS5 mRNA methyltransferase (MTase) domains. A library of 500 drug-like fragments was first screened by thermal-shift assay (TSA) leading to the identification of 36 and 32 fragment hits binding Hel and MTase from DENV, respectively. In a second stage, we set up a fragment-based X-ray crystallographic screening (FBS-X) in order to provide both validated fragment hits and structural binding information. No fragment hit was confirmed for DENV Hel. In contrast, a total of seven fragments were identified as DENV MTase binders and structures of MTase-fragment hit complexes were solved at resolution at least 2.0Å or better. All fragment hits identified contain either a five- or six-membered aromatic ring or both, and three novel binding sites were located on the MTase. To further characterize the fragment hits identified by TSA and FBS-X, we performed enzymatic assays to assess their inhibition effect on the N7- and 2'-O-MTase enzymatic activities: five of these fragment hits inhibit at least one of the two activities with IC50 ranging from 180μM to 9mM. This work validates the FBS-X strategy for identifying new anti-flaviviral hits targeting MTase, while Hel might not be an amenable target for fragment-based drug discovery (FBDD). This approach proved to be a fast and efficient screening method for FBDD target validation and discovery of starting hits for the development of higher affinity molecules that bind to novel allosteric sites. Copyright © 2014 Elsevier B.V. All rights reserved.

Diversity-Oriented Synthesis as a Strategy for Fragment Evolution against GSK3β.

PubMed

Wang, Yikai; Wach, Jean-Yves; Sheehan, Patrick; Zhong, Cheng; Zhan, Chenyang; Harris, Richard; Almo, Steven C; Bishop, Joshua; Haggarty, Stephen J; Ramek, Alexander; Berry, Kayla N; O'Herin, Conor; Koehler, Angela N; Hung, Alvin W; Young, Damian W

2016-09-08

Traditional fragment-based drug discovery (FBDD) relies heavily on structural analysis of the hits bound to their targets. Herein, we present a complementary approach based on diversity-oriented synthesis (DOS). A DOS-based fragment collection was able to produce initial hit compounds against the target GSK3β, allow the systematic synthesis of related fragment analogues to explore fragment-level structure-activity relationship, and finally lead to the synthesis of a more potent compound.

Ablation effects in oxygen-lead fragmentation at 2.1 GeV/nucleon

NASA Technical Reports Server (NTRS)

Townsend, L. W.

1984-01-01

The mechanism of particle evaporation was used to examine ablation effects in the fragmentation of 2.1 GeV/nucleon oxygen nuclei by lead targets. Following the initial abrasion process, the excited projectile prefragment is assumed to statistically decay in a manner analogous to that of a compound nucleus. The decay probabilities for the various particle emission channels are calculated by using the EVAP-4 Monte Carlo computer program. The input excitation energy spectrum for the prefragment is estimated from the geometric ""clean cut'' abrasion-ablation model. Isotope production cross sections are calculated and compared with experimental data and with the predictions from the standard geometric abrasion-ablation fragmentation model.

Fragment-Based Drug Design Facilitated by Protein-Templated Click Chemistry: Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin.

PubMed

Mondal, Milon; Unver, M Yagiz; Pal, Asish; Bakker, Matthijs; Berrier, Stephan P; Hirsch, Anna K H

2016-10-10

There is an urgent need for the development of efficient methodologies that accelerate drug discovery. We demonstrate that the strategic combination of fragment linking/optimization and protein-templated click chemistry is an efficient and powerful method that accelerates the hit-identification process for the aspartic protease endothiapepsin. The best binder, which inhibits endothiapepsin with an IC 50 value of 43 μm, represents the first example of triazole-based inhibitors of endothiapepsin. Our strategy could find application on a whole range of drug targets. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Comparison of fragments created by low- and hyper-velocity impacts

NASA Astrophysics Data System (ADS)

Hanada, T.; Liou, J.-C.

This paper summarizes two new satellite impact experiments. The objective of the experiments was to investigate the outcome of low- and hyper-velocity impacts on two identical target satellites. The first experiment was performed at a low-velocity of 1.5 km/s using a 40-g aluminum alloy sphere. The second experiment was performed at a hyper-velocity of 4.4 km/s using a 4-g aluminum alloy sphere. The target satellites were 15 cm × 15 cm × 15 cm in size and 800 g in mass. The ratios of impact energy to target mass for the two experiments were approximately the same. The target satellites were completely fragmented in both experiments, although there were some differences in the characteristics of the fragments. The projectile of the low-velocity impact experiment was partially fragmented while the projectile of the hyper-velocity impact experiment was completely fragmented beyond recognition. To date, approximately 1500 fragments from each impact experiment have been collected for detailed analysis. Each piece has been weighed, measured, and analyzed based on the analytic method used in the NASA Standard Breakup Model (2000 revision). These fragments account for about 95% of the target mass for both impact experiments. Preliminary analysis results will be presented in this paper.

Diversity-Oriented Synthesis as a Strategy for Fragment Evolution against GSK3β

PubMed Central

2016-01-01

Traditional fragment-based drug discovery (FBDD) relies heavily on structural analysis of the hits bound to their targets. Herein, we present a complementary approach based on diversity-oriented synthesis (DOS). A DOS-based fragment collection was able to produce initial hit compounds against the target GSK3β, allow the systematic synthesis of related fragment analogues to explore fragment-level structure–activity relationship, and finally lead to the synthesis of a more potent compound. PMID:27660690

Application of microtomography and image analysis to the quantification of fragmentation in ceramics after impact loading

NASA Astrophysics Data System (ADS)

Forquin, Pascal; Ando, Edward

2017-01-01

Silicon carbide ceramics are widely used in personal body armour and protective solutions. However, during impact, an intense fragmentation develops in the ceramic tile due to high-strain-rate tensile loadings. In this work, microtomography equipment was used to analyse the fragmentation patterns of two silicon carbide grades subjected to edge-on impact (EOI) tests. The EOI experiments were conducted in two configurations. The so-called open configuration relies on the use of an ultra-high-speed camera to visualize the fragmentation process with an interframe time set to 1 µs. The so-called sarcophagus configuration consists in confining the target in a metallic casing to avoid any dispersion of fragments. The target is infiltrated after impact so the final damage pattern is entirely scanned using X-ray tomography and a microfocus source. Thereafter, a three-dimensional (3D) segmentation algorithm was tested and applied in order to separate fragments in 3D allowing a particle size distribution to be obtained. Significant differences between the two specimens of different SiC grades were noted. To explain such experimental results, numerical simulations were conducted considering the Denoual-Forquin-Hild anisotropic damage model. According to the calculations, the difference of crack pattern in EOI tests is related to the population of defects within the two ceramics. This article is part of the themed issue 'Experimental testing and modelling of brittle materials at high strain rates'.

A simplified model for the assessment of the impact probability of fragments.

PubMed

Gubinelli, Gianfilippo; Zanelli, Severino; Cozzani, Valerio

2004-12-31

A model was developed for the assessment of fragment impact probability on a target vessel, following the collapse and fragmentation of a primary vessel due to internal pressure. The model provides the probability of impact of a fragment with defined shape, mass and initial velocity on a target of a known shape and at a given position with respect to the source point. The model is based on the ballistic analysis of the fragment trajectory and on the determination of impact probabilities by the analysis of initial direction of fragment flight. The model was validated using available literature data.

Neisseria Heparin Binding Antigen is targeted by the human alternative pathway C3-convertase

PubMed Central

Di Fede, Martina; Biagini, Massimiliano; Cartocci, Elena; Parillo, Carlo; Greco, Alessandra; Martinelli, Manuele; Marchi, Sara; Pezzicoli, Alfredo; Delany, Isabel

2018-01-01

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein specific for Neisseria and constitutes one of the three main protein antigens of the Bexsero vaccine. Meningococcal and human proteases, cleave NHBA protein upstream or downstream of a conserved Arg-rich region, respectively. The cleavage results in the release of the C-terminal portion of the protein. The C-terminal fragment originating from the processing of meningococcal proteases, referred to as C2 fragment, exerts a toxic effect on endothelial cells altering the endothelial permeability. In this work, we reported that recombinant C2 fragment has no influence on the integrity of human airway epithelial cell monolayers, consistent with previous findings showing that Neisseria meningitidis traverses the epithelial barrier without disrupting the junctional structures. We showed that epithelial cells constantly secrete proteases responsible for a rapid processing of C2 fragment, generating a new fragment that does not contain the Arg-rich region, a putative docking domain reported to be essential for C2-mediated toxic effect. Moreover, we found that the C3-convertase of the alternative complement pathway is one of the proteases responsible for this processing. Overall, our data provide new insights on the cleavage of NHBA protein during meningococcal infection. NHBA cleavage may occur at different stages of the infection, and it likely has a different role depending on the environment the bacterium is interacting with. PMID:29579105

A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification.

PubMed

Xiao, Kunyi; Liu, Juan; Chen, Hui; Zhang, Song; Kong, Jilie

2017-05-15

A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 10 5 cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Intermolecular Coulombic Decay (ICD) Occuring in Triatomic Molecular Dimer

NASA Astrophysics Data System (ADS)

Iskandar, Wael; Gatton, Averell; Gaire, Bishwanath; Champenois, Elio; Larsen, Kirk; Shivaram, Niranjan; Moradmand, Ali; Severt, Travis; Williams, Joshua; Slaughter, Daniel; Weber, Thorsten

2017-04-01

For over two decades, the production of ICD process has been extensively investigated theoretically and experimentally in different systems bounded by a week force (ex. van-der-Waals or Hydrogen force). Furthermore, the ICD process has been demonstrated a strong implication in biological system (DNA damage and DNA repair mechanism) because of the production of genotoxic low energy electrons during the decay cascade. Studying large complex system such as triatomic molecular dimer may be helpful for further exploration of ``Auger electron driven cancer therapy''. The present experiment investigates the dissociation dynamics happened in collision between a photons and CO2 dimer. We will focus more specifically on the CO2++CO2+ fragmentation channel and the detection in coincidence of the two ionic fragments and the two electrons will be done using a COld Target Recoil Ion Momentum Spectroscopy (COLTRIMS). The measurements of the Kinetic Energy Release of the two fragments and the relative angular distribution of the electrons in the molecular frame reveal that the ICD is the only mechanism responsible for the production of this fragmentation channel.

Collisional disruptions of rotating targets

NASA Astrophysics Data System (ADS)

Ševeček, Pavel; Broz, Miroslav

2017-10-01

Collisions are key processes in the evolution of the Main Asteroid Belt and impact events - i.e. target fragmentation and gravitational reaccumulation - are commonly studied by numerical simulations, namely by SPH and N-body methods. In our work, we extend the previous studies by assuming rotating targets and we study the dependence of resulting size-distributions on the pre-impact rotation of the target. To obtain stable initial conditions, it is also necessary to include the self-gravity already in the fragmentation phase which was previously neglected.To tackle this problem, we developed an SPH code, accelerated by SSE/AVX instruction sets and parallelized. The code solves the standard set of hydrodynamic equations, using the Tillotson equation of state, von Mises criterion for plastic yielding and scalar Grady-Kipp model for fragmentation. We further modified the velocity gradient by a correction tensor (Schäfer et al. 2007) to ensure a first-order conservation of the total angular momentum. As the intact target is a spherical body, its gravity can be approximated by a potential of a homogeneous sphere, making it easy to set up initial conditions. This is however infeasible for later stages of the disruption; to this point, we included the Barnes-Hut algorithm to compute the gravitational accelerations, using a multipole expansion of distant particles up to hexadecapole order.We tested the code carefully, comparing the results to our previous computations obtained with the SPH5 code (Benz and Asphaug 1994). Finally, we ran a set of simulations and we discuss the difference between the synthetic families created by rotating and static targets.

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

Avakian, Harut

Studies of the 3D structure of the nucleon encoded in Transverse Momentum Dependent distribution and fragmentation functions of partons and Generalized Parton Distributions are among the key objectives of the JLab 12 GeV upgrade and the Electron Ion Collider. Main challenges in extracting 3D partonic distributions from precision measurements of hard scattering processes include clear understanding of leading twist QCD fundamentals, higher twist effects, and also correlations of hadron production in target and current fragmentation regions. In this contribution we discuss some ongoing studies and future measurements of spin-orbit correlations at Jefferson Lab.

Target-projectile interaction during impact melting at Kamil Crater, Egypt

NASA Astrophysics Data System (ADS)

Fazio, Agnese; D'Orazio, Massimo; Cordier, Carole; Folco, Luigi

2016-05-01

In small meteorite impacts, the projectile may survive through fragmentation; in addition, it may melt, and chemically and physically interact with both shocked and melted target rocks. However, the mixing/mingling between projectile and target melts is a process still not completely understood. Kamil Crater (45 m in diameter; Egypt), generated by the hypervelocity impact of the Gebel Kamil Ni-rich ataxite on sandstone target, allows to study the target-projectile interaction in a simple and fresh geological setting. We conducted a petrographic and geochemical study of macroscopic impact melt lapilli and bombs ejected from the crater, which were collected during our geophysical campaign in February 2010. Two types of glasses constitute the impact melt lapilli and bombs: a white glass and a dark glass. The white glass is mostly made of SiO2 and it is devoid of inclusions. Its negligible Ni and Co contents suggest derivation from the target rocks without interaction with the projectile (<0.1 wt% of projectile contamination). The dark glass is a silicate melt with variable contents of Al2O3 (0.84-18.7 wt%), FeOT (1.83-61.5 wt%), and NiO (<0.01-10.2 wt%). The dark glass typically includes fragments (from few μm to several mm in size) of shocked sandstone, diaplectic glass, lechatelierite, and Ni-Fe metal blebs. The metal blebs are enriched in Ni compared to the Gebel Kamil meteorite. The dark glass is thus a mixture of target and projectile melts (11-12 wt% of projectile contamination). Based on recently proposed models for target-projectile interaction and for impact glass formation, we suggest a scenario for the glass formation at Kamil. During the transition from the contact and compression stage and the excavation stage, projectile and target liquids formed at their interface and chemically interact in a restricted zone. Projectile contamination affected only a shallow portion of the target rocks. The SiO2 melt that eventually solidified as white glass behaved as an immiscible liquid and did not interact with the projectile. During the excavation stage dark glass melt engulfed and coated the white glass melt, target fragments, and got stuck to iron meteorite shrapnel fragments. This model could also explain the common formation of white and dark glasses in small impact craters generated by iron bodies (e.g., Wabar).

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry.

PubMed

Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K H

2016-08-01

Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Fragment Screening and HIV Therapeutics

PubMed Central

Bauman, Joseph D.; Patel, Disha; Arnold, Eddy

2013-01-01

Fragment screening has proven to be a powerful alternative to traditional methods for drug discovery. Biophysical methods, such as X-ray crystallography, NMR spectroscopy, and surface plasmon resonance, are used to screen a diverse library of small molecule compounds. Although compounds identified via this approach have relatively weak affinity, they provide a good platform for lead development and are highly efficient binders with respect to their size. Fragment screening has been utilized for a wide-range of targets, including HIV-1 proteins. Here, we review the fragment screening studies targeting HIV-1 proteins using X-ray crystallography or surface plasmon resonance. These studies have successfully detected binding of novel fragments to either previously established or new sites on HIV-1 protease and reverse transcriptase. In addition, fragment screening against HIV-1 reverse transcriptase has been used as a tool to better understand the complex nature of ligand binding to a flexible target. PMID:21972022

Production of neutron-rich nuclei approaching r-process by gamma-induced fission of 238U at ELI-NP

NASA Astrophysics Data System (ADS)

Mei, Bo; Balabanski, Dimiter; Constantin, Paul; Anh Le, Tuan; Viet Cuong, Phan

2018-05-01

The investigation of neutron-rich exotic nuclei is crucial not only for nuclear physics but also for nuclear astrophysics. Experimentally, only few neutron-rich nuclei near the stability have been studied, however, most neutron-rich nuclei have not been measured due to their small production cross sections as well as short half-lives. At ELI-NP, gamma beams with high intensities will open new opportunities to investigate very neutron-rich fragments produced by photofission of 238U targets in a gas cell. Based on some simulations, a novel gas cell has been designed to produce, stop and extract 238U photofission fragments. The extraction time and efficiency of photofission fragments have been optimized by using SIMION simulations. According to these simulations, a high extraction efficiency and a short extraction time can be achieved for 238U photofission fragments in the gas cell, which will allow one to measure very neutron-rich fragments with short half-lives by using the IGISOL facility proposed at ELI-NP.

Screening a fragment cocktail library using ultrafiltration

PubMed Central

Shibata, Sayaka; Zhang, Zhongsheng; Korotkov, Konstantin V.; Delarosa, Jaclyn; Napuli, Alberto; Kelley, Angela M.; Mueller, Natasha; Ross, Jennifer; Zucker, Frank H.; Buckner, Frederick S.; Merritt, Ethan A.; Verlinde, Christophe L. M. J.; Van Voorhis, Wesley C.; Hol, Wim G. J.; Fan, Erkang

2011-01-01

Ultrafiltration provides a generic method to discover ligands for protein drug targets with millimolar to micromolar Kd, the typical range of fragment-based drug discovery. This method was tailored to a 96-well format, and cocktails of fragment-sized molecules, with molecular masses between 150 and 300 Da, were screened against medical structural genomics target proteins. The validity of the method was confirmed through competitive binding assays in the presence of ligands known to bind the target proteins. PMID:21750879

On moments of the multiplicity events of slow target fragments in relativistic Sulfur-ion collisions

NASA Astrophysics Data System (ADS)

Abdelsalam, A.; Kamel, S.; Rashed, N.; Sabry, N.

2014-07-01

A detailed study on the multiplicity characteristics of the slow target fragments emitted in relativistic heavy-ion collisions has been carried out at ELab = 3.7A and 200A GeV using 32S projectile. The beam energy dependence of the black particles produced in the full phase space of 32S-emulsion (32S-Em) interactions on the target size in terms of their moments (mean, variance, skewness and kurtosis) is investigated. The various order moments of target fragments emitted in the interactions of 32S beams with the heavy (AgBr) target nuclei are estimated in the forward (FHS) and backward (BHS) hemispheres. The investigated values of ratio of variance to mean at both energies show that the multiplicity distributions (MDs) are not Poissonian and the strongly correlated emission of target fragments are in the forward directions. The degree of anisotropic fragment emission and nature of correlation among the emitted fragments are investigated. The energy dependence of entropy is examined in both hemispheres. The entropy values normalized to average multiplicity are found to be energy independent. Scaling of MD of black particles produced in these interactions has been studied to verify the validity of scaling hypothesis via two scaling (Koba-Nielsen-Olesen (KNO)-scaling and Hegyi-scaling) functions. A simplified universal function has been used in each scaling to display the experimental data.

Effects of cryogenic temperature on dynamic fragmentation of laser shock-loaded metal foils

NASA Astrophysics Data System (ADS)

de Rességuier, T.; Lescoute, E.; Loison, D.; Chevalier, J. M.; Ducasse, F.

2011-12-01

Although shock-induced fracture and fragmentation of materials at low temperatures are issues of considerable interest for many applications, such as the protection from hypervelocity impacts in outer space or the ongoing development of high energy laser facilities aiming at inertial confinement fusion, little data can be found on the subject yet. In this paper, laser driven shock experiments are performed on gold and aluminum samples at both ambient and cryogenic (down to about 30 K) temperatures. Complementary techniques including transverse optical shadowgraphy, time-resolved velocity measurements, and post-recovery analyses are combined to assess the effects of target temperature upon the processes of microjetting, spallation, and dynamic punching, which are expected to govern fragments generation and ejection. The results indicate that cryogenic temperature tends to reduce the resistance to tensile and shear stresses, promotes brittle fracture, and leads to slightly higher fragments ejection velocities.

Electromagnetic Dissociation of Uranium in Heavy Ion Collisions at 120 Mev/a

NASA Astrophysics Data System (ADS)

Justice, Marvin Lealon

The heavy-ion induced electromagnetic dissociation (EMD) of a 120 MeV/A ^{238}U beam incident on five targets (^9Be, ^{27}Al, ^ {nat}Cu, ^{nat} Ag, and ^{nat}U) has been studied experimentally. Electromagnetic dissociation at this beam energy is essentially a two step process involving the excitation of a giant resonance followed by particle decay. At 120 MeV/A there is predicted to be a significant contribution (~25%) of the giant quadrupole resonance to the EMD cross sections. The specific exit channel which was looked at was projectile fission. The two fission fragments were detected in coincidence by an array of solid-state DeltaE-E detectors, allowing the charges of the fragments to be determined to within +/- .5 units. The events were sorted on the basis of the sums of the fragments' charges, acceptance corrections were applied, and total cross sections for the most peripheral events (i.e. those leading to charge sums of approximately 92) were determined. Electromagnetic fission at the beam energy of this experiment always leads to a true charge sum of 92. Due to the imperfect resolution of the detectors, charge sums of 91 and 93 were included in order to account for all of the electromagnetic fission events. The experimentally observed cross sections are due to nuclear interaction processes as well as electromagnetic processes. Under the conditions of this experiment, the cross sections for the beryllium target are almost entirely due to nuclear processes. The nuclear cross sections for the other four targets were determined by extrapolation from the beryllium data using a geometrical scaling model. After subtraction of the nuclear cross sections, the resulting electromagnetic cross sections are compared to theoretical calculations based on the equivalent photon approximation. Systematic uncertainties associated with the normalization of the data make quantitative comparisons with theory difficult, however. The systematic uncertainties are discussed and suggestions for improving the experiment are given.

The role of porosity and annealing in the impact fragmentation of an aluminum reactive material

NASA Astrophysics Data System (ADS)

Hooper, Joseph

2017-06-01

A reactive fragment has a unique structural requirement to survive explosive launch but then fragment catastrophically and combust upon impact. Suitable materials for this application tend to be metal composites with high ductility in compression but elastic-brittle behavior in tension. Characterizing the dynamic fragmentation of such materials is key for understanding their lethality. Here we consider a prototypical aluminum reactive frag material, formed via cold isostatic pressing of micron-scale powder followed by annealing. Samples were gun-launched into a target and recovered in a soft-catch medium of artificial snow, allowing for excellent recovery down to micron sizes and minimal contamination. Recovered fragment distributions were analyzed and compared to standard energy-balance theories. We study the effect of compaction pressure and annealing conditions on the fragmentation behavior at 500-800 m/s impacts, and find a particularly strong effect from short annealing periods. Though dynamic fracture occurs entirely along original particle boundaries in this material, recovery processes within the Al microstructure during annealing lead to a rapid decrease in the extent of fragmentation. This work was funded by the Office of Naval Research, program director Cliff Bedford.

Discovery of novel dengue virus NS5 methyltransferase non-nucleoside inhibitors by fragment-based drug design.

PubMed

Benmansour, Fatiha; Trist, Iuni; Coutard, Bruno; Decroly, Etienne; Querat, Gilles; Brancale, Andrea; Barral, Karine

2017-01-05

With the aim to help drug discovery against dengue virus (DENV), a fragment-based drug design approach was applied to identify ligands targeting a main component of DENV replication complex: the NS5 AdoMet-dependent mRNA methyltransferase (MTase) domain, playing an essential role in the RNA capping process. Herein, we describe the identification of new inhibitors developed using fragment-based, structure-guided linking and optimization techniques. Thermal-shift assay followed by a fragment-based X-ray crystallographic screening lead to the identification of three fragment hits binding DENV MTase. We considered linking two of them, which bind to proximal sites of the AdoMet binding pocket, in order to improve their potency. X-ray crystallographic structures and computational docking were used to guide the fragment linking, ultimately leading to novel series of non-nucleoside inhibitors of flavivirus MTase, respectively N-phenyl-[(phenylcarbamoyl)amino]benzene-1-sulfonamide and phenyl [(phenylcarbamoyl)amino]benzene-1-sulfonate derivatives, that show a 10-100-fold stronger inhibition of 2'-O-MTase activity compared to the initial fragments. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Effects of target fragmentation on evaluation of LET spectra from space radiations: implications for space radiation protection studies

NASA Technical Reports Server (NTRS)

Cucinotta, F. A.; Wilson, J. W.; Shinn, J. L.; Badavi, F. F.; Badhwar, G. D.

1996-01-01

We present calculations of linear energy transfer (LET) spectra in low earth orbit from galactic cosmic rays and trapped protons using the HZETRN/BRYNTRN computer code. The emphasis of our calculations is on the analysis of the effects of secondary nuclei produced through target fragmentation in the spacecraft shield or detectors. Recent improvements in the HZETRN/BRYNTRN radiation transport computer code are described. Calculations show that at large values of LET (> 100 keV/micrometer) the LET spectra seen in free space and low earth orbit (LEO) are dominated by target fragments and not the primary nuclei. Although the evaluation of microdosimetric spectra is not considered here, calculations of LET spectra support that the large lineal energy (y) events are dominated by the target fragments. Finally, we discuss the situation for interplanetary exposures to galactic cosmic rays and show that current radiation transport codes predict that in the region of high LET values the LET spectra at significant shield depths (> 10 g/cm2 of Al) is greatly modified by target fragments. These results suggest that studies of track structure and biological response of space radiation should place emphasis on short tracks of medium charge fragments produced in the human body by high energy protons and neutrons.

Fusion-fission and quasifission of superheavy systems with Z =110 -116 formed in 48Ca-induced reactions

NASA Astrophysics Data System (ADS)

Kozulin, E. M.; Knyazheva, G. N.; Itkis, I. M.; Itkis, M. G.; Bogachev, A. A.; Chernysheva, E. V.; Krupa, L.; Hanappe, F.; Dorvaux, O.; Stuttgé, L.; Trzaska, W. H.; Schmitt, C.; Chubarian, G.

2014-11-01

Background: In heavy-ion-induced reactions the mechanism leading to the formation of the compound nucleus and the role of quasifission is still not clear. Purpose: Investigation of the quasifission process of superheavy composite systems with Z =110 -116 and comparison with properties of fusion-fission and quasifission of lighter composite systems. Method: Mass and energy distributions of fissionlike fragments formed in the reactions 48Ca+232Th, 238U , 244Pu , and 248Cm at energies near the Coulomb barrier have been measured using the double-arm time-of-flight spectrometer CORSET at the U-400 cyclotron of the FLNR JINR. Results: The most probable fragment masses as well as total kinetic energies and their dispersions in dependence on the interaction energies and ion-target combinations have been studied for asymmetric and symmetric fragments formed in the reactions. The capture cross sections were obtained for the reactions 48Ca+244Pu and 248Cm . The lower limits for fission barriers of 283 -286Cn , 289 -292Fl , and 293 -296Lv compound nuclei were estimated. Conclusions: Analysis of the properties of symmetric fragments has shown that a significant part of these fragments may be attributed to fusion-fission process for the reactions 48Ca +238U , 244Pu , and 248Cm .

Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors.

PubMed

Thangavelu, Bharani; Bhansali, Pravin; Viola, Ronald E

2015-10-15

Aspartate-β-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the aspartate metabolic pathway which leads to the biosynthesis of several essential amino acids and some important metabolites. This pathway is crucial for many metabolic processes in plants and microbes like bacteria and fungi, but is absent in mammals. Therefore, the key microbial enzymes involved in this pathway are attractive potential targets for development of new antibiotics with novel modes of action. The ASADH enzyme family shares the same substrate binding and active site catalytic groups; however, the enzymes from representative bacterial and fungal species show different inhibition patterns when previously screened against low molecular weight inhibitors identified from fragment library screening. In the present study several approaches, including fragment based drug discovery (FBDD), inhibitor docking, kinetic, and structure-activity relationship (SAR) studies have been used to guide ASADH inhibitor development. Elaboration of a core structure identified by FBDD has led to the synthesis of low micromolar inhibitors of the target enzyme, with high selectivity introduced between the Gram-negative and Gram-positive orthologs of ASADH. This new set of structures open a novel direction for the development of inhibitors against this validated drug-target enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gene transfer of Hodgkin cell lines via multivalent anti-CD30 scFv displaying bacteriophage.

PubMed

Chung, Yoon-Suk A; Sabel, Katja; Krönke, Martin; Klimka, Alexander

2008-04-16

The display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. After uptake of the phage, their internal single-stranded DNA is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. This opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low. Here we demonstrate the display of an anti-CD30 single chain variable fragment fused to the major coat protein pVIII on the surface of bacteriophage. These phage particles showed an improved binding and transduction efficiency of CD30 positive Hodgkin-lymphoma cells, compared to bacteriophage with the anti-CD30 single chain variable fragment fused to the minor coat protein pIII. We can conclude from the results that the postulated multivalency of the anti-CD30-pVIII displaying bacteriophage combined with disseminated display of the anti-CD30 scFv on the whole particle surface is responsible for the improved gene transfer rate. These results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors.

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome

PubMed Central

ORBAN, TAMAS I.; IZAURRALDE, ELISA

2005-01-01

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21–22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5′ mRNA fragments generated by RISC cleavage are rapidly degraded from their 3′ ends by the exosome, whereas the 3′ fragments are degraded from their 5′ ends by XRN1. Exosome-mediated decay of the 5′ fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation. PMID:15703439

incaRNAfbinv: a web server for the fragment-based design of RNA sequences

PubMed Central

Drory Retwitzer, Matan; Reinharz, Vladimir; Ponty, Yann; Waldispühl, Jérôme; Barash, Danny

2016-01-01

Abstract In recent years, new methods for computational RNA design have been developed and applied to various problems in synthetic biology and nanotechnology. Lately, there is considerable interest in incorporating essential biological information when solving the inverse RNA folding problem. Correspondingly, RNAfbinv aims at including biologically meaningful constraints and is the only program to-date that performs a fragment-based design of RNA sequences. In doing so it allows the design of sequences that do not necessarily exactly fold into the target, as long as the overall coarse-grained tree graph shape is preserved. Augmented by the weighted sampling algorithm of incaRNAtion, our web server called incaRNAfbinv implements the method devised in RNAfbinv and offers an interactive environment for the inverse folding of RNA using a fragment-based design approach. It takes as input: a target RNA secondary structure; optional sequence and motif constraints; optional target minimum free energy, neutrality and GC content. In addition to the design of synthetic regulatory sequences, it can be used as a pre-processing step for the detection of novel natural occurring RNAs. The two complementary methodologies RNAfbinv and incaRNAtion are merged together and fully implemented in our web server incaRNAfbinv, available at http://www.cs.bgu.ac.il/incaRNAfbinv. PMID:27185893

Implicit proactive interference, age, and automatic versus controlled retrieval strategies.

PubMed

Ikier, Simay; Yang, Lixia; Hasher, Lynn

2008-05-01

We assessed the extent to which implicit proactive interference results from automatic versus controlled retrieval among younger and older adults. During a study phase, targets (e.g., "ALLERGY") either were or were not preceded by nontarget competitors (e.g., "ANALOGY"). After a filled interval, the participants were asked to complete word fragments, some of which cued studied words (e.g., "A_L_ _GY"). Retrieval strategies were identified by the difference in response speed between a phase containing fragments that cued only new words and a phase that included a mix of fragments cuing old and new words. Previous results were replicated: Proactive interference was found in implicit memory, and the negative effects were greater for older than for younger adults. Novel findings demonstrate two retrieval processes that contribute to interference: an automatic one that is age invariant and a controlled process that can reduce the magnitude of the automatic interference effects. The controlled process, however, is used effectively only by younger adults. This pattern of findings potentially explains age differences in susceptibility to proactive interference.

Fragment-based approaches to TB drugs.

PubMed

Marchetti, Chiara; Chan, Daniel S H; Coyne, Anthony G; Abell, Chris

2018-02-01

Tuberculosis is an infectious disease associated with significant mortality and morbidity worldwide, particularly in developing countries. The rise of antibiotic resistance in Mycobacterium tuberculosis (Mtb) urgently demands the development of new drug leads to tackle resistant strains. Fragment-based methods have recently emerged at the forefront of pharmaceutical development as a means to generate more effective lead structures, via the identification of fragment molecules that form weak but high quality interactions with the target biomolecule and subsequent fragment optimization. This review highlights a number of novel inhibitors of Mtb targets that have been developed through fragment-based approaches in recent years.

Secondary radiation measurements for particle therapy applications: nuclear fragmentation produced by 4He ion beams in a PMMA target.

PubMed

Marafini, M; Paramatti, R; Pinci, D; Battistoni, G; Collamati, F; De Lucia, E; Faccini, R; Frallicciardi, P M; Mancini-Terracciano, C; Mattei, I; Muraro, S; Piersanti, L; Rovituso, M; Rucinski, A; Russomando, A; Sarti, A; Sciubba, A; Solfaroli Camillocci, E; Toppi, M; Traini, G; Voena, C; Patera, V

2017-02-21

Nowadays there is a growing interest in particle therapy treatments exploiting light ion beams against tumors due to their enhanced relative biological effectiveness and high space selectivity. In particular promising results are obtained by the use of 4 He projectiles. Unlike the treatments performed using protons, the beam ions can undergo a fragmentation process when interacting with the atomic nuclei in the patient body. In this paper the results of measurements performed at the Heidelberg Ion-Beam Therapy center are reported. For the first time the absolute fluxes and the energy spectra of the fragments-protons, deuterons, and tritons-produced by 4 He ion beams of 102, 125 and 145 MeV u -1 energies on a poly-methyl methacrylate target were evaluated at different angles. The obtained results are particularly relevant in view of the necessary optimization and review of the treatment planning software being developed for clinical use of 4 He beams in clinical routine and the relative bench-marking of Monte Carlo algorithm predictions.

TRIC: an automated alignment strategy for reproducible protein quantification in targeted proteomics.

PubMed

Röst, Hannes L; Liu, Yansheng; D'Agostino, Giuseppe; Zanella, Matteo; Navarro, Pedro; Rosenberger, George; Collins, Ben C; Gillet, Ludovic; Testa, Giuseppe; Malmström, Lars; Aebersold, Ruedi

2016-09-01

Next-generation mass spectrometric (MS) techniques such as SWATH-MS have substantially increased the throughput and reproducibility of proteomic analysis, but ensuring consistent quantification of thousands of peptide analytes across multiple liquid chromatography-tandem MS (LC-MS/MS) runs remains a challenging and laborious manual process. To produce highly consistent and quantitatively accurate proteomics data matrices in an automated fashion, we developed TRIC (http://proteomics.ethz.ch/tric/), a software tool that utilizes fragment-ion data to perform cross-run alignment, consistent peak-picking and quantification for high-throughput targeted proteomics. TRIC reduced the identification error compared to a state-of-the-art SWATH-MS analysis without alignment by more than threefold at constant recall while correcting for highly nonlinear chromatographic effects. On a pulsed-SILAC experiment performed on human induced pluripotent stem cells, TRIC was able to automatically align and quantify thousands of light and heavy isotopic peak groups. Thus, TRIC fills a gap in the pipeline for automated analysis of massively parallel targeted proteomics data sets.

Cumulative Damage in Strength-Dominated Collisions of Rocky Asteroids: Rubble Piles and Brick Piles

NASA Technical Reports Server (NTRS)

Housen, Kevin

2009-01-01

Laboratory impact experiments were performed to investigate the conditions that produce large-scale damage in rock targets. Aluminum cylinders (6.3 mm diameter) impacted basalt cylinders (69 mm diameter) at speeds ranging from 0.7 to 2.0 km/s. Diagnostics included measurements of the largest fragment mass, velocities of the largest remnant and large fragments ejected from the periphery of the target, and X-ray computed tomography imaging to inspect some of the impacted targets for internal damage. Significant damage to the target occurred when the kinetic energy per unit target mass exceeded roughly 1/4 of the energy required for catastrophic shattering (where the target is reduced to one-half its original mass). Scaling laws based on a rate-dependent strength were developed that provide a basis for extrapolating the results to larger strength-dominated collisions. The threshold specific energy for widespread damage was found to scale with event size in the same manner as that for catastrophic shattering. Therefore, the factor of four difference between the two thresholds observed in the lab also applies to larger collisions. The scaling laws showed that for a sequence of collisions that are similar in that they produce the same ratio of largest fragment mass to original target mass, the fragment velocities decrease with increasing event size. As a result, rocky asteroids a couple hundred meters in diameter should retain their large ejecta fragments in a jumbled rubble-pile state. For somewhat larger bodies, the ejection velocities are sufficiently low that large fragments are essentially retained in place, possibly forming ordered "brick-pile" structures.

Application of Fragment Based Drug Discovery to Membrane Proteins: Biophysical Identification of Ligands of the Integral Membrane Enzyme DsbB

PubMed Central

Früh, Virginie; Zhou, Yunpeng; Chen, Dan; Loch, Caroline; Eiso, AB; Grinkova, Yelena N.; Verheij, Herman; Sligar, Stephen G; Bushweller, John H.; Siegal, Gregg

2014-01-01

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. PMID:20797617

Fragment-based protein-protein interaction antagonists of a viral dimeric protease

PubMed Central

Gable, Jonathan E.; Lee, Gregory M.; Acker, Timothy M.; Hulce, Kaitlin R.; Gonzalez, Eric R.; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J.; Craik, Charles S.

2016-01-01

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose response determination was performed as a confirmation screen and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed via NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80% of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogs. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. PMID:26822284

Identification of lipid- and protein-based binders in paintings by direct on-plate wet chemistry and matrix-assisted laser desorption ionization mass spectrometry.

PubMed

Calvano, Cosima Damiana; van der Werf, Inez Dorothé; Palmisano, Francesco; Sabbatini, Luigia

2015-01-01

Direct on-target plate processing of small (ca. 100 μg) fragments of paint samples for MALDI-MS identification of lipid- and protein-based binders is described. Fragments were fixed on a conventional stainless steel target plate by colloidal graphite followed by in situ fast tryptic digestion and matrix addition. The new protocol was first developed on paint replicas composed of chicken egg, collagen, and cow milk mixed with inorganic pigments and then successfully applied on historical paint samples taken from a fifteenth century Italian panel painting. The present work contributes a step forward in the simplification of binder identification in very small paint samples since no conventional solvent extraction is required, speeding up the whole sample preparation to 10 min and reducing lipid/protein loss.

DrugECs: An Ensemble System with Feature Subspaces for Accurate Drug-Target Interaction Prediction

PubMed Central

Jiang, Jinjian; Wang, Nian; Zhang, Jun

2017-01-01

Background Drug-target interaction is key in drug discovery, especially in the design of new lead compound. However, the work to find a new lead compound for a specific target is complicated and hard, and it always leads to many mistakes. Therefore computational techniques are commonly adopted in drug design, which can save time and costs to a significant extent. Results To address the issue, a new prediction system is proposed in this work to identify drug-target interaction. First, drug-target pairs are encoded with a fragment technique and the software “PaDEL-Descriptor.” The fragment technique is for encoding target proteins, which divides each protein sequence into several fragments in order and encodes each fragment with several physiochemical properties of amino acids. The software “PaDEL-Descriptor” creates encoding vectors for drug molecules. Second, the dataset of drug-target pairs is resampled and several overlapped subsets are obtained, which are then input into kNN (k-Nearest Neighbor) classifier to build an ensemble system. Conclusion Experimental results on the drug-target dataset showed that our method performs better and runs faster than the state-of-the-art predictors. PMID:28744468

β-Decay Study of the rp-Process Nucleus ^96Cd

NASA Astrophysics Data System (ADS)

Becerril, Ana; Amthor, A.; Baumann, T.; Bazin, D.; Crawford, H.; Estrade, A.; Gade, A.; Ginter, T.; Guess, C.; Hausmann, M.; Hitt, G.; Lorusso, G.; Mantica, P.; Matos, M.; Meharchand, R.; Minamisono, K.; Montes, F.; Pereira, J.; Perdikakis, G.; Pinter, J.; Portillo, M.; Schatz, H.; Smith, K.; Stoker, J.; Zegers, R.

2008-10-01

The half-life of ^96Cd, one of the major waiting points along the reaction path of the rp-process [1] has been measured at NSCL. Nuclei of interest were produced by fragmentation of a 120 MeV/u ^112Sn primary beam on a Be target and selected with the A1900 fragment separator in conjunction with the RF Fragment Separator [2]. The experimental setup, which consisted on the NSCL β-Counting System [3] and the Segmented Germanium Array [4], permitted the correlation of implants and decays as well as the detection of both prompt and β-delayed γ-rays from implanted ions. Details of the experiment and results will be presented and their implications discussed. [1] H. Schatz et al., Phys. Rep. 294, 167 1998 [2] D. Gorelov et al. PAC 2005, Knoxville, TN, May 16-20 [3] J. Prisciandaro et al., NIM A 505, 140 2003 [4] W. Mueller et al., NIM A 466, 492 2001

Site Identification by Ligand Competitive Saturation (SILCS) simulations for fragment-based drug design.

PubMed

Faller, Christina E; Raman, E Prabhu; MacKerell, Alexander D; Guvench, Olgun

2015-01-01

Fragment-based drug design (FBDD) involves screening low molecular weight molecules ("fragments") that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind nonoverlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy.The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is "soaked" in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called "FragMaps" can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine "Grid Free Energies (GFEs)," which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities.

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

PubMed Central

Mondal, Milon; Radeva, Nedyalka; Fanlo‐Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard

2016-01-01

Abstract Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization. PMID:27400756

Bioisosteric Replacements Extracted from High-Quality Structures in the Protein Databank.

PubMed

Seddon, Matthew P; Cosgrove, David A; Gillet, Valerie J

2018-03-20

Bioisosterism is an important concept in the lead optimisation phase of drug discovery where the aim is to make modifications to parts of a molecule in order to improve some properties while maintaining others. We present an analysis of bioisosteric fragments extracted from the ligands in an established data set consisting of 121 protein targets. A pairwise analysis is carried out of all ligands for a given target. The ligands are fragmented using the BRICS fragmentation scheme and a pair of fragments is deemed to be bioisosteric if they occupy a similar volume of the protein binding site. We consider two levels of generality, one which does not consider the number of attachment points in the fragments and a more restricted case in which both fragments are required to have the same number of attachments. We investigate the extent to which the bioisosteric pairs that are found are common across different target. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tetraplex PCR assay involving double gene-sites discriminates beef and buffalo in Malaysian meat curry and burger products.

PubMed

Hossain, M A Motalib; Ali, Md Eaqub; Hamid, Sharifah Bee Abd; Hossain, S M Azad; Asing; Nizar, Nina Naquiah Ahmad; Uddin, Mohammad Nasir; Ali, Lokman; Asaduzzaman, Md; Akanda, Md Jahurul Haque

2017-06-01

Replacement of beef by buffalo and vice versa is frequent in global markets, but their authentication is challenging in processed foods due to the fragmentation of most biomarkers including DNA. The shortening of target sequences through use of two target sites might ameliorate assay reliability because it is highly unlikely that both targets will be lost during food processing. For the first time, we report a tetraplex polymerase chain reaction (PCR) assay targeting two different DNA regions in beef (106 and 120-bp) and buffalo (90 and 138-bp) mitochondrial genes to discriminate beef and buffalo in processed foods. All targets were stable under boiling, autoclaving and microwave cooking conditions. A survey in Malaysian markets revealed 71% beef curries contained buffalo but there was no buffalo in beef burgers. The assay detected down to 0.01ng DNA and 1% meat in admixed and burger products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Target fragmentation in radiobiology

NASA Technical Reports Server (NTRS)

Wilson, John W.; Cucinotta, Francis A.; Shinn, Judy L.; Townsend, Lawrence W.

1993-01-01

Nuclear reactions in biological systems produce low-energy fragments of the target nuclei seen as local high events of linear energy transfer (LET). A nuclear-reaction formalism is used to evaluate the nuclear-induced fields within biosystems and their effects within several biological models. On the basis of direct ionization interaction, one anticipates high-energy protons to have a quality factor and relative biological effectiveness (RBE) of unity. Target fragmentation contributions raise the effective quality factor of 10 GeV protons to 3.3 in reasonable agreement with RBE values for induced micronuclei in bean sprouts. Application of the Katz model indicates that the relative increase in RBE with decreasing exposure observed in cell survival experiments with 160 MeV protons is related solely to target fragmentation events. Target fragment contributions to lens opacity given an RBE of 1.4 for 2 GeV protons in agreement with the work of Lett and Cox. Predictions are made for the effective RBE for Harderian gland tumors induced by high-energy protons. An exposure model for lifetime cancer risk is derived from NCRP 98 risk tables, and protraction effects are examined for proton and helium ion exposures. The implications of dose rate enhancement effects on space radiation protection are considered.

High voltage fragmentation of composites from secondary raw materials - Potential and limitations.

PubMed

Leißner, T; Hamann, D; Wuschke, L; Jäckel, H-G; Peuker, U A

2018-04-01

The comminution of composites for liberation of valuable components is a costly and energy-intensive process within the recycling of spent products. It therefore is continuously studied and optimized. In addition to conventional mechanical comminution innovative new principles for size reduction have been developed. One is the use of high voltage (HV) pulses, which is known to be a technology selectively liberating along phase boundaries. This technology offers the advantage of targeted liberation, preventing overgrinding of the material and thus improving the overall processing as well as product quality. In this study, the high voltage fragmentation of three different non-brittle composites (galvanized plastics, carbon fibre composites, electrode foils from Li-ion batteries) was investigated. The influence of pulse rate, number of pulses and filling level on the liberation and efficiency of comminution is discussed. Using the guideline VDI 2225 HV, fragmentation is compared to conventional mechanical comminution with respect to numerous criteria such as cost, throughput, energy consumption, availability and scalability. It was found that at current state of development, HV fragmentation cannot compete with mechanical comminution beyond laboratory scale. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mitochondrial fragmentation in excitotoxicity requires ROCK activation.

PubMed

Martorell-Riera, Alejandro; Segarra-Mondejar, Marc; Reina, Manuel; Martínez-Estrada, Ofelia M; Soriano, Francesc X

2015-01-01

Mitochondria morphology constantly changes through fission and fusion processes that regulate mitochondrial function, and it therefore plays a prominent role in cellular homeostasis. Cell death progression is associated with mitochondrial fission. Fission is mediated by the mainly cytoplasmic Drp1, which is activated by different post-translational modifications and recruited to mitochondria to perform its function. Our research and other studies have shown that in the early moments of excitotoxic insult Drp1 must be nitrosylated to mediate mitochondrial fragmentation in neurons. Nonetheless, mitochondrial fission is a multistep process in which filamentous actin assembly/disassembly and myosin-mediated mitochondrial constriction play prominent roles. Here we establish that in addition to nitric oxide production, excitotoxicity-induced mitochondrial fragmentation also requires activation of the actomyosin regulator ROCK. Although ROCK1 has been shown to phosphorylate and activate Drp1, experiments using phosphor-mutant forms of Drp1 in primary cortical neurons indicate that in excitotoxic conditions, ROCK does not act directly on Drp1 to mediate fission, but may act on the actomyosin complex. Thus, these data indicate that a wider range of signaling pathways than those that target Drp1 are amenable to be inhibited to prevent mitochondrial fragmentation as therapeutic option.

Computational Hydrocode Study of Target Damage due to Fragment-Blast Impact

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

Hatch-Aguilar, T; Najjar, F; Szymanski, E

2011-03-24

A target's terminal ballistic effects involving explosively generated fragments, along with the original blast, are of critical importance for many different security and safety related applications. Personnel safety and protective building design are but a few of the practical disciplines that can gain from improved understanding combined loading effects. Traditionally, any engineering level analysis or design effort involving explosions would divide the target damage analysis into two correspondingly critical areas: blast wave and fragment related impact effects. The hypothesis of this paper lies in the supposition that a linear combination of a blast-fragment loading, coupled with an accurate target responsemore » description, can lead to a non-linear target damage effect. This non-linear target response could then stand as the basis of defining what a synergistic or combined frag-blast loading might actually look like. The table below, taken from Walters, et. al. categorizes some of the critical parameters driving any combined target damage effect and drives the evaluation of results. Based on table 1 it becomes clear that any combined frag-blast analysis would need to account for the target response matching similar ranges for the mechanics described above. Of interest are the critical times upon which a blast event or fragment impact loading occurs relative to the target's modal response. A blast, for the purposes of this paper is defined as the sudden release of chemical energy from a given material (henceforth referred to as an energetic material) onto its surrounding medium. During the coupling mechanism a discrete or discontinuous shockwave is generated. This shockwave travels outward from the source transferring energy and momentum to any surrounding objects including personnel and engineering structures. From an engineering perspective blast effects are typically characterized by way of physical characteristics such as Peak Pressure (PP), Time of Arrival (TOA), Pressure-Impulse (PI) and Time of Duration (TD). Other peculiarities include the radial decrease in pressure from the source, any fireball size measurement, and subsequent increase in temperature from the passing of the shockwave through the surrounding medium. In light of all of these metrics, the loading any object receives from a blast event becomes intricately connected to the distance between itself and the source. Because of this, a clear distinction is made between close-in effects and those from a source far away from the object of interest. Explosively generated fragments on the other hand are characterized by means of their localized damage potential. Metrics such as whether the fragment penetrates or perforates a given object is quantified as well as other variables including fragment's residual velocity, % kinetic energy decrease, residual fragment mass and other exit criteria. A fragment launched under such violent conditions could easily be traveling at speeds in excess of 2500 ft/s. Given these speeds it is conceivable to imagine how any given fragment could deliver a concentrated load to a target and penetrates through walls, vehicles or even the protection systems of nearby personnel. This study will focus on the individual fragment-target impact event with the hopes of expanding it to eventually include statistical procedures. Since this is a modeling excursion into the combined frag-blast target damage effects the numerical methods used to frame this problem become important in-so-far as the simulations are done in a consistent manner. For this study a Finite-Element based Hydrocode solution called ALE3D (ALE=Arbitrary Lagrangian-Eulerian) was utilized. ALE3D is developed by Lawrence Livermore National Laboratory (Livermore, CA), and as this paper will show, successfully implemented a converged ALE formulation including as many of the different aspects needed to query the synergistic damage on a given target. Further information on the modeling setup is included.« less

Fragment-Based Phenotypic Lead Discovery: Cell-Based Assay to Target Leishmaniasis.

PubMed

Ayotte, Yann; Bilodeau, François; Descoteaux, Albert; LaPlante, Steven R

2018-05-02

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment-based phenotypic lead discovery (FPLD) combines aspects of traditional fragment-based lead discovery (FBLD), which involves the screening of small-molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug-like compounds in cell-based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low-molecular-weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone-marrow-derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The multiple roles of computational chemistry in fragment-based drug design

NASA Astrophysics Data System (ADS)

Law, Richard; Barker, Oliver; Barker, John J.; Hesterkamp, Thomas; Godemann, Robert; Andersen, Ole; Fryatt, Tara; Courtney, Steve; Hallett, Dave; Whittaker, Mark

2009-08-01

Fragment-based drug discovery (FBDD) represents a change in strategy from the screening of molecules with higher molecular weights and physical properties more akin to fully drug-like compounds, to the screening of smaller, less complex molecules. This is because it has been recognised that fragment hit molecules can be efficiently grown and optimised into leads, particularly after the binding mode to the target protein has been first determined by 3D structural elucidation, e.g. by NMR or X-ray crystallography. Several studies have shown that medicinal chemistry optimisation of an already drug-like hit or lead compound can result in a final compound with too high molecular weight and lipophilicity. The evolution of a lower molecular weight fragment hit therefore represents an attractive alternative approach to optimisation as it allows better control of compound properties. Computational chemistry can play an important role both prior to a fragment screen, in producing a target focussed fragment library, and post-screening in the evolution of a drug-like molecule from a fragment hit, both with and without the available fragment-target co-complex structure. We will review many of the current developments in the area and illustrate with some recent examples from successful FBDD discovery projects that we have conducted.

Impact experiments of porous gypsum-glass bead mixtures simulating parent bodies of ordinary chondrites: Implications for re-accumulation processes related to rubble-pile formation

NASA Astrophysics Data System (ADS)

Yasui, M.; Arakawa, M.

2011-12-01

Most of asteroids are expected to be impact fragments produced by collisions among planetesimals or rubble-pile bodies produced by re-accumulation of fragments. In order to study the formation processes of asteroids, it is necessary to examine the collisional disruption and re-accumulation conditions of planetesimals. Most of meteorites recovered on the Earth are ordinary chondrites (OCs). The OCs have the components of millimeter-sized round grains (chondrules) and submicron-sized dusts (matrix). So, the planetesimals forming the parent bodies of OCs could be mainly composed of chondrules and matrix. Therefore, we conducted impact experiments with porous gypsum mixed with glass beads having the spherical shape with various diameters simulating chondrules, and examined the effect of chondrules on the ejecta velocity and the impact strength. The targets included glass beads with a diameter ranging from 100 μm to 3 mm and the volume fraction was 0.6, similar to that of ordinary chondrites, which is about 0.65-0.75. We also prepared the porous gypsum sample without glass bead to examine the effect of volume fraction. Nylon projectiles with the diameters of 10 mm and 2 mm were impacted at 60-180 m/s by a single-stage gas gun and at about 4 km/s by a two-stage light gas gun, respectively. After the shot, we measured the mass of the recovered fragments to calculate the impact strength Q defined by Q=mpVi^2/2(mp+Mt), where Vi is the impact velocity, and mp and Mt are the mass of projectile and target, respectively. The collisional disruption of the target was observed by a high-speed video camera to measure the ejecta velocity. The antipodal velocity Va increased with the increase of Q, irrespective of glass bead size and volume fraction. However, the Va for low-velocity collisions at 60-180 m/s was an order magnitude larger than that for high-velocity collisions at 4 km/s. The velocities of fragments ejected from two corners on the impact surface of the target Vc-g measured in the center of the mass system, were independent on the target materials. The impact strength of the mixture target was found to range from 56 to 116 J/kg depending on the glass bead size, and was several times smaller than that of the gypsum target, 446 J/kg in low-velocity collisions. The impact strengths of the 100 μm bead target and the gypsum target strongly depended on the impact velocity: those obtained in high-velocity collisions were several times greater than those obtained in low-velocity collisions. The obtained results of Vc-g were compared to the escape velocity of chondrule-including planetesimals (CiPs) to study the conditions for the formation of rubble-pile bodies after the catastrophic disruption. The fragments of CiPs for catastrophic disruption could be re-accumulated at the radius of a body larger than 3 km, irrespective of chondrule size included in the CiPs, which is rather smaller than that for basalt bodies. Thus, we suggested that there were more parent bodies of OCs having a rubble-pile structure.

Fragment-Based Protein-Protein Interaction Antagonists of a Viral Dimeric Protease.

PubMed

Gable, Jonathan E; Lee, Gregory M; Acker, Timothy M; Hulce, Kaitlin R; Gonzalez, Eric R; Schweigler, Patrick; Melkko, Samu; Farady, Christopher J; Craik, Charles S

2016-04-19

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80 % of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Ligand-observed Mass Spectrometry Approach Integrated into the Fragment Based Lead Discovery Pipeline

PubMed Central

Chen, Xin; Qin, Shanshan; Chen, Shuai; Li, Jinlong; Li, Lixin; Wang, Zhongling; Wang, Quan; Lin, Jianping; Yang, Cheng; Shui, Wenqing

2015-01-01

In fragment-based lead discovery (FBLD), a cascade combining multiple orthogonal technologies is required for reliable detection and characterization of fragment binding to the target. Given the limitations of the mainstream screening techniques, we presented a ligand-observed mass spectrometry approach to expand the toolkits and increase the flexibility of building a FBLD pipeline especially for tough targets. In this study, this approach was integrated into a FBLD program targeting the HCV RNA polymerase NS5B. Our ligand-observed mass spectrometry analysis resulted in the discovery of 10 hits from a 384-member fragment library through two independent screens of complex cocktails and a follow-up validation assay. Moreover, this MS-based approach enabled quantitative measurement of weak binding affinities of fragments which was in general consistent with SPR analysis. Five out of the ten hits were then successfully translated to X-ray structures of fragment-bound complexes to lay a foundation for structure-based inhibitor design. With distinctive strengths in terms of high capacity and speed, minimal method development, easy sample preparation, low material consumption and quantitative capability, this MS-based assay is anticipated to be a valuable addition to the repertoire of current fragment screening techniques. PMID:25666181

Software Exploit Prevention and Remediation via Software Memory Protection

DTIC Science & Technology

2009-05-01

trampolines that are necessary. Trampolines are pieces of code emitted into the fragment cache to transfer con- trol back to Strata. Most control...transfer instructions (CTIs) are initially linked to trampolines (unless the transfer target already exists in the fragment cache). Once a CTI’s target...instruction becomes available in the fragment cache, the CTI is linked directly to the destination, avoiding future uses of the trampoline . This

Fragmentation Cross Sections of 290 and 400 MeV/nucleon 12C Beamson Elemental Targets

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

Zeitlin, C.; Guetersloh, S.; Heilbronn, L.

Charge-changing and fragment production cross sections at 0circ have been obtained for interactions of 290 MeV/nucleon and 400MeV/nucleon carbon beams with C, CH2, Al, Cu, Sn, and Pb targets. Thesebeams are relevant to cancer therapy, space radiation, and the productionof radioactive beams. We compare to previously published results using Cand CH2 targets at similar beam energies. Due to ambiguities arising fromthe presence of multiple fragments on many events, previous publicationshave reported only cross sections for B and Be fragments. In this work wehave extracted cross sections for all fragment species, using dataobtained at three distinct values of angular acceptance, supplementedmore » bydata taken with the detector stack placed off the beam axis. A simulationof the experiment with the PHITS Monte Carlo code shows fair agreementwith the data obtained with the large acceptance detectors, but agreementis poor at small acceptance. The measured cross sections are alsocompared to the predictions of the one-dimensional cross section modelsEPAX2 and NUCFRG2; the latter is presently used in NASA's space radiationtransport calculations. Though PHITS and NUCFRG2 reproduce thecharge-changing cross sections with reasonable accuracy, none of themodels is able to accurately predict the fragment cross sections for allfragment species and target materials.« less

Extension of the BRYNTRN code to monoenergetic light ion beams

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Wilson, John W.; Badavi, Francis F.

1994-01-01

A monoenergetic version of the BRYNTRN transport code is extended to beam transport of light ions (H-2, H-3, He-3, and He-4) in shielding materials (thick targets). The redistribution of energy in nuclear reactions is included in transport solutions that use nuclear fragmentation models. We also consider an equilibrium target-fragment spectrum for nuclei with mass number greater than four to include target fragmentation effects in the linear energy transfer (LET) spectrum. Illustrative results for water and aluminum shielding, including energy and LET spectra, are discussed for high-energy beams of H-2 and He-4.

Apparatus and method for producing fragment-free openings

DOEpatents

Cherry, Christopher R.

2001-01-01

An apparatus and method for explosively penetrating hardened containers such as steel drums without producing metal fragmentation is disclosed. The apparatus can be used singularly or in combination with water disrupters and other disablement tools. The apparatus is mounted in close proximity to the target and features a main sheet explosive that is initiated at least three equidistant points along the sheet's periphery. A buffer material is placed between the sheet explosive and the target. As a result, the metallic fragments generated from the detonation of the detonator are attenuated so that no fragments from the detonator are transferred to the target. As a result, an opening can be created in containers such as steel drums through which access to the IED is obtained to defuse it with projectiles or fluids.

Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response▿

PubMed Central

Glowacka, Ilona; Bertram, Stephanie; Müller, Marcel A.; Allen, Paul; Soilleux, Elizabeth; Pfefferle, Susanne; Steffen, Imke; Tsegaye, Theodros Solomon; He, Yuxian; Gnirss, Kerstin; Niemeyer, Daniela; Schneider, Heike; Drosten, Christian; Pöhlmann, Stefan

2011-01-01

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion. PMID:21325420

One-step assembly and targeted integration of multigene constructs assisted by the I-SceI meganuclease in Saccharomyces cerevisiae

PubMed Central

Kuijpers, Niels GA; Chroumpi, Soultana; Vos, Tim; Solis-Escalante, Daniel; Bosman, Lizanne; Pronk, Jack T; Daran, Jean-Marc; Daran-Lapujade, Pascale

2013-01-01

In vivo assembly of overlapping fragments by homologous recombination in Saccharomyces cerevisiae is a powerful method to engineer large DNA constructs. Whereas most in vivo assembly methods reported to date result in circular vectors, stable integrated constructs are often preferred for metabolic engineering as they are required for large-scale industrial application. The present study explores the potential of combining in vivo assembly of large, multigene expression constructs with their targeted chromosomal integration in S. cerevisiae. Combined assembly and targeted integration of a ten-fragment 22-kb construct to a single chromosomal locus was successfully achieved in a single transformation process, but with low efficiency (5% of the analyzed transformants contained the correctly assembled construct). The meganuclease I-SceI was therefore used to introduce a double-strand break at the targeted chromosomal locus, thus to facilitate integration of the assembled construct. I-SceI-assisted integration dramatically increased the efficiency of assembly and integration of the same construct to 95%. This study paves the way for the fast, efficient, and stable integration of large DNA constructs in S. cerevisiae chromosomes. PMID:24028550

Laboratory experiments on the impact disruption of iron meteorites at temperature of near-Earth space

NASA Astrophysics Data System (ADS)

Katsura, Takekuni; Nakamura, Akiko M.; Takabe, Ayana; Okamoto, Takaya; Sangen, Kazuyoshi; Hasegawa, Sunao; Liu, Xun; Mashimo, Tsutomu

2014-10-01

Iron meteorites and some M-class asteroids are generally understood to be fragments that were originally part of cores of differentiated planetesimals or part of local melt pools on primitive bodies. The parent bodies of iron meteorites may have formed in the terrestrial planet region, from which they were then scattered into the main belt (Bottke, W.F., Nesvorný, D., Grimm, R.E., Morbidelli, A., O'Brien, D.P. [2006]. Nature 439, 821-824). Therefore, a wide range of collisional events at different mass scales, temperatures, and impact velocities would have occurred between the time when the iron was segregated and the impact that eventually exposed the iron meteorites to interplanetary space. In this study, we performed impact disruption experiments of iron meteorite specimens as projectiles or targets at room temperature to increase understanding of the disruption process of iron bodies in near-Earth space. Our iron specimens (as projectiles or targets) were almost all smaller in size than their counterparts (as targets or projectiles, respectively). Experiments of impacts of steel specimens were also conducted for comparison. The fragment mass distribution of the iron material was different from that of rocks. In the iron fragmentation, a higher percentage of the mass was concentrated in larger fragments, probably due to the ductile nature of the material at room temperature. The largest fragment mass fraction f was dependent not only on the energy density but also on the size d of the specimen. We assumed a power-law dependence of the largest fragment mass fraction to initial peak pressure P0 normalized by a dynamic strength, Y, which was defined to be dependent on the size of the iron material. A least squares fit to the data of iron meteorite specimens resulted in the following relationship: f∝∝d, indicating a large size dependence of f. Additionally, the deformation of the iron materials in high-velocity shots was found to be most significant when the initial pressure greatly exceeded the dynamic strength of the material.

Microorganism mediated liquid fuels

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

Troiano, Richard

Herein disclosed is a method for producing liquid hydrocarbon product, the method comprising disintegrating a hydrocarbon source; pretreating the disintegrated hydrocarbon source; solubilizing the disintegrated hydrocarbon source to form a slurry comprising a reactant molecule of the hydrocarbon source; admixing a biochemical liquor into the slurry, wherein the biochemical liquor comprises at least one conversion enzyme configured to facilitate bond selective photo-fragmentation of said reactant molecule of the hydrocarbon source, to form liquid hydrocarbons via enzyme assisted bond selective photo-fragmentation, wherein said conversion enzyme comprises reactive sites configured to restrict said reactant molecule such that photo-fragmentation favorably targets a preselectedmore » internal bond of said reactant molecule; separating the liquid hydrocarbons from the slurry, wherein contaminants remain in the slurry; and enriching the liquid hydrocarbons to form a liquid hydrocarbon product. Various aspects of such method/process are also discussed.« less

Study of the peculiarities of multiparticle production via event-by-event analysis in asymmetric nucleus-nucleus interactions

NASA Astrophysics Data System (ADS)

Fedosimova, Anastasiya; Gaitinov, Adigam; Grushevskaya, Ekaterina; Lebedev, Igor

2017-06-01

In this work the study on the peculiarities of multiparticle production in interactions of asymmetric nuclei to search for unusual features of such interactions, is performed. A research of long-range and short-range multiparticle correlations in the pseudorapidity distribution of secondary particles on the basis of analysis of individual interactions of nuclei of 197 Au at energy 10.7 AGeV with photoemulsion nuclei, is carried out. Events with long-range multiparticle correlations (LC), short-range multiparticle correlations (SC) and mixed type (MT) in pseudorapidity distribution of secondary particles, are selected by the Hurst method in accordance with Hurst curve behavior. These types have significantly different characteristics. At first, they have different fragmentation parameters. Events of LC type are processes of full destruction of the projectile nucleus, in which multicharge fragments are absent. In events of mixed type several multicharge fragments of projectile nucleus are discovered. Secondly, these two types have significantly different multiplicity distribution. The mean multiplicity of LC type events is significantly more than in mixed type events. On the basis of research of the dependence of multiplicity versus target-nuclei fragments number for events of various types it is revealed, that the most considerable multiparticle correlations are observed in interactions of the mixed type, which correspond to the central collisions of gold nuclei and nuclei of CNO-group, i.e. nuclei with strongly asymmetric volume, nuclear mass, charge, etc. Such events are characterised by full destruction of the target-nucleus and the disintegration of the projectile-nucleus on several multi-charged fragments.

A distributed computational search strategy for the identification of diagnostics targets: application to finding aptamer targets for methicillin-resistant staphylococci.

PubMed

Flanagan, Keith; Cockell, Simon; Harwood, Colin; Hallinan, Jennifer; Nakjang, Sirintra; Lawry, Beth; Wipat, Anil

2014-06-30

The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

Laser Subdivision of the Genesis Concentrator Target Sample 60000

NASA Technical Reports Server (NTRS)

Lauer, Howard V., Jr.; Burkett, P. J.; Rodriquez, M. C.; Nakamura-Messenger, K.; Clemett, S. J.; Gonzales, C. P.; Allton, J. H.; McNamara, K. M.; See, T. H.

2013-01-01

The Genesis Allocation Committee received a request for 1 square centimeter of the diamond-like-carbon (DLC) concentrator target for the analysis of solar wind nitrogen isotopes. The target consists of a single crystal float zone (FZ) silicon substrate having a thickness on the order of 550 micrometers with a 1.5-3.0 micrometer-thick coating of DLC on the exposed surface. The solar wind is implanted shallowly in the front side DLC. The original target was a circular quadrant with a radius of 3.1 cm; however, the piece did not survive intact when the spacecraft suffered an anomalous landing upon returning to Earth on September 8, 2004. An estimated 75% of the DLC target was recovered in at least 18 fragments. The largest fragment, Genesis sample 60000, has been designated for this allocation and is the first sample to be subdivided using our laser scribing system Laser subdivision has associated risks including thermal diffusion of the implant if heating occurs and unintended breakage during cleavage. A careful detailed study and considerable subdividing practice using non-flight FZ diamond on silicon, DOS, wafers has considerably reduced the risk of unplanned breakage during the cleaving process. In addition, backside scribing reduces the risk of possible thermal excursions affecting the implanted solar wind, implanted shallowly in the front side DLC.

A distributed computational search strategy for the identification of diagnostics targets: Application to finding aptamer targets for methicillin-resistant staphylococci.

PubMed

Flanagan, Keith; Cockell, Simon; Harwood, Colin; Hallinan, Jennifer; Nakjang, Sirintra; Lawry, Beth; Wipat, Anil

2014-06-01

The rapid and cost-effective identification of bacterial species is crucial, especially for clinical diagnosis and treatment. Peptide aptamers have been shown to be valuable for use as a component of novel, direct detection methods. These small peptides have a number of advantages over antibodies, including greater specificity and longer shelf life. These properties facilitate their use as the detector components of biosensor devices. However, the identification of suitable aptamer targets for particular groups of organisms is challenging. We present a semi-automated processing pipeline for the identification of candidate aptamer targets from whole bacterial genome sequences. The pipeline can be configured to search for protein sequence fragments that uniquely identify a set of strains of interest. The system is also capable of identifying additional organisms that may be of interest due to their possession of protein fragments in common with the initial set. Through the use of Cloud computing technology and distributed databases, our system is capable of scaling with the rapidly growing genome repositories, and consequently of keeping the resulting data sets up-to-date. The system described is also more generically applicable to the discovery of specific targets for other diagnostic approaches such as DNA probes, PCR primers and antibodies.

Mechanistic studies on a sequential PDT protocol

NASA Astrophysics Data System (ADS)

Kessel, David

2016-03-01

A low (~LD15) PDT dose resulting in selective lysosomal photodamage can markedly promote photokilling by subsequent photodamage targeted to mitochondria. Experimental data are consistent with the proposal that cleavage of the autophagyassociated protein ATG5 to a pro-apoptotic fragment is responsible for this effect. This process is known to be dependent on the proteolytic activity of calpain. We have proposed that Ca2+ released from photodamaged lysosomes is the trigger for ATG5 cleavage. We can now document the conversion of ATG5 to the truncated form after lysosomal photodamage. Photofrin, a photosensitizer that targets both mitochondria and lysosomes, can be used for either phase of the sequential PDT process. The ability of Photofrin to target both loci may explain the well-documented efficacy of this agent.

Fragmentation of relativistic nuclei in peripheral interactions in nuclear track emulsion

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

Artemenkov, D. A., E-mail: artemenkov@lhe.jinr.ru; Bradnova, V.; Chernyavsky, M. M.

2008-09-15

The technique of nuclear track emulsions is used to explore the fragmentation of light relativistic nuclei down to the most peripheral interactions: nuclear 'white' stars. A complete pattern of the relativistic dissociation of a 8B nucleus with target fragment accompaniment is presented. Relativistic dissociation {sup 9}Be {yields} 2{alpha} is explored using significant statistics, and a relative contribution of {sup 8}Be decays from 0+ and 2+ states is established. Target fragment accompaniments are shown for relativistic fragmentation {sup 14}N {yields} 3He +H and {sup 22}Ne {yields} 5He. The leading role of the electromagnetic dissociation on heavy nuclei with respect to breakupsmore » on target protons is demonstrated in all these cases. It is possible to conclude that the peripheral dissociation of relativistic nuclei in nuclear track emulsion is a unique tool to study many-body systems composed of the lightest nuclei and nucleons in the energy scale relevant for nuclear astrophysics.« less

SHOP: a method for structure-based fragment and scaffold hopping.

PubMed

Fontaine, Fabien; Cross, Simon; Plasencia, Guillem; Pastor, Manuel; Zamora, Ismael

2009-03-01

A new method for fragment and scaffold replacement is presented that generates new families of compounds with biological activity, using GRID molecular interaction fields (MIFs) and the crystal structure of the targets. In contrast to virtual screening strategies, this methodology aims only to replace a fragment of the original molecule, maintaining the other structural elements that are known or suspected to have a critical role in ligand binding. First, we report a validation of the method, recovering up to 95% of the original fragments searched among the top-five proposed solutions, using 164 fragment queries from 11 diverse targets. Second, six key customizable parameters are investigated, concluding that filtering the receptor MIF using the co-crystallized ligand atom type has the greatest impact on the ranking of the proposed solutions. Finally, 11 examples using more realistic scenarios have been performed; diverse chemotypes are returned, including some that are similar to compounds that are known to bind to similar targets.

The ways and means of fragment-based drug design.

PubMed

Doak, Bradley C; Norton, Raymond S; Scanlon, Martin J

2016-11-01

Fragment-based drug design (FBDD) has emerged as a mainstream approach for the rapid and efficient identification of building blocks that can be used to develop high-affinity ligands against protein targets. One of the strengths of FBDD is the relative ease and low cost of the primary screen to identify fragments that bind. However, the fragments that emerge from primary screens often have low affinities, with K D values in the high μM to mM range, and a significant challenge for FBDD is to develop the initial fragments into more potent ligands. Successful fragment elaboration often requires co-structures of the fragments bound to their target proteins, as well as a range of biophysical and biochemical assays to track potency and efficacy. These challenges have led to the development of specific chemical strategies for the elaboration of weakly-binding fragments into more potent "hits" and lead compounds. In this article we review different approaches that have been employed to meet these challenges and describe some of the strategies that have resulted in several fragment-derived compounds entering clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

Is Semantic Priming (Ir)rational? Insights from the Speeded Word Fragment Completion Task

ERIC Educational Resources Information Center

Heyman, Tom; Hutchison, Keith A.; Storms, Gert

2016-01-01

Semantic priming, the phenomenon that a target is recognized faster if it is preceded by a semantically related prime, is a well-established effect. However, the mechanisms producing semantic priming are subject of debate. Several theories assume that the underlying processes are controllable and tuned to prime utility. In contrast, purely…

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

PubMed

Blaazer, Antoni R; Orrling, Kristina M; Shanmugham, Anitha; Jansen, Chimed; Maes, Louis; Edink, Ewald; Sterk, Geert Jan; Siderius, Marco; England, Paul; Bailey, David; de Esch, Iwan J P; Leurs, Rob

2015-01-01

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization. © 2014 Society for Laboratory Automation and Screening.

Targeting of a chlamydial protease impedes intracellular bacterial growth.

PubMed

Christian, Jan G; Heymann, Julia; Paschen, Stefan A; Vier, Juliane; Schauenburg, Linda; Rupp, Jan; Meyer, Thomas F; Häcker, Georg; Heuer, Dagmar

2011-09-01

Chlamydiae are obligate intracellular bacteria that propagate in a cytosolic vacuole. Recent work has shown that growth of Chlamydia induces the fragmentation of the Golgi apparatus (GA) into ministacks, which facilitates the acquisition of host lipids into the growing inclusion. GA fragmentation results from infection-associated cleavage of the integral GA protein, golgin-84. Golgin-84-cleavage, GA fragmentation and growth of Chlamydia trachomatis can be blocked by the peptide inhibitor WEHD-fmk. Here we identify the bacterial protease chlamydial protease-like activity factor (CPAF) as the factor mediating cleavage of golgin-84 and as the target of WEHD-fmk-inhibition. WEHD-fmk blocked cleavage of golgin-84 as well as cleavage of known CPAF targets during infection with C. trachomatis and C. pneumoniae. The same effect was seen when active CPAF was expressed in non-infected cells and in a cell-free system. Ectopic expression of active CPAF in non-infected cells was sufficient for GA fragmentation. GA fragmentation required the small GTPases Rab6 and Rab11 downstream of CPAF-activity. These results define CPAF as the first protein that is essential for replication of Chlamydia. We suggest that this role makes CPAF a potential anti-infective therapeutic target.

Biological effectiveness of nuclear fragments produced by high-energy protons interacting in tissues near the bone- soft tissue interface

NASA Astrophysics Data System (ADS)

Shavers, Mark Randall

1999-12-01

High-energy protons in the galactic cosmic rays (GCR)-or generated by nuclear interactions of GCR heavy-ions with material-are capable of penetrating great thicknesses of shielding to irradiate humans in spacecraft or in lunar or Martian habitats. As protons interact with the nuclei of the elemental constituents of soft tissue and bone, low energy nuclei-target fragments-are emitted into the cells responsible for bone development and maintenance and for hematopoiesis. Leukemogenesis is the principal endpoint of concern because it is the most likely deleterious effect, and it has a short latency period and comparatively low survival rate, although other myelo- proliferative disorders and osteosarcoma also may be induced. A one-dimensional proton-target fragment transport model was used to calculate the energy spectra of fragments produced in bone and soft tissue, and present in marrow cavities at distances from a bone interface. In terms of dose equivalent, the target fragments are as significant as the incident protons. An average radiation quality factor was found to be between 1.8 and 2.6. Biological response to the highly non- uniform energy deposition of the target fragments is such that an alternative approach to conventional predictive risk assessment is needed. Alternative procedures are presented. In vitro cell response and relative biological effectiveness were calculated from the radial dose distribution of each fragment produced by 1-GeV protons using parameters of a modified Ion-Gamma- Kill (IGK) model of radiation action. The modelled endpoints were survival of C3H10t 1/2 and V79 cells, neoplastic transformation of C3H10t1/2 cells, and mutation of the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in V79 cells. The dose equivalent and cell responses increased by 10% or less near the interface. Since RBE increases with decreasing dose in the IGK model, comparisons with quality factors were made at dose levels 0.01 <= D [Gy] <= 2. Applying average quality factors derived herein to GCR exposures results in a <= 5% increase of in average quality. Calculated RBEs indicate that accepted quality factors for high-energy protons may be too low due to the relatively high effectiveness of the low-charged target fragments. Derived RBEs for target fragments increase the calculated biological effectiveness of GCR by 20% to 180%.

Search for unbound nuclides and beam/fragment optics with the MoNA/LISA segmented target at NSCL

NASA Astrophysics Data System (ADS)

Gueye, Paul; Frank, Nathan; Thoennessen, Michael; Redpath, Thomas; MoNA Collaboration

2017-09-01

A multi-layered Si/Be segmented target consisting of three 700 mg/cm2 thick Be9 slabs and four 140 microns Si detectors was used by the MoNA Collaboration at the National Superconducting Cyclotron Laboratory of Michigan State University to study the O26 lifetime. This target provides unprecedented information on the incident beams and fragments (energy loss and position), thus allowing for better determination of the incident and outgoing energies and momenta of the detected particles compare to previous experiments conducted at this facility. With the availability of a newly developed Geant4 Monte Carlo simulation of the full N2 vault, we will present and discuss the performances of this target. Search for unbound nuclides and beam/fragment optics with the MoNA/LISA segmented target at NSCL.

Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.

PubMed

Huang, Wenlin; Zhang, Zhongsheng; Ranade, Ranae M; Gillespie, J Robert; Barros-Álvarez, Ximena; Creason, Sharon A; Shibata, Sayaka; Verlinde, Christophe L M J; Hol, Wim G J; Buckner, Frederick S; Fan, Erkang

2017-06-15

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC 50 of 4nM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Site Identification by Ligand Competitive Saturation (SILCS) Simulations for Fragment-Based Drug Design

PubMed Central

Faller, Christina E.; Raman, E. Prabhu; MacKerell, Alexander D.; Guvench, Olgun

2015-01-01

Fragment-based drug design (FBDD) involves screening low molecular weight molecules (“fragments”) that correspond to functional groups found in larger drug-like molecules to determine their binding to target proteins or nucleic acids. Based on the principle of thermodynamic additivity, two fragments that bind non-overlapping nearby sites on the target can be combined to yield a new molecule whose binding free energy is the sum of those of the fragments. Experimental FBDD approaches, like NMR and X-ray crystallography, have proven very useful but can be expensive in terms of time, materials, and labor. Accordingly, a variety of computational FBDD approaches have been developed that provide different levels of detail and accuracy. The Site Identification by Ligand Competitive Saturation (SILCS) method of computational FBDD uses all-atom explicit-solvent molecular dynamics (MD) simulations to identify fragment binding. The target is “soaked” in an aqueous solution with multiple fragments having different identities. The resulting computational competition assay reveals what small molecule types are most likely to bind which regions of the target. From SILCS simulations, 3D probability maps of fragment binding called “FragMaps” can be produced. Based on the probabilities relative to bulk, SILCS FragMaps can be used to determine “Grid Free Energies (GFEs),” which provide per-atom contributions to fragment binding affinities. For essentially no additional computational overhead relative to the production of the FragMaps, GFEs can be used to compute Ligand Grid Free Energies (LGFEs) for arbitrarily complex molecules, and these LGFEs can be used to rank-order the molecules in accordance with binding affinities. PMID:25709034

Fragmentation of {sup 14}N, {sup 16}O, {sup 20}Ne, and {sup 24}Mg nuclei at 290 to 1000 MeV/nucleon

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

Zeitlin, C.; Miller, J.; Guetersloh, S.

We report fragmentation cross sections measured at 0 deg. for beams of {sup 14}N, {sup 16}O, {sup 20}Ne, and {sup 24}Mg ions, at energies ranging from 290 MeV/nucleon to 1000 MeV/nucleon. Beams were incident on targets of C, CH{sub 2}, Al, Cu, Sn, and Pb, with the C and CH{sub 2} target data used to obtain hydrogen-target cross sections. Using methods established in earlier work, cross sections obtained with both large-acceptance and small-acceptance detectors are extracted from the data and, when necessary, corrected for acceptance effects. The large-acceptance data yield cross sections for fragments with charges approximately half of themore » beam charge and above, with minimal corrections. Cross sections for lighter fragments are obtained from small-acceptance spectra, with more significant, model-dependent corrections that account for the fragment angular distributions. Results for both charge-changing and fragment production cross sections are compared to the predictions of the Los Alamos version of the quark gluon string model (LAQGSM) as well as the NASA Nuclear Fragmentation (NUCFRG2) model and the Particle and Heavy Ion Transport System (PHITS) model. For all beams and targets, cross sections for fragments as light as He are compared to the models. Estimates of multiplicity-weighted helium production cross sections are obtained from the data and compared to PHITS and LAQGSM predictions. Summary statistics show that the level of agreement between data and predictions is slightly better for PHITS than for either NUCFRG2 or LAQGSM.« less

Deformation and Fracture Behavior of Steel Projectiles Impact AD95 Ceramic Targets-Experimental Investigation

NASA Astrophysics Data System (ADS)

Wei, Gang; Zhang, Wei

2013-06-01

The deformation and fracture behavior of steel projectile impacting ceramic target is an interesting investigation topic. The deformation and failure behavior of projectile and target was investigated experimentally in the normal impact by different velocities. Lab-scale ballistic tests of AD95 ceramic targets with 20 mm thickness against two different hardness 38CrSi steel projectiles with 7.62 mm diameter have been conducted at a range of velocities from 100 to 1000 m/s. Experimental results show that, with the impact velocity increasing, for the soft projectiles, the deformation and fracture modes were mushrooming, shear cracking, petalling and fragmentation(with large fragments and less number), respectively; for the hard projectiles there are three deformation and fracture modes: mushrooming, shearing cracking and fragmentation(with small fragments and large number). All projectiles were rebound after impact. But, with the velocity change, the target failure modes have changed. At low velocity, only radial cracks were found; then circumferential cracks appeared with the increasing velocity; the ceramic cone occurred when the velocity reached 400 m/s above, and manifested in two forms: front surface intact at lower velocity and perforated at higher velocity. The higher velocity, the fragment size is smaller and more uniform distribution. The difference of ceramic target damage is not obvious after impacted by two kinds of projectiles with different hardness at the same velocity. National Natural Science Foundation of China (No.: 11072072).

Investigation of the heavy nuclei fission with anomalously high values of the fission fragments total kinetic energy

NASA Astrophysics Data System (ADS)

Khryachkov, Vitaly; Goverdovskii, Andrei; Ketlerov, Vladimir; Mitrofanov, Vecheslav; Sergachev, Alexei

2018-03-01

Binary fission of 232Th and 238U induced by fast neutrons were under intent investigation in the IPPE during recent years. These measurements were performed with a twin ionization chamber with Frisch grids. Signals from the detector were digitized for further processing with a specially developed software. It results in information of kinetic energies, masses, directions and Bragg curves of registered fission fragments. Total statistics of a few million fission events were collected during each experiment. It was discovered that for several combinations of fission fragment masses their total kinetic energy was very close to total free energy of the fissioning system. The probability of such fission events for the fast neutron induced fission was found to be much higher than for spontaneous fission of 252Cf and thermal neutron induced fission of 235U. For experiments with 238U target the energy of incident neutrons were 5 MeV and 6.5 MeV. Close analysis of dependence of fission fragment distribution on compound nucleus excitation energy gave us some explanation of the phenomenon. It could be a process in highly excited compound nucleus which leads the fissioning system from the scission point into the fusion valley with high probability.

Optical model analyses of galactic cosmic ray fragmentation in hydrogen targets

NASA Technical Reports Server (NTRS)

Townsend, Lawrence W.

1993-01-01

Quantum-mechanical optical model methods for calculating cross sections for the fragmentation of galactic cosmic ray nuclei by hydrogen targets are presented. The fragmentation cross sections are calculated with an abrasion-ablation collision formalism. Elemental and isotopic cross sections are estimated and compared with measured values for neon, sulfur, and calcium ions at incident energies between 400A MeV and 910A MeV. Good agreement between theory and experiment is obtained.

The role of electron transfer in DNA building blocks: Evaluation of strand breaks and their implications

NASA Astrophysics Data System (ADS)

Almeida, Diogo Alexandre Fialho de

Radiation-induced damage to biological systems, both direct and indirect processes, has increasingly come under scrutiny by the international scientific community due to recent findings that electrons are a very effective agent in damaging DNA/RNA. Indeed, much remains to be discovered regarding the exact physico-chemical processes that occur in the nascent stages of DNA/RNA damage by incident radiation. However, it is also known that electrons do not exist freely in the physiological medium, but rather solvated and/or pre-solvated states. This leads to the need for new techniques that can better explore the damaging role of "bound" electrons to DNA/RNA. The work presented in this thesis consists on the study of electron transfer in collisions of atomic species with molecules of biological relevance. In order to study these processes, two experimental setups were used. One setup consists of a crossed beam experiment where a neutral potassium beam is created and made to collide with an effusive molecular target beam. The anionic products that stem from electron transfer in potassium atom to the molecular target collisions are then extracted and time-of-flight (TOF) mass analysed. In the second setup a beam of anionic species is formed and made to collide with a molecular target. Collisions with three different anionic beams were performed (H-, O- and OH-), as well as with different simple organic molecules, by measuring the positive and negative ion fragmentation patterns with a quadrupole mass spectrometer (QMS). A comparison between these two collisional systems can greatly help to understand the underlying mechanisms of the electron transfer processes. Finally, studies of potassium collisions with sugar surrogates D-Ribose and THF were performed. These studies show very different fragmentation patterns from DEA, although in the case of THF, it is suggested that the initially accessed states are the same as in DEA. With these studies was also possible to show for the first time collision induced site and bond selectivity breaking, where the electron is transferred into a given state of the acceptor molecule and the resulting fragmentation pathways are exclusive to the initial anionic state. Furthermore, the role of the potassium cation post collisionwas explored and indeed its presence is suggested to induce at least partial suppression of auto-detachment. The implications that ensue from this degradation are analysed in the light of the obtained fragmentation patterns.

Characteristics study of projectile's lightest fragment for 84Kr36-emulsion interaction at around 1 A GeV

NASA Astrophysics Data System (ADS)

Marimuthu, N.; Singh, V.; Inbanathan, S. S. R.

2017-04-01

In this article, we present the results of our investigations on the projectile's lightest fragment (proton) multiplicity and probability distributions with 84Kr36 emulsion collision at around 1 A GeV. The multiplicity and normalized multiplicity of projectile's lightest fragment (proton) are correlated with the compound particles, shower particles, black particles, grey particles; alpha (helium nucleus) fragments and heavily ionizing charged particles. It is found that projectile's lightest fragment (proton) is strongly correlated with compound particles and shower particles rather than other particles and the average multiplicity of projectile's lightest fragment (proton) increases with increasing compound, shower and heavily ionizing charge particles. Normalized projectile's lightest fragment (proton) is strongly correlated with compound particles, shower particles and heavily ionizing charge particles. The multiplicity distribution of the projectile's lightest fragment (proton) emitted in the 84Kr36 + emulsion interaction at around 1 A GeV with different target has been well explained by KNO scaling. The mean multiplicity of projectile's lightest fragments (proton) depends on the mass number of the projectile and does not significantly dependent of the projectile energy. The mean multiplicity of projectile's lightest fragment (proton) increases with increasing the target mass number.

A Comparison of the SOCIT and DebriSat Experiments

NASA Technical Reports Server (NTRS)

Ausay, Erick; Blake, Brandon; Boyle, Colleen; Cornejo, Alex; Horn, Alexa; Palma, Kirsten; Pistella, Frank; Sato, Taishi; Todd, Naromi; Zimmerman, Jeffrey;

Effects of Target Fragmentation on Evaluation of LET Spectra From Space Radiation in Low-Earth Orbit (LEO) Environment: Impact on SEU Predictions

NASA Technical Reports Server (NTRS)

Shinn, J. L.; Cucinotta, F. A.; Badhwar, G. D.; ONeill, P. M.; Badavi, F. F.

1995-01-01

Recent improvements in the radiation transport code HZETRN/BRYNTRN and galactic cosmic ray environmental model have provided an opportunity to investigate the effects of target fragmentation on estimates of single event upset (SEU) rates for spacecraft memory devices. Since target fragments are mostly of very low energy, an SEU prediction model has been derived in terms of particle energy rather than linear energy transfer (LET) to account for nonlinear relationship between range and energy. Predictions are made for SEU rates observed on two Shuttle flights, each at low and high inclination orbit. Corrections due to track structure effects are made for both high energy ions with track structure larger than device sensitive volume and for low energy ions with dense track where charge recombination is important. Results indicate contributions from target fragments are relatively important at large shield depths (or any thick structure material) and at low inclination orbit. Consequently, a more consistent set of predictions for upset rates observed in these two flights is reached when compared to an earlier analysis with CREME model. It is also observed that the errors produced by assuming linear relationship in range and energy in the earlier analysis have fortuitously canceled out the errors for not considering target fragmentation and track structure effects.

Development of a homogeneous immunoassay system using protein A fusion fragmented Renilla luciferase.

PubMed

Mie, Masayasu; Thuy, Ngo Phan Bich; Kobatake, Eiry

2012-03-07

A homogeneous immunoassay system was developed using fragmented Renilla luciferase (Rluc). The B domain of protein A was fused to two Rluc fragments. When complexes between an antibody and fragmented Rluc fusion proteins bind to target molecules, the Rluc fragments come into close proximity and the luminescence activity of fragmented Rluc is restored by complementation. As proof-of-principle, this fragmented Rluc system was used to detect E. coli homogeneously using an anti-E. coli antibody.

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

PubMed Central

Nomura, Yayoi; Sato, Yumi; Suno, Ryoji; Horita, Shoichiro

2016-01-01

Abstract Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies. PMID:27595817

Two neutron correlations in photo-fission

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

Dale, D. S.; Kosinov, O.; Forest, T.

2016-01-01

A large body of experimental work has established the strong kinematical correlation between fission fragments and fission neutrons. Here, we report on the progress of investigations of the potential for strong two neutron correlations arising from the nearly back-to-back nature of the two fission fragments that emit these neutrons in the photo-fission process. In initial measurements, a pulsed electron linear accelerator was used to generate bremsstrahlung photons that impinged upon an actinide target, and the energy and opening angle distributions of coincident neutrons were measured using a large acceptance neutron detector array. A planned comprehensive set of measurements of twomore » neutron correlations in the photo-fission of actinides is expected to shed light on several fundamental aspects of the fission process including the multiplicity distributions associated with the light and heavy fission fragments, the nuclear temperatures of the fission fragments, and the mass distribution of the fission fragments as a function of energy released. In addition to these measurements providing important nuclear data, the unique kinematics of fission and the resulting two neutron correlations have the potential to be the basis for a new tool to detect fissionable materials. A key technical challenge of this program arises from the need to perform coincidence measurements with a low duty factor, pulsed electron accelerator. This has motivated the construction of a large acceptance neutron detector array, and the development of data analysis techniques to directly measure uncorrelated two neutron backgrounds.« less

Repurposing Suzuki Coupling Reagents as a Directed Fragment Library Targeting Serine Hydrolases and Related Enzymes.

PubMed

Lanier, Marion; Cole, Derek C; Istratiy, Yelena; Klein, Michael G; Schwartz, Phillip A; Tjhen, Richard; Jennings, Andy; Hixon, Mark S

2017-06-22

Serine hydrolases are susceptible to potent reversible inhibition by boronic acids. Large collections of chemically diverse boronic acid fragments are commercially available because of their utility in coupling chemistry. We repurposed the approximately 650 boronic acid reagents in our collection as a directed fragment library targeting serine hydrolases and related enzymes. Highly efficient hits (LE > 0.6) often result. The utility of the approach is illustrated with the results against autotaxin, a phospholipase implicated in cardiovascular disease.

Accelerate the transition of radioisotopes and unwanted weapons-grade ^239Pu into stable nuclei with a system of high frequency modulation for a net energy gain

NASA Astrophysics Data System (ADS)

Pamfiloff, Eugene

2006-10-01

A process of high frequency stimulation of nucleons can be utilized for the accelerated fission, decay or controlled transition of unstable isotopes. ^238U could be persuaded to transition promptly into the stable ^206Pb isotope, where a portion of the total mass difference of 29873.802 MeV per nucleus becomes available energy. The proposals of this paper describe an effective system for nuclei stimulation configured to accelerate such a series of 14 transitions over several milliseconds, instead of 4.47 x 10^9 years. Positive ions or ionized capsules of fuel suspended by magnetic fields and subjected to the system of correlated frequency modulation of multiple beam lines, tailored to the specific target, will emit sufficient energy to stimulate subsequent targets. The system can be applied to all radioisotopes, nuclear waste product isotopes such as ^239Pu, and a variety of other suitable unstable or stable nuclei. Through the proposed confinement system and application of high frequency stimulation in the 10^22 to 10^24 Hz regime, the change in mass can be applied to both the fragmentation of subsequent, periodically injected targets, and the production of heat, making a continuous supply of energy possible. The system allows the particle fragmentation process to be brought into the lab and provides potential solutions to the safe disposal of fissile material.

Accelerate the transition of radioisotopes and unwanted weapons-grade ^239Pu into stable nuclei with a system of high frequency modulation for a net energy gain

NASA Astrophysics Data System (ADS)

Pamfiloff, Eugene

2006-11-01

A process of high frequency stimulation of nucleons can be utilized for the accelerated fission, decay or controlled transition of unstable isotopes. ^238U could be persuaded to transition promptly into the stable ^206Pb isotope, where a portion of the total mass difference of 29873.802 MeV per nucleus becomes available energy. The proposals of this paper describe an effective system for nuclei stimulation configured to accelerate such a series of 14 transitions over several milliseconds, instead of 4.47 x 10^9 years. Positive ions or ionized capsules of fuel suspended by magnetic fields and subjected to the system of correlated frequency modulation of multiple beam lines, tailored to the specific target, will emit sufficient energy to stimulate subsequent targets. The system can be applied to all radioisotopes, nuclear waste product isotopes such as ^239Pu, and a variety of other suitable unstable or stable nuclei. Through the proposed confinement system and application of high frequency stimulation in the 10^22 to 10^24 Hz regime, the change in mass can be applied to both the fragmentation of subsequent, periodically injected targets, and the production of heat, making a continuous supply of energy possible. The system allows the particle fragmentation process to be brought into the lab and provides potential solutions to the safe disposal of fissile material.

Dual Fragment Impact of PBX Charges

NASA Astrophysics Data System (ADS)

Haskins, Peter; Briggs, Richard; Leeming, David; White, Nathan; Cheese, Philip; DE&S MoD UK Team; Ordnance Test Solutions Ltd Team

2017-06-01

Fragment impact can pose a significant hazard to many systems containing explosives or propellants. Testing for this threat is most commonly carried out using a single fragment. However, it can be argued that an initial fragment strike (or strikes) could sensitise the energetic material to subsequent impacts, which may then lead to a more violent reaction than would have been predicted based upon single fragment studies. To explore this potential hazard we have developed the capability to launch 2 fragments from the same gun at a range of velocities, and achieve impacts on an acceptor charge with good control over the spatial and temporal separation of the strikes. In this paper we will describe in detail the experimental techniques we have used, both to achieve the dual fragment launch and observe the acceptor charge response. In addition, we will describe the results obtained against PBX filled explosive targets; discuss the mechanisms controlling the target response and their significance for vulnerability assessment. Results of these tests have clearly indicated the potential for detonation upon the second strike, at velocities well below those needed for shock initiation by a single fragment.

Universal odd-even staggering in isotopic fragmentation and spallation cross sections of neutron-rich fragments

NASA Astrophysics Data System (ADS)

Mei, B.; Tu, X. L.; Wang, M.

2018-04-01

An evident odd-even staggering (OES) in fragment cross sections has been experimentally observed in many fragmentation and spallation reactions. However, quantitative comparisons of this OES effect in different reaction systems are still scarce for neutron-rich nuclei near the neutron drip line. By employing a third-order difference formula, the magnitudes of this OES in extensive experimental cross sections are systematically investigated for many neutron-rich nuclei with (N -Z ) from 1 to 23 over a broad range of atomic numbers (Z ≈3 -50 ). A comparison of these magnitude values extracted from fragment cross sections measured in different fragmentation and spallation reactions with a large variety of projectile-target combinations over a wide energy range reveals that the OES magnitude is almost independent of the projectile-target combinations and the projectile energy. The weighted average of these OES magnitudes derived from cross sections accurately measured in different reaction systems is adopted as the evaluation value of the OES magnitude. These evaluated OES magnitudes are recommended to be used in fragmentation and spallation models to improve their predictions for fragment cross sections.

Forward-backward emission of target evaporated fragments in high energy nucleus-nucleus collisions

NASA Astrophysics Data System (ADS)

Zhang, Zhi; Ma, Tian-Li; Zhang, Dong-Hai

2015-10-01

The multiplicity distribution, multiplicity moment, scaled variance, entropy and reduced entropy of target evaporated fragments emitted in forward and backward hemispheres in 12 A GeV 4He, 3.7 A GeV 16O, 60 A GeV 16O, 1.7 A GeV 84Kr and 10.7 A GeV 197Au -induced emulsion heavy target (AgBr) interactions are investigated. It is found that the multiplicity distribution of target evaporated fragments emitted in both forward and backward hemispheres can be fitted by a Gaussian distribution. The multiplicity moments of target evaporated particles emitted in the forward and backward hemispheres increase with the order of the moment q, and the second-order multiplicity moment is energy independent over the entire energy range for all the interactions in the forward and backward hemisphere. The scaled variance, a direct measure of multiplicity fluctuations, is close to one for all the interactions, which indicate a correlation among the produced particles. The entropy of target evaporated fragments emitted in both forward and backward hemispheres are the same within experimental errors. Supported by National Science Foundation of China (11075100), Natural Science Foundation of Shanxi Province (2011011001-2) and the Shanxi Provincial Foundation for Returned Overseas Chinese Scholars, (2011-058)

Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach

PubMed Central

2018-01-01

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers. PMID:29529862

Targeting Ligandable Pockets on Plant Homeodomain (PHD) Zinc Finger Domains by a Fragment-Based Approach.

PubMed

Amato, Anastasia; Lucas, Xavier; Bortoluzzi, Alessio; Wright, David; Ciulli, Alessio

2018-04-20

Plant homeodomain (PHD) zinc fingers are histone reader domains that are often associated with human diseases. Despite this, they constitute a poorly targeted class of readers, suggesting low ligandability. Here, we describe a successful fragment-based campaign targeting PHD fingers from the proteins BAZ2A and BAZ2B as model systems. We validated a pool of in silico fragments both biophysically and structurally and solved the first crystal structures of PHD zinc fingers in complex with fragments bound to an anchoring pocket at the histone binding site. The best-validated hits were found to displace a histone H3 tail peptide in competition assays. This work identifies new chemical scaffolds that provide suitable starting points for future ligand optimization using structure-guided approaches. The demonstrated ligandability of the PHD reader domains could pave the way for the development of chemical probes to drug this family of epigenetic readers.

A Novel Method of Aircraft Detection Based on High-Resolution Panchromatic Optical Remote Sensing Images.

PubMed

Wang, Wensheng; Nie, Ting; Fu, Tianjiao; Ren, Jianyue; Jin, Longxu

2017-05-06

In target detection of optical remote sensing images, two main obstacles for aircraft target detection are how to extract the candidates in complex gray-scale-multi background and how to confirm the targets in case the target shapes are deformed, irregular or asymmetric, such as that caused by natural conditions (low signal-to-noise ratio, illumination condition or swaying photographing) and occlusion by surrounding objects (boarding bridge, equipment). To solve these issues, an improved active contours algorithm, namely region-scalable fitting energy based threshold (TRSF), and a corner-convex hull based segmentation algorithm (CCHS) are proposed in this paper. Firstly, the maximal variance between-cluster algorithm (Otsu's algorithm) and region-scalable fitting energy (RSF) algorithm are combined to solve the difficulty of targets extraction in complex and gray-scale-multi backgrounds. Secondly, based on inherent shapes and prominent corners, aircrafts are divided into five fragments by utilizing convex hulls and Harris corner points. Furthermore, a series of new structure features, which describe the proportion of targets part in the fragment to the whole fragment and the proportion of fragment to the whole hull, are identified to judge whether the targets are true or not. Experimental results show that TRSF algorithm could improve extraction accuracy in complex background, and that it is faster than some traditional active contours algorithms. The CCHS is effective to suppress the detection difficulties caused by the irregular shape.

Dissociation of biomolecules in liquid environments during fast heavy-ion irradiation

NASA Astrophysics Data System (ADS)

Nomura, Shinji; Tsuchida, Hidetsugu; Kajiwara, Akihiro; Yoshida, Shintaro; Majima, Takuya; Saito, Manabu

2017-12-01

The effect of aqueous environment on fast heavy-ion radiation damage of biomolecules was studied by comparative experiments using liquid- and gas-phase amino acid targets. Three types of amino acids with different chemical structures were used: glycine, proline, and hydroxyproline. Ion-induced reaction products were analyzed by time-of-flight secondary-ion mass spectrometry. The results showed that fragments from the amino acids resulting from the C—Cα bond cleavage were the major products for both types of targets. For liquid-phase targets, specific products originating from chemical reactions in solutions were observed. Interestingly, multiple dissociated atomic fragments were negligible for the liquid-phase targets. We found that the ratio of multifragment to total fragment ion yields was approximately half of that for gas-phase targets. This finding agreed with the results of other studies on biomolecular cluster targets. It is concluded that the suppression of molecular multifragmentation is caused by the energy dispersion to numerous water molecules surrounding the biomolecular solutes.

Electrospray mass spectrometry of NeuAc oligomers associated with the C fragment of the tetanus toxin

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

Prieto, M C; Whittal, R M; Baldwin, M A

2005-04-03

The Clostridial neurotoxins, botulinum and tetanus, gain entry into neuronal cells by protein recognition involving cell specific binding sites. The sialic or N-acetylneuraminic acid (NeuAc) residues of gangliosides attached to the surface of motor neurons are the suspected recognition and interaction points with Clostridial neurotoxins, although not necessarily the only ones. We have used electrospray ionization mass spectrometry (ESIMS) to examine formation of complexes between the tetanus toxin C fragment, or targeting domain, and carbohydrates containing NeuAc groups to determine how NeuAc residues contribute to ganglioside binding. ESI-MS was used to rapidly and efficiently measure dissociation constants for a numbermore » of related NeuAc-containing carbohydrates and NeuAc oligomers, information that has helped identify the structural features of gangliosides that determine their binding to tetanus toxin. The strength of the interactions between the C fragment and (NeuAc){sub n}, are consistent with the topography of the targeting domain of tetanus toxin and the nature of its carbohydrate binding sites. The results suggest that the targeting domain of tetanus toxin contains two binding sites that can accommodate NeuAc (or a dimer). This study also shows that NeuAc must play an important role in ganglioside binding and molecular recognition, a process critical for normal cell function and one frequently exploited by toxins, bacteria and viruses to facilitate their entrance into cells.« less

Fragment based drug discovery: practical implementation based on ¹⁹F NMR spectroscopy.

PubMed

Jordan, John B; Poppe, Leszek; Xia, Xiaoyang; Cheng, Alan C; Sun, Yax; Michelsen, Klaus; Eastwood, Heather; Schnier, Paul D; Nixey, Thomas; Zhong, Wenge

2012-01-26

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.

Robustness of optimal random searches in fragmented environments

NASA Astrophysics Data System (ADS)

Wosniack, M. E.; Santos, M. C.; Raposo, E. P.; Viswanathan, G. M.; da Luz, M. G. E.

2015-05-01

The random search problem is a challenging and interdisciplinary topic of research in statistical physics. Realistic searches usually take place in nonuniform heterogeneous distributions of targets, e.g., patchy environments and fragmented habitats in ecological systems. Here we present a comprehensive numerical study of search efficiency in arbitrarily fragmented landscapes with unlimited visits to targets that can only be found within patches. We assume a random walker selecting uniformly distributed turning angles and step lengths from an inverse power-law tailed distribution with exponent μ . Our main finding is that for a large class of fragmented environments the optimal strategy corresponds approximately to the same value μopt≈2 . Moreover, this exponent is indistinguishable from the well-known exact optimal value μopt=2 for the low-density limit of homogeneously distributed revisitable targets. Surprisingly, the best search strategies do not depend (or depend only weakly) on the specific details of the fragmentation. Finally, we discuss the mechanisms behind this observed robustness and comment on the relevance of our results to both the random search theory in general, as well as specifically to the foraging problem in the biological context.

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies.

PubMed

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Sun, Guohui; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2.

Insights into the Impact of Linker Flexibility and Fragment Ionization on the Design of CK2 Allosteric Inhibitors: Comparative Molecular Dynamics Simulation Studies

PubMed Central

Zhou, Yue; Zhang, Na; Qi, Xiaoqian; Tang, Shan; Zhao, Lijiao; Zhong, Rugang; Peng, Yongzhen

2018-01-01

Protein kinase is a novel therapeutic target for human diseases. The off-target and side effects of ATP-competitive inhibitors preclude them from the clinically relevant drugs. The compounds targeting the druggable allosteric sites outside the highly conversed ATP binding pocket have been identified as promising alternatives to overcome current barriers of ATP-competitive inhibitors. By simultaneously interacting with the αD region (new allosteric site) and sub-ATP binding pocket, the attractive compound CAM4066 was named as allosteric inhibitor of CK2α. It has been demonstrated that the rigid linker and non-ionizable substituted fragment resulted in significant decreased inhibitory activities of compounds. The molecular dynamics simulations and energy analysis revealed that the appropriate coupling between the linker and pharmacophore fragments were essential for binding of CAM4066 with CK2α. The lower flexible linker of compound 21 lost the capability of coupling fragments A and B to αD region and positive area, respectively, whereas the methyl benzoate of fragment B induced the re-orientated Pre-CAM4066 with the inappropriate polar interactions. Most importantly, the match between the optimized linker and pharmacophore fragments is the challenging work of fragment-linking based drug design. These results provide rational clues to further structural modification and development of highly potent allosteric inhibitors of CK2. PMID:29301250

Fragment-based screen against HIV protease.

PubMed

Perryman, Alexander L; Zhang, Qing; Soutter, Holly H; Rosenfeld, Robin; McRee, Duncan E; Olson, Arthur J; Elder, John E; Stout, C David

2010-03-01

We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 A resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the 'exo site' adjacent to the Gly(16)Gly(17)Gln(18)loop where the amide of Gly(17)is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys(14)and Leu(63). Another fragment, indole-6-carboxylic acid, binds on the 'outside/top of the flap' via hydrophobic contacts with Trp(42), Pro(44), Met(46), and Lys(55), a hydrogen bond with Val(56), and a salt-bridge with Arg(57). 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.

Fragment-Based Screen against HIV Protease

PubMed Central

Perryman, A. L.; Zhang, Q.; Soutter, H. H.; Rosenfeld, R.; McRee, D. E.; Olson, A. J.; Elder, J. E.; Stout, C. D.

2009-01-01

We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the ‘exo site’ adjacent to the Gly16Gly17Gln18 loop where the amide of Gly17 is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys14 and Leu63. Another fragment, indole-6-carboxylic acid, binds on the ‘outside/top of the flap’ via hydrophobic contacts with Trp42, Pro44, Met46, and Lys55, a hydrogen bond with Val56, and a salt-bridge with Arg57. 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target. PMID:20659109

Targeting incentives to reduce habitat fragmentation

Treesearch

David Lewis; Andrew Plantinga; Junjie Wu

2009-01-01

This article develops a theoretical model to analyze the spatial targeting of incentives for the restoration of forested landscapes when wildlife habitat can be enhanced by reducing fragmentation. The key theoretical result is that the marginal net benefits of increasing forest can be convex, in which case corner solutions--converting either none or all of the...

A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI.

PubMed

Shinozuka, Hiroshi; Cogan, Noel O I; Shinozuka, Maiko; Marshall, Alexis; Kay, Pippa; Lin, Yi-Han; Spangenberg, German C; Forster, John W

2015-04-11

Fragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine. A semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2'-deoxycytidine-5'-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2'-deoxycytidine-5'-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation. The proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing.

Detection of rabbit and hare processed material in compound feeds by TaqMan real-time PCR.

PubMed

Pegels, N; López-Calleja, I; García, T; Martín, R; González, I

2013-01-01

Food and feed traceability has become a priority for governments due to consumer demand for comprehensive and integrated safety policies. In the present work, a TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for specific detection of rabbit and hare material in animal feeds and pet foods. The technique is based on the use of three species-specific primer/probe detection systems targeting three 12S rRNA gene fragments: one from rabbit species, another one from hare species and a third fragment common to rabbit and hare (62, 102 and 75 bp length, respectively). A nuclear 18S rRNA PCR system, detecting a 77-bp amplicon, was used as positive amplification control. Assay performance and sensitivity were assessed through the analysis of a batch of laboratory-scale feeds treated at 133°C at 3 bar for 20 min to reproduce feed processing conditions dictated by European regulations. Successful detection of highly degraded rabbit and hare material was achieved at the lowest target concentration assayed (0.1%). Furthermore, the method was applied to 96 processed commercial pet food products to determine whether correct labelling had been used at the market level. The reported real-time PCR technique detected the presence of rabbit tissues in 80 of the 96 samples analysed (83.3%), indicating a possible labelling fraud in some pet foods. The real-time PCR method reported may be a useful tool for traceability purposes within the framework of feed control.

A Bioinorganic Approach to Fragment-Based Drug Discovery Targeting Metalloenzymes.

PubMed

Cohen, Seth M

2017-08-15

Metal-dependent enzymes (i.e., metalloenzymes) make up a large fraction of all enzymes and are critically important in a wide range of biological processes, including DNA modification, protein homeostasis, antibiotic resistance, and many others. Consequently, metalloenzymes represent a vast and largely untapped space for drug development. The discovery of effective therapeutics that target metalloenzymes lies squarely at the interface of bioinorganic and medicinal chemistry and requires expertise, methods, and strategies from both fields to mount an effective campaign. In this Account, our research program that brings together the principles and methods of bioinorganic and medicinal chemistry are described, in an effort to bridge the gap between these fields and address an important class of medicinal targets. Fragment-based drug discovery (FBDD) is an important drug discovery approach that is particularly well suited for metalloenzyme inhibitor development. FBDD uses relatively small but diverse chemical structures that allow for the assembly of privileged molecular collections that focus on a specific feature of the target enzyme. For metalloenzyme inhibition, the specific feature is rather obvious, namely, a metal-dependent active site. Surprisingly, prior to our work, the exploration of diverse molecular fragments for binding the metal active sites of metalloenzymes was largely unexplored. By assembling a modest library of metal-binding pharmacophores (MBPs), we have been able to find lead hits for many metalloenzymes and, from these hits, develop inhibitors that act via novel mechanisms of action. A specific case study on the use of this strategy to identify a first-in-class inhibitor of zinc-dependent Rpn11 (a component of the proteasome) is highlighted. The application of FBDD for the development of metalloenzyme inhibitors has raised several other compelling questions, such as how the metalloenzyme active site influences the coordination chemistry of bound fragments, how one can identify the best fragments for a given metalloenzyme, and many others. Among the most significant, and concerning, questions for metalloenzyme inhibition are those that reside around issues of specificity and whether metalloenzyme inhibitors can be as selective and specific as other small molecule inhibitors (i.e., compounds that inhibit enzymes that do not utilize a metal at their active site). This also leads to the question of whether metalloenzyme inhibitors might interfere more broadly with the metallome. Efforts to address these and related questions are discussed, with the expectation that our findings will illuminate some of these topics, alleviate some of these concerns, and encourage greater interest in this important, undervalued class of drug targets.

"Solar-Wind-Rich" Howardite: True Regolith vs. CM-Implanted Components

NASA Technical Reports Server (NTRS)

Cartwright, Julia A.; Mittlefehldt, D. W.; Herrin, J. S.; Hermann, S.; Ott, U.

2011-01-01

Howardite, eucrite and diogenite meteorites (collectively HED) likely originate from asteroid 4-Vesta [1], one of two asteroids targeted by NASA s Dawn mission. Many howardites (polymict breccias of E and D material) contain "regolithic" features, including impact-melt clasts, fragmental breccia clasts, and carbonaceous chondrite fragments. True regolithic nature can be determined through noble gas analysis, as Solar Wind (SW) is implanted into the upper-most surfaces of solar system bodies. Whilst previous work [2] suggested that high siderophile element contents (e.g. Ni of 300-1200 g/g) were regolith indicators, we found no obvious correlation between SW and these indicators in our initial howardite noble gas analyses [3]. We observed CM-like fragments in a number of our howardites, whose textures suggest late addition to the breccia assemblage [4]. As typical CMs contain mixtures of SW (in matrix) and planetary (in clasts) components [5], we investigate the dominance of such components in SW-rich howardites. This will help deter-mine the extent of implanted SW in HED grains vs. SW and planetary gases from CM fragments, and allow better understanding of regolith processes

Study on the Failure and Energy Absorption Mechanism of Multilayer Explosively Welded Plates Impacted by Spherical Fragments

NASA Astrophysics Data System (ADS)

Zhou, N.; Wang, J. X.; Tang, S. Z.; Tao, Q. C.; Wang, M. X.

2018-01-01

A stereomicroscope, microscopic metallograph, scanning electron microscope, and the ANSYS/LS-DYNA 3D finite-element code were employed to investigate the failure and energy absorption mechanism of two-layer steel/aluminum and three-layer steel/aluminum/steel and aluminum/steel/aluminum explosively welded composite plates impacted by spherical fragments. The effects of layer number, target order, and the combination state of interfaces on the failure and energy absorption mechanism are analyzed based on experimental and numerical results. Results showed that the effect of the combination state of interfaces on the failure mode was pronounced the most compared with other factors. The failure mechanism of the front and middle plates were shearing and plugging, and that of rear plate was ductile deformation when the tied interface failed by tension (or by shearing and plugging when the interface combination remained connected). A narrow adiabatic shear band was formed in the locally yielding plate damaged by shearing and plugging during the penetration process. The amount of energy needed to completely perforate the three-layer composite target was greater than that for a two-layer composite target with the same areal density and total thickness. The protective performance of the steel/aluminum/steel target was better than that of the aluminum/steel/aluminum target with the same areal density.

Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean.

PubMed

Zhang, Haibo; Yang, Litao; Guo, Jinchao; Li, Xiang; Jiang, Lingxi; Zhang, Dabing

2008-07-23

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs.

The tetraspanin CD63 regulates ESCRT-independent and dependent endosomal sorting during melanogenesis

PubMed Central

van Niel, Guillaume; Charrin, Stéphanie; Simoes, Sabrina; Romao, Maryse; Rochin, Leila; Saftig, Paul; Marks, Michael S.; Rubinstein, Eric; Raposo, Graça

2011-01-01

Summary Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for numerous physiological processes including lysosome-related organelle (LRO) biogenesis. PMEL – a component of melanocyte LROs (melanosomes) – is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis. PMID:21962903

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves

PubMed Central

Jutras, Philippe V.; Marusic, Carla; Lonoce, Chiara; Deflers, Carole; Goulet, Marie-Claire; Benvenuto, Eugenio; Donini, Marcello

2016-01-01

The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories. PMID:27893815

Simulation of impact ballistic of Cu-10wt%Sn frangible bullet using smoothed particle hydrodynamics

NASA Astrophysics Data System (ADS)

Hidayat, Mas Irfan P.; Widyastuti, Simaremare, Peniel

2018-04-01

Frangible bullet is designed to disintegrate upon impact against a hard target. Understanding the impact response and performance of frangible bullet is therefore of highly interest. In this paper, simulation of impact ballistic of Cu-IOwt%Sn frangible bullet using smoothed particle hydrodynamics (SPH) method is presented. The frangible bullet is impacted against a hard, cylindrical stainless steel target. Effect of variability of the frangible bullet material properties due to the variation of sintering temperature in its manufacturing process to the bullet frangibility factor (FF) is investigated numerically. In addition, the bullet kinetic energy during impact as well as its ricochet and fragmentation are also examined and simulated. Failure criterion based upon maximum strain is employed in the simulation. It is shown that the SPH simulation can produce good estimation for kinetic energy of bullet after impact, thus giving the FF prediction with respect to the variation of frangible bullet material properties. In comparison to explicit finite element (FE) simulation, in which only material/element deletion is shown, convenience in showing frangible bullet fragmentation is shown using the SPH simulation. As a result, the effect of sintering temperature to the way of the frangible bullet fragmented can be also observed clearly.

Differential cross-sections measurements for hadrontherapy: 50 MeV/A 12C reactions on H, C, O, Al and natTi targets

NASA Astrophysics Data System (ADS)

Divay, C.; Colin, J.; Cussol, D.; Finck, Ch.; Karakaya, Y.; Labalme, M.; Rousseau, M.; Salvador, S.; Vanstalle, M.

2017-09-01

In order to keep the benefits of a carbon treatment, the dose and biological effects induced by secondary fragments must be taken into account when simulating the treatment plan. These Monte-Carlo simulations codes are done using nuclear models that are constrained by experimental data. It is hence necessary to have precise measurements of the production rates of these fragments all along the beam path and for its whole energy range. In this context, a series of experiments aiming to measure the double differential fragmentation cross-sections of carbon on thin targets of medical interest has been started by our collaboration. In March 2015, an experiment was performed with a 50 MeV/nucleon 12C beam at GANIL. During this experiment, energy and angular differential cross-section distributions on H, C, O, Al and natTi have been measured. In the following, the experimental set-up and analysis process are briefly described and some experimental results are presented. Comparisons between several exit channel models from Phits and Geant4 show great discrepancies with the experimental data. Finally, the homemade Sliipie model is briefly presented and preliminary results are compared to the data with a promising outcome.

Dose equivalent near the bone-soft tissue interface from nuclear fragments produced by high-energy protons

NASA Technical Reports Server (NTRS)

Shavers, M. R.; Poston, J. W.; Cucinotta, F. A.; Wilson, J. W.

1996-01-01

During manned space missions, high-energy nucleons of cosmic and solar origin collide with atomic nuclei of the human body and produce a broad linear energy transfer spectrum of secondary particles, called target fragments. These nuclear fragments are often more biologically harmful than the direct ionization of the incident nucleon. That these secondary particles increase tissue absorbed dose in regions adjacent to the bone-soft tissue interface was demonstrated in a previous publication. To assess radiological risks to tissue near the bone-soft tissue interface, a computer transport model for nuclear fragments produced by high energy nucleons was used in this study to calculate integral linear energy transfer spectra and dose equivalents resulting from nuclear collisions of 1-GeV protons transversing bone and red bone marrow. In terms of dose equivalent averaged over trabecular bone marrow, target fragments emitted from interactions in both tissues are predicted to be at least as important as the direct ionization of the primary protons-twice as important, if recently recommended radiation weighting factors and "worst-case" geometry are used. The use of conventional dosimetry (absorbed dose weighted by aa linear energy transfer-dependent quality factor) as an appropriate framework for predicting risk from low fluences of high-linear energy transfer target fragments is discussed.

Structure-Based Identification of Inhibitory Fragments Targeting the p300/CBP-Associated Factor Bromodomain

PubMed Central

2016-01-01

The P300/CBP-associated factor plays a central role in retroviral infection and cancer development, and the C-terminal bromodomain provides an opportunity for selective targeting. Here, we report several new classes of acetyl-lysine mimetic ligands ranging from mM to low micromolar affinity that were identified using fragment screening approaches. The binding modes of the most attractive fragments were determined using high resolution crystal structures providing chemical starting points and structural models for the development of potent and selective PCAF inhibitors. PMID:26731131

A comparison of DNA fragmentation methods - Applications for the biochip technology.

PubMed

Sapojnikova, Nelly; Asatiani, Nino; Kartvelishvili, Tamar; Asanishvili, Lali; Zinkevich, Vitaly; Bogdarina, Irina; Mitchell, Julian; Al-Humam, Abdulmohsen

2017-08-20

The efficiency of hybridization signal detection in a biochip is affected by the method used for test DNA preparation, such as fragmentation, amplification and fluorescent labelling. DNA fragmentation is the commonest methods used and it is recognised as a critical step in biochip analysis. Currently methods used for DNA fragmentation are based either on sonication or on the enzymatic digestion. In this study, we compared the effect of different types of enzymatic DNA fragmentations, using DNase I to generate ssDNA breaks, NEBNext dsDNA fragmentase and SaqAI restrictase, on DNA labelling. DNA from different Desulfovibrio species was used as a substrate for these enzymes. Of the methods used, DNA fragmented by NEBNext dsDNA Fragmentase digestion was subsequently labelled with the greatest efficiency. As a result of this, the use of this enzyme to fragment target DNA increases the sensitivity of biochip-based detection significantly, and this is an important consideration when determining the presence of targeted DNA in ecological and medical samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Dissecting fragment-based lead discovery at the von Hippel-Lindau protein:hypoxia inducible factor 1α protein-protein interface.

PubMed

Van Molle, Inge; Thomann, Andreas; Buckley, Dennis L; So, Ernest C; Lang, Steffen; Crews, Craig M; Ciulli, Alessio

2012-10-26

Fragment screening is widely used to identify attractive starting points for drug design. However, its potential and limitations to assess the tractability of often challenging protein:protein interfaces have been underexplored. Here, we address this question by means of a systematic deconstruction of lead-like inhibitors of the pVHL:HIF-1α interaction into their component fragments. Using biophysical techniques commonly employed for screening, we could only detect binding of fragments that violate the Rule of Three, are more complex than those typically screened against classical druggable targets, and occupy two adjacent binding subsites at the interface rather than just one. Analyses based on ligand and group lipophilicity efficiency of anchored fragments were applied to dissect the individual subsites and probe for binding hot spots. The implications of our findings for targeting protein interfaces by fragment-based approaches are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Numerical Simulation of Ballistic Impact on Particulate Composite Target using Discrete Element Method: 1-D and 2-D Models

NASA Astrophysics Data System (ADS)

Nair, Rajesh P.; Lakshmana Rao, C.

2014-01-01

Ballistic impact (BI) is a study that deals with a projectile hitting a target and observing its effects in terms of deformation and fragmentation of the target. The Discrete Element Method (DEM) is a powerful numerical technique used to model solid and particulate media. Here, an attempt is made to simulate the BI process using DEM. 1-D DEM for BI is developed and depth of penetration (DOP) is obtained. The DOP is compared with results obtained from 2-D DEM. DEM results are found to match empirical results. Effects of strain rate sensitivity of the material response on DOP are also simulated.

A Novel Method of Aircraft Detection Based on High-Resolution Panchromatic Optical Remote Sensing Images

PubMed Central

Wang, Wensheng; Nie, Ting; Fu, Tianjiao; Ren, Jianyue; Jin, Longxu

2017-01-01

In target detection of optical remote sensing images, two main obstacles for aircraft target detection are how to extract the candidates in complex gray-scale-multi background and how to confirm the targets in case the target shapes are deformed, irregular or asymmetric, such as that caused by natural conditions (low signal-to-noise ratio, illumination condition or swaying photographing) and occlusion by surrounding objects (boarding bridge, equipment). To solve these issues, an improved active contours algorithm, namely region-scalable fitting energy based threshold (TRSF), and a corner-convex hull based segmentation algorithm (CCHS) are proposed in this paper. Firstly, the maximal variance between-cluster algorithm (Otsu’s algorithm) and region-scalable fitting energy (RSF) algorithm are combined to solve the difficulty of targets extraction in complex and gray-scale-multi backgrounds. Secondly, based on inherent shapes and prominent corners, aircrafts are divided into five fragments by utilizing convex hulls and Harris corner points. Furthermore, a series of new structure features, which describe the proportion of targets part in the fragment to the whole fragment and the proportion of fragment to the whole hull, are identified to judge whether the targets are true or not. Experimental results show that TRSF algorithm could improve extraction accuracy in complex background, and that it is faster than some traditional active contours algorithms. The CCHS is effective to suppress the detection difficulties caused by the irregular shape. PMID:28481260

GlyQ-IQ: Glycomics Quintavariate-Informed Quantification with High-Performance Computing and GlycoGrid 4D Visualization

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

Kronewitter, Scott R.; Slysz, Gordon W.; Marginean, Ioan

2014-05-31

Dense LC-MS datasets have convoluted extracted ion chromatograms with multiple chromatographic peaks that cloud the differentiation between intact compounds with their overlapping isotopic distributions, peaks due to insource ion fragmentation, and noise. Making this differentiation is critical in glycomics datasets because chromatographic peaks correspond to different intact glycan structural isomers. The GlyQ-IQ software is targeted chromatography centric software designed for chromatogram and mass spectra data processing and subsequent glycan composition annotation. The targeted analysis approach offers several key advantages to LC-MS data processing and annotation over traditional algorithms. A priori information about the individual target’s elemental composition allows for exactmore » isotope profile modeling for improved feature detection and increased sensitivity by focusing chromatogram generation and peak fitting on the isotopic species in the distribution having the highest intensity and data quality. Glycan target annotation is corroborated by glycan family relationships and in source fragmentation detection. The GlyQ-IQ software is developed in this work (Part 1) and was used to profile N-glycan compositions from human serum LC-MS Datasets. The companion manuscript GlyQ-IQ Part 2 discusses developments in human serum N-glycan sample preparation, glycan isomer separation, and glycan electrospray ionization. A case study is presented to demonstrate how GlyQ-IQ identifies and removes confounding chromatographic peaks from high mannose glycan isomers from human blood serum. In addition, GlyQ-IQ was used to generate a broad N-glycan profile from a high resolution (100K/60K) nESI-LS-MS/MS dataset including CID and HCD fragmentation acquired on a Velos Pro Mass spectrometer. 101 glycan compositions and 353 isomer peaks were detected from a single sample. 99% of the GlyQ-IQ glycan-feature assignments passed manual validation and are backed with high resolution mass spectra and mass accuracies less than 7 ppm.« less

Lipid Profiling of the Arabidopsis Hypersensitive Response Reveals Specific Lipid Peroxidation and Fragmentation Processes: Biogenesis of Pimelic and Azelaic Acid1[C][W

PubMed Central

Zoeller, Maria; Stingl, Nadja; Krischke, Markus; Fekete, Agnes; Waller, Frank; Berger, Susanne; Mueller, Martin J.

2012-01-01

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids. PMID:22822212

Lipid profiling of the Arabidopsis hypersensitive response reveals specific lipid peroxidation and fragmentation processes: biogenesis of pimelic and azelaic acid.

PubMed

Zoeller, Maria; Stingl, Nadja; Krischke, Markus; Fekete, Agnes; Waller, Frank; Berger, Susanne; Mueller, Martin J

2012-09-01

Lipid peroxidation (LPO) is induced by a variety of abiotic and biotic stresses. Although LPO is involved in diverse signaling processes, little is known about the oxidation mechanisms and major lipid targets. A systematic lipidomics analysis of LPO in the interaction of Arabidopsis (Arabidopsis thaliana) with Pseudomonas syringae revealed that LPO is predominantly confined to plastid lipids comprising galactolipid and triacylglyceride species and precedes programmed cell death. Singlet oxygen was identified as the major cause of lipid oxidation under basal conditions, while a 13-lipoxygenase (LOX2) and free radical-catalyzed lipid oxidation substantially contribute to the increase upon pathogen infection. Analysis of lox2 mutants revealed that LOX2 is essential for enzymatic membrane peroxidation but not for the pathogen-induced free jasmonate production. Despite massive oxidative modification of plastid lipids, levels of nonoxidized lipids dramatically increased after infection. Pathogen infection also induced an accumulation of fragmented lipids. Analysis of mutants defective in 9-lipoxygenases and LOX2 showed that galactolipid fragmentation is independent of LOXs. We provide strong in vivo evidence for a free radical-catalyzed galactolipid fragmentation mechanism responsible for the formation of the essential biotin precursor pimelic acid as well as of azelaic acid, which was previously postulated to prime the immune response of Arabidopsis. Our results suggest that azelaic acid is a general marker for LPO rather than a general immune signal. The proposed fragmentation mechanism rationalizes the pathogen-induced radical amplification and formation of electrophile signals such as phytoprostanes, malondialdehyde, and hexenal in plastids.

Monoclonal antibody fragment removal mediated by mixed mode resins.

PubMed

O'Connor, Ellen; Aspelund, Matthew; Bartnik, Frank; Berge, Mark; Coughlin, Kelly; Kambarami, Mutsa; Spencer, David; Yan, Huiming; Wang, William

2017-05-26

Efforts to increase monoclonal antibody expression in cell culture can result in the presence of fragmented species requiring removal in downstream processing. Capto adhere, HEA Hypercel, and PPA Hypercel anion exchange/hydrophobic interaction mixed mode resins were evaluated for their fragment removal capabilities and found to separate large hinge IgG1 antibody fragment (LHF) from monomer. Removal of greater than 75% of LHF population occurred at pH 8 and low conductivity. The mechanism of fragment removal was investigated in two series of experiments. The first experimental series consisted of comparison to chromatographic behavior on corresponding single mode resins. Both single mode anion exchange and hydrophobic interaction resins failed to separate LHF. The second experimental series studied the impact of phase modifiers, ethylene glycol, urea, and arginine on the mixed mode mediated removal. The addition of ethylene glycol decreased LHF removal by half. Further decreases in LHF separation were seen upon incubation with urea and arginine. Therefore, it was discovered that the purification is the result of a mixed mode phenomena dominated by hydrophobic interaction and hydrogen bonding effects. The site of interaction between the LHF and mixed mode resin was determined by chemical labeling of lysine residues with sulfo-NHS acetate. The labeling identified the antibody hinge and light chain regions as mediating the fragment separation. Sequence analysis showed that under separation conditions, a hydrophobic proline patch and hydrogen bonding serine and threonine residues mediate the hinge interaction with the Capto adhere ligand. Additionally, a case study is presented detailing the optimization of fragment removal using Capto adhere resin to achieve purity and yield targets in a manufacturing facility. This study demonstrated that mixed mode resins can be readily integrated into commercial antibody platform processes when additional chromatographic abilities are required. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

A dual-targeting PDGFRbeta/VEGF-A molecule assembled from stable antibody fragments demonstrates anti-angiogenic activity in vitro and in vivo.

PubMed

Mabry, Robert; Gilbertson, Debra G; Frank, Amanda; Vu, Tuyen; Ardourel, Dan; Ostrander, Craig; Stevens, Brenda; Julien, Susan; Franke, Secil; Meengs, Brent; Brody, Jennifer; Presnell, Scott; Hamacher, Nels B; Lantry, Megan; Wolf, Anitra; Bukowski, Tom; Rosler, Robert; Yen, Cindy; Anderson-Haley, Monica; Brasel, Kenneth; Pan, Qi; Franklin, Hank; Thompson, Penny; Dodds, Mike; Underwood, Sara; Peterson, Scott; Sivakumar, Pallavur V; Snavely, Mark

2010-01-01

Targeting angiogenesis is a promising approach to the treatment of solid tumors and age-related macular degeneration (AMD). Inhibition of vascularization has been validated by the successful marketing of monoclonal antibodies (mAbs) that target specific growth factors or their receptors, but there is considerable room for improvement in existing therapies. Combination of mAbs targeting both the VEGF and PDGF pathways has the potential to increase the efficacy of anti-angiogenic therapy without the accompanying toxicities of tyrosine kinase inhibitors and the inability to combine efficiently with traditional chemotherapeutics. However, development costs and regulatory issues have limited the use of combinatorial approaches for the generation of more efficacious treatments. The concept of mediating disease pathology by targeting two antigens with one therapeutic was proposed over two decades ago. While mAbs are particularly suitable candidates for a dual-targeting approach, engineering bispecificity into one molecule can be difficult due to issues with expression and stability, which play a significant role in manufacturability. Here, we address these issues upstream in the process of developing a bispecific antibody (bsAb). Single-chain antibody fragments (scFvs) targeting PDGFRbeta and VEGF-A were selected for superior stability. The scFvs were fused to both termini of human Fc to generate a bispecific, tetravalent molecule. The resulting molecule displays potent activity, binds both targets simultaneously, and is stable in serum. The assembly of a bsAb using stable monomeric units allowed development of an anti-PDGFRB/VEGF-A antibody capable of attenuating angiogenesis through two distinct pathways and represents an efficient method for rapid engineering of dual-targeting molecules.

Antagonistic Effects of BACE1 and APH1B-γ-Secretase Control Axonal Guidance by Regulating Growth Cone Collapse.

PubMed

Barão, Soraia; Gärtner, Annette; Leyva-Díaz, Eduardo; Demyanenko, Galina; Munck, Sebastian; Vanhoutvin, Tine; Zhou, Lujia; Schachner, Melitta; López-Bendito, Guillermina; Maness, Patricia F; De Strooper, Bart

2015-09-01

ΒACE1 is the major drug target for Alzheimer's disease, but we know surprisingly little about its normal function in the CNS. Here, we show that this protease is critically involved in semaphorin 3A (Sema3A)-mediated axonal guidance processes in thalamic and hippocampal neurons. An active membrane-bound proteolytic CHL1 fragment is generated by BACE1 upon Sema3A binding. This fragment relays the Sema3A signal via ezrin-radixin-moesin (ERM) proteins to the neuronal cytoskeleton. APH1B-γ-secretase-mediated degradation of this fragment stops the Sema3A-induced collapse and sensitizes the growth cone for the next axonal guidance cue. Thus, we reveal a cycle of proteolytic activity underlying growth cone collapse and restoration used by axons to find their correct trajectory in the brain. Our data also suggest that BACE1 and γ-secretase inhibition have physiologically opposite effects in this process, supporting the idea that combination therapy might attenuate some of the side effects associated with these drugs. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Extracellular matrix fragmentation in young, healthy cartilaginous tissues.

PubMed

Craddock, R J; Hodson, N W; Ozols, M; Shearer, T; Hoyland, J A; Sherratt, M J

2018-02-09

Although the composition and structure of cartilaginous tissues is complex, collagen II fibrils and aggrecan are the most abundant assemblies in both articular cartilage (AC) and the nucleus pulposus (NP) of the intervertebral disc (IVD). Whilst structural heterogeneity of intact aggrecan ( containing three globular domains) is well characterised, the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months), bovine AC and NP tissues, it was shown that, whilst the ultrastructure of intact aggrecan was tissue-dependent, most molecules (AC: 95 %; NP: 99.5 %) were fragmented (lacking one or more globular domains). Fragments were significantly smaller and more structurally heterogeneous in the NP compared with the AC (molecular area; AC: 8543 nm2; NP: 4625 nm2; p < 0.0001). In contrast, fibrillar collagen appeared structurally intact and tissue-invariant. Molecular fragmentation is considered indicative of a pathology; however, these young, skeletally mature tissues were histologically and mechanically (reduced modulus: AC: ≈ 500 kPa; NP: ≈ 80 kPa) comparable to healthy tissues and devoid of notable gelatinase activity (compared with rat dermis). As aggrecan fragmentation was prevalent in neonatal bovine AC (99.5 % fragmented, molecular area: 5137 nm2) as compared with mature AC (95.0 % fragmented, molecular area: 8667 nm2), it was hypothesised that targeted proteolysis might be an adaptive process that modified aggrecan packing (as simulated computationally) and, hence, tissue charge density, mechanical properties and porosity. These observations provided a baseline against which pathological and/or age-related fragmentation of aggrecan could be assessed and suggested that new strategies might be required to engineer constructs that mimic the mechanical properties of native cartilaginous tissues.

Discovery of novel drugs for promising targets.

PubMed

Martell, Robert E; Brooks, David G; Wang, Yan; Wilcoxen, Keith

2013-09-01

Once a promising drug target is identified, the steps to actually discover and optimize a drug are diverse and challenging. The goal of this study was to provide a road map to navigate drug discovery. Review general steps for drug discovery and provide illustrating references. A number of approaches are available to enhance and accelerate target identification and validation. Consideration of a variety of potential mechanisms of action of potential drugs can guide discovery efforts. The hit to lead stage may involve techniques such as high-throughput screening, fragment-based screening, and structure-based design, with informatics playing an ever-increasing role. Biologically relevant screening models are discussed, including cell lines, 3-dimensional culture, and in vivo screening. The process of enabling human studies for an investigational drug is also discussed. Drug discovery is a complex process that has significantly evolved in recent years. © 2013 Elsevier HS Journals, Inc. All rights reserved.

Differential cross section measurements for hadron therapy: 50 MeV/nucleon 12C reactions on H, C, O, Al, and natTi targets

NASA Astrophysics Data System (ADS)

Divay, C.; Colin, J.; Cussol, D.; Finck, Ch.; Karakaya, Y.; Labalme, M.; Rousseau, M.; Salvador, S.; Vanstalle, M.

2017-04-01

During a carbon therapy treatment, the beam undergoes inelastic nuclear reactions leading to the production of secondary fragments. These nuclear interactions tend to delocate a part of the dose into healthy tissues and create a mixed radiation field. In order to accurately estimate the dose deposited into the tissues, the production rate of these fragments all along the beam path have to be taken into account. But the double differential carbon fragmentation cross sections are not well known in the energy range needed for a treatment (up to 400 MeV/nucleon). Therefore, a series of experiments aiming to measure the double differential fragmentation cross sections of carbon on thin targets of medical interest has been started by our collaboration. In March 2015 we performed an experiment to study the fragmentation of a 50 MeV/nucleon 12C beam on thin targets at GANIL. During this experiment, energy and angular cross-section distributions on H, C, O, Al, and natTi have been measured. The experimental set-up will be detailed as well as the systematic error study and all the experimental results will be presented.

Developments in SPR Fragment Screening.

PubMed

Chavanieu, Alain; Pugnière, Martine

2016-01-01

Fragment-based approaches have played an increasing role alongside high-throughput screening in drug discovery for 15 years. The label-free biosensor technology based on surface plasmon resonance (SPR) is now sensitive and informative enough to serve during primary screens and validation steps. In this review, the authors discuss the role of SPR in fragment screening. After a brief description of the underlying principles of the technique and main device developments, they evaluate the advantages and adaptations of SPR for fragment-based drug discovery. SPR can also be applied to challenging targets such as membrane receptors and enzymes. The high-level of immobilization of the protein target and its stability are key points for a relevant screening that can be optimized using oriented immobilized proteins and regenerable sensors. Furthermore, to decrease the rate of false negatives, a selectivity test may be performed in parallel on the main target bearing the binding site mutated or blocked with a low-off-rate ligand. Fragment-based drug design, integrated in a rational workflow led by SPR, will thus have a predominant role for the next wave of drug discovery which could be greatly enhanced by new improvements in SPR devices.

[Microbial community structure in bio-ceramics and biological activated carbon analyzed by PCR-SSCP technique].

PubMed

Liu, Xiao-Lin; Liu, Wen-Jun

2007-04-01

Analyses of microbial community structure in bio-ceramics (BC) and biological activated carbon (BAC), which widely used in drinking water treatment were performed by polymerase-chain-reaction-single-strand-conformation-polymorphism (PCR-SSCP) targeted eubacterial 16S ribosomal RNA gene. Microorganisms on bio-ceramics and biological activated carbon were detached by ultrasonic, culturing on R2A and LB agar, respectively, followed by genome DNA extracting. Results show that larger than 10 kb genome DNA could be extracted from all the samples except the BAC samples processed by ultrasonic. However, quantities of the extracted DNA were different. 408 bp gene fragments were observed after PCR using the extracted genome DNA as templates. These gene fragments were digested with lambda exonuclease followed by SSCP electrophoresis. Same SSCP profiles were observed between ultrasonic eluting, R2A and LB agar culturing. The identity of the segment from bio-ceramics with uncultured Pseudomonas sp. Clone FTL201 16S rDNA (GenBank, AF509293.1) fragment was 92%, and identities of the two segments from BAC with Bacillus sp. JH19 16S rDNA (GenBank , DQ232748.1) fragment and Bacterium VA-S-11 16S rDNA (GenBank, AY395279.1) fragment were 100% and 99%, respectively.

Simulation of dependence of the cross section of deuterons beam fragmentation into cumulative pions and protons on the mass of the target nucleus

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

Litvinenko, A. G., E-mail: alitvin@jinr.ru; Litvinenko, E. I.

2015-03-15

We have studied the mechanisms influencing production of cumulative pions and protons in the fragmentation of the incident deuterons into cumulative pions and protons emitted at zero angle. We argue that the peripheral dependence on the atomic mass of the target nucleus, which was obtained in the experiments for medium and heavy nuclei, can be explained by scattering on target nucleons without introducing additional parameters.

Virtual Screening of Peptide and Peptidomimetic Fragments Targeted to Inhibit Bacterial Dithiol Oxidase DsbA.

PubMed

Duprez, Wilko; Bachu, Prabhakar; Stoermer, Martin J; Tay, Stephanie; McMahon, Róisín M; Fairlie, David P; Martin, Jennifer L

2015-01-01

Antibacterial drugs with novel scaffolds and new mechanisms of action are desperately needed to address the growing problem of antibiotic resistance. The periplasmic oxidative folding system in Gram-negative bacteria represents a possible target for anti-virulence antibacterials. By targeting virulence rather than viability, development of resistance and side effects (through killing host native microbiota) might be minimized. Here, we undertook the design of peptidomimetic inhibitors targeting the interaction between the two key enzymes of oxidative folding, DsbA and DsbB, with the ultimate goal of preventing virulence factor assembly. Structures of DsbB--or peptides--complexed with DsbA revealed key interactions with the DsbA active site cysteine, and with a hydrophobic groove adjacent to the active site. The present work aimed to discover peptidomimetics that target the hydrophobic groove to generate non-covalent DsbA inhibitors. The previously reported structure of a Proteus mirabilis DsbA active site cysteine mutant, in a non-covalent complex with the heptapeptide PWATCDS, was used as an in silico template for virtual screening of a peptidomimetic fragment library. The highest scoring fragment compound and nine derivatives were synthesized and evaluated for DsbA binding and inhibition. These experiments discovered peptidomimetic fragments with inhibitory activity at millimolar concentrations. Although only weakly potent relative to larger covalent peptide inhibitors that interact through the active site cysteine, these fragments offer new opportunities as templates to build non-covalent inhibitors. The results suggest that non-covalent peptidomimetics may need to interact with sites beyond the hydrophobic groove in order to produce potent DsbA inhibitors.

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking.

PubMed

Fan, Lihua; Shuai, Jiangbing; Zeng, Ruoxue; Mo, Hongfei; Wang, Suhua; Zhang, Xiaofeng; He, Yongqiang

2017-12-01

Genome fragment enrichment (GFE) method was applied to identify host-specific bacterial genetic markers that differ among different fecal metagenomes. To enrich for swine-specific DNA fragments, swine fecal DNA composite (n = 34) was challenged against a DNA composite consisting of cow, human, goat, sheep, chicken, duck and goose fecal DNA extracts (n = 83). Bioinformatic analyses of 384 non-redundant swine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode metabolism-associated, cellular processes and information storage and processing. After challenged against fecal DNA extracted from different animal sources, four sequences from the clone libraries targeting two Bacteroidales- (genes 1-38 and 3-53), a Clostridia- (gene 2-109) as well as a Bacilli-like sequence (gene 2-95), respectively, showed high specificity to swine feces based on PCR analysis. Host-specificity and host-sensitivity analysis confirmed that oligonucleotide primers and probes capable of annealing to select Bacteroidales-like sequences (1-38 and 3-53) exhibited high specificity (>90%) in quantitative PCR assays with 71 fecal DNAs from non-target animal sources. The two assays also demonstrated broad distributions of corresponding genetic markers (>94% positive) among 72 swine feces. After evaluation with environmental water samples from different areas, swine-targeted assays based on two Bacteroidales-like GFE sequences appear to be suitable quantitative tracing tools for swine fecal pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Accelerate the transition of radioisotopes or unwanted weapons-grade ^239Pu into stable nuclei with a system of high frequency modulation for a net energy gain

NASA Astrophysics Data System (ADS)

Pamfiloff, Eugene

2006-10-01

A process of high frequency stimulation of nucleons can be utilized for the accelerated fission, decay or controlled transition of unstable isotopes. For example ^238U could be persuaded to transition promptly into ^206Pb, where portions of the total mass difference of 29873.802 MeV per nucleus becomes available energy. The proposals of this paper describe an effective system for nuclei stimulation configured to accelerate such a series of 14 transitions over several milliseconds, instead of 4.47 x 10^9 years. Positive ions or ionized capsules of fuel suspended by magnetic fields and subjected to the system of correlated frequency modulation of multiple beam lines, tailored to the specific target, will emit sufficient energy to stimulate subsequent targets. The system can be applied to all radioisotopes, including ^232Th, nuclear waste product isotopes such as ^239Pu, and a variety of other suitable unstable or stable nuclei. Through the proposed confinement system and application of high frequency stimulation in the 10^22 to 10^24 Hz regime, the change in rest mass can be applied to both the fragmentation of subsequent, periodically injected targets, and the production of heat, making a continuous supply of energy possible. The system allows the particle fragmentation process to be brought into the laboratory and provides potential solutions to the safe disposal of fissile material.

Target fragments in collisions of 1.8 GeV/nucleon Fe-56 nuclei with photoemulsion nuclei, and the cascade-evaporation model

NASA Technical Reports Server (NTRS)

Dudkin, V. E.; Kovalev, E. E.; Nefedov, N. A.; Antonchik, V. A.; Bogdanov, S. D.; Ostroumov, V. I.; Benton, E. V.; Crawford, H. J.

1995-01-01

Nuclear photographic emulsion is used to study the dependence of the characteristics of target-nucleus fragments on the masses and impact parameters of interacting nuclei. The data obtained are compared in all details with the calculation results made in terms of the Dubna version of the cascade-evaporation model (DCM).

In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study

PubMed Central

2014-01-01

Background Patients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours. Methods The fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup. Results Both species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys. Conclusions We conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies. PMID:24598405

Microwave-Accelerated Method for Ultra-Rapid Extraction of Neisseria gonorrhoeae DNA for Downstream Detection

PubMed Central

Melendez, Johan H.; Santaus, Tonya M.; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A.; Geddes, Chris D.

2016-01-01

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by the detection of the genomic target often involving PCR-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (GC) DNA. Our approach is based on the use of highly-focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the present study, we show that highly focused microwaves at 2.45 GHz, using 12.3 mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification in less than 10 minutes total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward towards the development of a point-of-care (POC) platform for detection of gonorrhea infections. PMID:27325503

The fragmentation of 510 MeV/nucleon iron-56 in polyethylene. II. Comparisons between data and a model

NASA Technical Reports Server (NTRS)

Zeitlin, C.; Heilbronn, L.; Miller, J.; Schimmerling, W.; Townsend, L. W.; Tripathi, R. K.; Wilson, J. W.

1996-01-01

The results of a Monte Carlo model for calculating fragment fluences and LET spectra are compared to data taken with 600 MeV/nucleon iron ions incident on an accelerator beamline configured for irradiation of biological samples, with no target and with 2, 5 and 8 cm of polyethylene. The model uses a multi-generation nuclear fragmentation code, coupled with a formulation of ionization energy loss based on the Bethe-Bloch equation. In the region where the data are reliable and the experimental acceptance is well understood, many of the features of the experimental spectra are well replicated by the model. To obtain good agreement with the experimental data, the model must allow for at least two generations of fragment production in the target.

An analysis of the wounding factors of four different shapes of fragments.

PubMed

Ma, Y Y; Feng, T S; Fu, R X; Li, M

1988-01-01

The wounding characteristics to a biological target of four typical shapes of fragments (square, triangular, cylindrical, and spherical) with masses of less than 1 gram and velocities between 460 and 1,500 m/s are studied in this paper. The following conclusions about the effects of the wounding factors, such as energy transfer, velocity, mass, and shape of fragment are presented: 1) For given target characteristics, the important wounding factors of fragments are impact velocity, mass, and shape, and of these velocity is the most important. 2) Besides direct effects, the fragment velocity has great influence on far-reaching, indirect wounding effects. When velocity increases, it not only increases the size of direct wound, but also the rate of indirect bone fracture. 3) The rate of energy transfer is affected by fragment shape, and it is also a decreasing function of mass. 4) Under the same conditions there are differences in wounding effectiveness among the four fragment shapes, the triangular with a comparatively high wounding effectiveness, followed by the square, cylindrical, and spherical. The types of wound channels are also different, the cylindrical and spherical making a "through" type, the square and triangular making a "blind-tube" type.

Fragmenting networks by targeting collective influencers at a mesoscopic level.

PubMed

Kobayashi, Teruyoshi; Masuda, Naoki

2016-11-25

A practical approach to protecting networks against epidemic processes such as spreading of infectious diseases, malware, and harmful viral information is to remove some influential nodes beforehand to fragment the network into small components. Because determining the optimal order to remove nodes is a computationally hard problem, various approximate algorithms have been proposed to efficiently fragment networks by sequential node removal. Morone and Makse proposed an algorithm employing the non-backtracking matrix of given networks, which outperforms various existing algorithms. In fact, many empirical networks have community structure, compromising the assumption of local tree-like structure on which the original algorithm is based. We develop an immunization algorithm by synergistically combining the Morone-Makse algorithm and coarse graining of the network in which we regard a community as a supernode. In this way, we aim to identify nodes that connect different communities at a reasonable computational cost. The proposed algorithm works more efficiently than the Morone-Makse and other algorithms on networks with community structure.

Fragmenting networks by targeting collective influencers at a mesoscopic level

NASA Astrophysics Data System (ADS)

Kobayashi, Teruyoshi; Masuda, Naoki

2016-11-01

A practical approach to protecting networks against epidemic processes such as spreading of infectious diseases, malware, and harmful viral information is to remove some influential nodes beforehand to fragment the network into small components. Because determining the optimal order to remove nodes is a computationally hard problem, various approximate algorithms have been proposed to efficiently fragment networks by sequential node removal. Morone and Makse proposed an algorithm employing the non-backtracking matrix of given networks, which outperforms various existing algorithms. In fact, many empirical networks have community structure, compromising the assumption of local tree-like structure on which the original algorithm is based. We develop an immunization algorithm by synergistically combining the Morone-Makse algorithm and coarse graining of the network in which we regard a community as a supernode. In this way, we aim to identify nodes that connect different communities at a reasonable computational cost. The proposed algorithm works more efficiently than the Morone-Makse and other algorithms on networks with community structure.

Fragmenting networks by targeting collective influencers at a mesoscopic level

PubMed Central

Kobayashi, Teruyoshi; Masuda, Naoki

2016-01-01

A practical approach to protecting networks against epidemic processes such as spreading of infectious diseases, malware, and harmful viral information is to remove some influential nodes beforehand to fragment the network into small components. Because determining the optimal order to remove nodes is a computationally hard problem, various approximate algorithms have been proposed to efficiently fragment networks by sequential node removal. Morone and Makse proposed an algorithm employing the non-backtracking matrix of given networks, which outperforms various existing algorithms. In fact, many empirical networks have community structure, compromising the assumption of local tree-like structure on which the original algorithm is based. We develop an immunization algorithm by synergistically combining the Morone-Makse algorithm and coarse graining of the network in which we regard a community as a supernode. In this way, we aim to identify nodes that connect different communities at a reasonable computational cost. The proposed algorithm works more efficiently than the Morone-Makse and other algorithms on networks with community structure. PMID:27886251

Production mechanism of new neutron-rich heavy nuclei in the 136Xe +198Pt reaction

NASA Astrophysics Data System (ADS)

Li, Cheng; Wen, Peiwei; Li, Jingjing; Zhang, Gen; Li, Bing; Xu, Xinxin; Liu, Zhong; Zhu, Shaofei; Zhang, Feng-Shou

2018-01-01

The multinucleon transfer reaction of 136Xe +198Pt at Elab = 7.98 MeV/nucleon is investigated by using the improved quantum molecular dynamics model. The quasielastic, deep-inelastic, and quasifission collision mechanisms are studied via analyzing the angular distributions of fragments and the energy dissipation processes during the collisions. The measured isotope production cross sections of projectile-like fragments are reasonably well reproduced by the calculation of the ImQMD model together with the GEMINI code. The isotope production cross sections for the target-like fragments and double differential cross sections of 199Pt, 203Pt, and 208Pt are calculated. It is shown that about 50 new neutron-rich heavy nuclei can be produced via deep-inelastic collision mechanism, where the production cross sections are from 10-3 to 10-6 mb. The corresponding emission angle and the kinetic energy for these new neutron-rich nuclei locate at 40∘-60∘ and 100-200 MeV, respectively.

Fragmentation of structural energetic materials: implications for performance

NASA Astrophysics Data System (ADS)

Aydelotte, B.; Braithwaite, C. H.; Thadhani, N. N.

2014-05-01

Fragmentation results for structural energetic materials based on intermetallic forming mixtures are reviewed and the implications of the fragment populations are discussed. Cold sprayed Ni+Al and explosively compacted mixtures of Ni+Al+W and Ni+Al+W+Zr powders were fabricated into ring shaped samples and explosively fragmented. Ring velocity was monitored and fragments were soft captured in order to study the fragmentation process. It was determined that the fragments produced by these structural energetic materials are much smaller than those typically produced by ductile metals such as steel or aluminum. This has implications for combustion processes that may occur subsequent to the fragmentation process.

Survival of the impactor during hypervelocity collisions - II. An analogue for high-porosity targets

NASA Astrophysics Data System (ADS)

Avdellidou, C.; Price, M. C.; Delbo, M.; Cole, M. J.

2017-01-01

We investigated how a target's porosity affects the outcome of a collision, with respect to the impactor's fate. Laboratory impact experiments using peridot projectiles were performed at a speed range between 0.3 and 3.0 km s-1, on to high-porosity water-ice (40 per cent) and fine-grained calcium carbonate (70 per cent) targets. We report that the amount of implanted material in the target body increases with increasing target's porosity, while the size frequency distribution of the projectile's ejecta fragments becomes steeper. A supplementary Raman study showed no sign of change of the Raman spectra of the recovered olivine projectile fragments indicate minimal physical change.

A framework to optimize the restoration and retention of large mature forest tracts in managed boreal landscapes.

PubMed

Bouchard, Mathieu; Garet, Jérôme

The decreasing abundance of mature forests and their fragmentation have been identified as major threats for the preservation of biodiversity in managed landscapes. In this study, we developed a multi-level framework to coordinate forest harvestings so as to optimize the retention or restoration of large mature forest tracts in managed forests. We used mixed-integer programming for this optimization, and integrated realistic management assumptions regarding stand yield and operational harvest constraints. The model was parameterized for eastern Canadian boreal forests, where clear-cutting is the main silvicultural system, and is used to examine two hypotheses. First, we tested if mature forest tract targets had more negative impacts on wood supplies when implemented in landscapes that are very different from targeted conditions. Second, we tested the hypothesis that using more partial cuts can be useful to attenuate the negative impacts of mature forest targets on wood supplies. The results indicate that without the integration of an explicit mature forest tract target, the optimization leads to relatively high fragmentation levels. Forcing the retention or restoration of large mature forest tracts on 40% of the landscapes had negative impacts on wood supplies in all types of landscapes, but these impacts were less important in landscapes that were initially fragmented. This counter-intuitive result is explained by the presence in the models of an operational constraint that forbids diffuse patterns of harvestings, which are more costly. Once this constraint is applied, the residual impact of the mature forest tract target is low. The results also indicate that partial cuts are of very limited use to attenuate the impacts of mature forest tract targets on wood supplies in highly fragmented landscapes. Partial cuts are somewhat more useful in landscapes that are less fragmented, but they have to be well coordinated with clearcut schedules in order to contribute efficiently to conservation objectives. This modeling framework could easily be adapted and parameterized to test hypotheses or to optimize restoration schedules in landscapes where issues such as forest fragmentation and the abundance of mature or old-growth forests are a concern.

Effects of Word and Fragment Writing during L2 Vocabulary Learning

ERIC Educational Resources Information Center

Barcroft, Joe

2007-01-01

This study examined how writing (copying) target words and word fragments affects intentional second language (L2) vocabulary learning. English-speaking first-semester learners of Spanish attempted to learn 24 Spanish nouns via word-picture repetition in three conditions: (1) word writing, (2) fragment writing, and (3) no writing. After the…

Dynamic Failure and Fragmentation of a Hot-Pressed Boron Carbide

NASA Astrophysics Data System (ADS)

Sano, Tomoko; Vargas-Gonzalez, Lionel; LaSalvia, Jerry; Hogan, James David

2017-12-01

This study investigates the failure and fragmentation of a hot-pressed boron carbide during high rate impact experiments. Four impact experiments are performed using a composite-backed target configuration at similar velocities, where two of the impact experiments resulted in complete target penetration and two resulted in partial penetration. This paper seeks to evaluate and understand the dynamic behavior of the ceramic that led to either the complete or partial penetration cases, focusing on: (1) surface and internal failure features of fragments using optical, scanning electron, and transmission electron microscopy, and (2) fragment size analysis using state-of-the-art particle-sizing technology that informs about the consequences of failure. Detailed characterization of the mechanical properties and the microstructure is also performed. Results indicate that transgranular fracture was the primary mode of failure in this boron carbide material, and no stress-induced amorphization features were observed. Analysis of the fragment sizes for the partial and completely penetrated experiments revealed a possible correlation between larger fragment sizes and impact performance. The results will add insight into designing improved advanced ceramics for impact protection applications.

Fission fragment yields from heavy-ion-induced reactions measured with a fragment separator

NASA Astrophysics Data System (ADS)

Tarasov, O. B.; Delaune, O.; Farget, F.; Morrissey, D. J.; Amthor, A. M.; Bastin, B.; Bazin, D.; Blank, B.; Cacéres, L.; Chbihi, A.; Fernández-Dominguez, B.; Grévy, S.; Kamalou, O.; Lukyanov, S. M.; Mittig, W.; Pereira, J.; Perrot, L.; Saint-Laurent, M.-G.; Savajols, H.; Sherrill, B. M.; Stodel, C.; Thomas, J. C.; Villari, A. C.

2018-04-01

The systematic study of fission fragment yields under different initial conditions has provided valuable experimental data for benchmarking models of fission product yields. Nuclear reactions using inverse kinematics coupled to the use of a high-resolution spectrometer with good fragment identification are shown here to be a powerful tool to measure the inclusive isotopic yields of fission fragments. In-flight fusion-fission was used in this work to produce secondary beams of neutron-rich isotopes in the collisions of a 238U beam at 24 MeV/u with 9Be and 12C targets at GANIL using the LISE3 fragment separator. Unique identification of the A, Z, and atomic charge state, q, of fission products was attained with the Δ E- TKE-B ρ- ToF measurement technique. Mass, and atomic number distributions are reported for the two reactions. The results show the importance of different reaction mechanisms in the two cases. The optimal target material for higher yields of neutron-rich high- Z isotopes produced in fusion-fission reactions as a function of projectile energy is discussed.

Recombinant human antibody fragment against tetanus toxoid produced by phage display.

PubMed

Neelakantam, B; Sridevi, N V; Shukra, A M; Sugumar, P; Samuel, S; Rajendra, L

2014-03-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen.

Ultrasensitive electrochemical detection of DNA based on Zn²⁺ assistant DNA recycling followed with hybridization chain reaction dual amplification.

PubMed

Qian, Yong; Wang, Chunyan; Gao, Fenglei

2015-01-15

A new strategy to combine Zn(2+) assistant DNA recycling followed with hybridization chain reaction dual amplification was designed for highly sensitive electrochemical detection of target DNA. A gold electrode was used to immobilize molecular beacon (MB) as the recognition probe and perform the amplification procedure. In the presence of the target DNA, the hairpin probe 1 was opened, and the DNAzyme was liberated from the caged structure. The activated DNAzyme hybridized with the MB and catalyzed its cleavage in the presence of Zn(2+) cofactor and resulting in a free DNAzyme strand. Finally, each target-induced activated DNAzyme underwent many cycles triggering the cleavage of MB, thus forming numerous MB fragments. The MB fragments triggered the HCR and formed a long double-helix DNA structure. Because both H1 and H2 were labeled by biotin, a lot of SA-ALP was captured on the electrode surface, thus catalyzing a silver deposition process for electrochemical stripping analysis. This novel cascade signal amplification strategy can detect target DNA down to the attomolar level with a dynamic range spanning 6 orders of magnitude. This highly sensitive and specific assay has a great potential to become a promising DNA quantification method in biomedical research and clinical diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Attrition of limestone by impact loading in fluidized beds

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

Fabrizio Scala; Fabio Montagnaro; Piero Salatino

2007-09-15

The present study addresses limestone attrition and fragmentation associated with impact loading, a process which may occur extensively in various regions of fluidized bed (FB) combustors/gasifiers, primarily the jetting region of the bottom bed, the exit region of the riser, and the cyclone. An experimental protocol for the characterization of the propensity of limestone to undergo attrition/fragmentation by impact loading is reported. The application of the protocol is demonstrated with reference to an Italian limestone whose primary fragmentation and attrition by surface wear have already been characterized in previous studies. The experimental procedure is based on the characterization of themore » amount and particle size distribution of the debris generated upon the impact of samples of sorbent particles against a target. Experiments were carried out at a range of particle impact velocities between 10 and 45 m/s, consistent with jet velocities corresponding to typical pressure drops across FB gas distributors. The protocol has been applied to either raw or preprocessed limestone samples. In particular, the effect of calcination, sulfation, and calcination/recarbonation cycles on the impact damage suffered by sorbent particles has been assessed. The measurement of particle voidage and pore size distribution by mercury intrusion was also accomplished to correlate fragmentation with the structural properties of the sorbent samples. Fragmentation by impact loading of the limestone is significant. Lime displays the largest propensity to undergo impact damage, followed by the sorbent sulfated to exhaustion, the recarbonated sorbent, and the raw limestone. Fragmentation of the raw limestone and of the sulfated lime follows a pattern typical of the failure of brittle materials. The fragmentation behavior of lime and recarbonated lime better conforms to a disintegration failure mode, with an extensive generation of very fine fragments. 27 refs., 9 figs. 1 tab.« less

Application of the fragment molecular orbital method analysis to fragment-based drug discovery of BET (bromodomain and extra-terminal proteins) inhibitors.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Ueda, Kazuyoshi

2017-06-01

The molecular interactions of inhibitors of bromodomains (BRDs) were investigated. BRDs are protein interaction modules that recognizing ε-N-acetyl-lysine (εAc-Lys) motifs found in histone tails and are promising protein-protein interaction (PPI) targets. First, we analyzed a peptide ligand containing εAc-Lys to evaluate native PPIs. We then analyzed tetrahydroquinazoline-6-yl-benzensulfonamide derivatives found by fragment-based drug design (FBDD) and examined their interactions with the protein compared with the peptide ligand in terms of the inter-fragment interaction energy. In addition, we analyzed benzodiazepine derivatives that are high-affinity ligands for BRDs and examined differences in the CH/π interactions of the amino acid residues. We further surveyed changes in the charges of the amino acid residues among individual ligands, performed pair interaction energy decomposition analysis and estimated the water profile within the ligand binding site. Thus, useful insights for drug design were provided. Through these analyses and considerations, we show that the FMO method is a useful drug design tool to evaluate the process of FBDD and to explore PPI inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets.

PubMed

Linardy, Evelyn M; Erskine, Simon M; Lima, Nicole E; Lonergan, Tina; Mokany, Elisa; Todd, Alison V

2016-01-15

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Extracellular Matrix Modulation: Optimizing Skin Care and Rejuvenation Procedures.

PubMed

Widgerow, Alan D; Fabi, Sabrina G; Palestine, Roberta F; Rivkin, Alexander; Ortiz, Arisa; Bucay, Vivian W; Chiu, Annie; Naga, Lina; Emer, Jason; Chasan, Paul E

2016-04-01

Normal aging and photoaging of the skin are chronic processes that progress gradually. The extracellular matrix (ECM), constituting over 70% of the skin, is the central hub for repair and regeneration of the skin. As such, the ECM is the area where changes related to photodamage are most evident. Degradation of the ECM with fragmentation of proteins significantly affects cross talk and signaling between cells, the matrix, and its constituents. The accumulation of collagen fragments, amorphous elastin agglutinations, and abnormal cross-linkages between the collagen fragments impedes the ECM from its normal repair and regenerative capacity, which manifests as wrinkled, non-elastic skin. Similar to how the chronic wound healing process requires wound bed preparation before therapeutic intervention, treatment of chronic aging of the skin would likely benefit from a "skin bed preparation" to optimize the outcome of rejuvenation procedures and skin maintenance programs. This involves introducing agents that can combat stress-induced oxidation, proteasome dysfunction, and non-enzymatic cross linkages involved in glycation end products, to collectively modulate this damaged ECM, and upregulate neocollagenesis and elastin production. Agents of particular interest are matrikines, peptides originating from the fragmentation of matrix proteins that exhibit a wide range of biological activities. Peptides of this type (tripeptide and hexapeptide) are incorporated in ALASTIN™ Skin Nectar with TriHex™ technology (ALASTIN Skincare, Inc., Carlsbad, CA), which is designed to target ECM modulation with a goal of optimizing results following invasive and non-invasive dermal rejuvenating procedures.

Target-directed Dynamic Combinatorial Chemistry: A Study on Potentials and Pitfalls as Exemplified on a Bacterial Target.

PubMed

Frei, Priska; Pang, Lijuan; Silbermann, Marleen; Eriş, Deniz; Mühlethaler, Tobias; Schwardt, Oliver; Ernst, Beat

2017-08-25

Target-directed dynamic combinatorial chemistry (DCC) is an emerging technique for the efficient identification of inhibitors of pharmacologically relevant targets. In this contribution, we present an application for a bacterial target, the lectin FimH, a crucial virulence factor of uropathogenic E. coli being the main cause of urinary tract infections. A small dynamic library of acylhydrazones was formed from aldehydes and hydrazides and equilibrated at neutral pH in presence of aniline as nucleophilic catalyst. The major success factors turned out to be an accordingly adjusted ratio of scaffolds and fragments, an adequate sample preparation prior to HPLC analysis, and the data processing. Only then did the ranking of the dynamic library constituents correlate well with affinity data. Furthermore, as a support of DCC applications especially to larger libraries, a new protocol for improved hit identification was established. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Experiments and scaling laws for catastrophic collisions. [of asteroids

NASA Technical Reports Server (NTRS)

Fujiwara, A.; Cerroni, P.; Davis, D.; Ryan, E.; Di Martino, M.

1989-01-01

The existing data on shattering impacts are reviewed using natural silicate, ice, and cement-mortar targets. A comprehensive data base containing the most important parameters describing these experiments was prepared. The collisional energy needed to shatter consolidated homogeneous targets and the ensuing fragment size distributions have been well studied experimentally. However, major gaps exist in the data on fragment velocity and rotational distributions, as well as collisional energy partitioning for these targets. Current scaling laws lead to predicted outcomes of asteroid collisions that are inconsistent with interpretations of astronomical data.

Detection of pork adulteration in processed meat by species-specific PCR-QIAxcel procedure based on D-loop and cytb genes.

PubMed

Barakat, Hassan; El-Garhy, Hoda A S; Moustafa, Mahmoud M A

2014-12-01

Detection of pork meat adulteration in "halal" meat products is a crucial issue in the fields of modern food inspection according to implementation of very strict procedures for halal food labelling. Present study aims at detecting and quantifying pork adulteration in both raw and cooked manufactured sausages. This is by applying an optimized species-specific PCR procedure followed by QIAxcel capillary electrophoresis system. Manufacturing experiment was designed by incorporating pork with beef meat at 0.01 to 10 % substitution levels beside beef and pork sausages as negative and positive controls, respectively. Subsequently, sausages were divided into raw and cooked sausages then subjected to DNA extraction. Results indicated that PCR amplifications of mitochondrial D-loop and cytochrome b (cytb) genes by porcine-specific primers produced 185 and 117 bp pork-specific DNA fragments in sausages, respectively. No DNA fragments were detected when PCR was applied on beef sausage DNA confirming primers specificity. For internal control, a 141-bp DNA fragment of eukaryotic 18S ribosomal RNA (rRNA) gene was amplified from pork and beef DNA templates. Although PCR followed by either QIAxcel or agarose techniques were efficient for targeted DNA fragments differentiation even as low as 0.01 % (pork/meat: w/w). For proficiency, adequacy, and performance, PCR-QIA procedure is highly sensitive, a time-saver, electronically documented, mutagenic-reagent free, of little manual errors, accurate in measuring PCR fragments length, and quantitative data supplier. In conclusion, it can be suggested that optimized PCR-QAI is considered as a rapid and sensitive method for routine pork detection and quantification in raw or processed meat.

Combining NMR and X-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective BACE-1 inhibitors.

PubMed

Wyss, Daniel F; Wang, Yu-Sen; Eaton, Hugh L; Strickland, Corey; Voigt, Johannes H; Zhu, Zhaoning; Stamford, Andrew W

2012-01-01

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade. We review here how we have used highly structure-driven fragment-based approaches to complement more traditional lead discovery to tackle high priority targets and those struggling for leads. Combining biomolecular nuclear magnetic resonance (NMR), X-ray crystallography, and molecular modeling with structure-assisted chemistry and innovative biology as an integrated approach for FBDD can solve very difficult problems, as illustrated in this chapter. Here, a successful FBDD campaign is described that has allowed the development of a clinical candidate for BACE-1, a challenging CNS drug target. Crucial to this achievement were the initial identification of a ligand-efficient isothiourea fragment through target-based NMR screening and the determination of its X-ray crystal structure in complex with BACE-1, which revealed an extensive H-bond network with the two active site aspartate residues. This detailed 3D structural information then enabled the design and validation of novel, chemically stable and accessible heterocyclic acylguanidines as aspartic acid protease inhibitor cores. Structure-assisted fragment hit-to-lead optimization yielded iminoheterocyclic BACE-1 inhibitors that possess desirable molecular properties as potential therapeutic agents to test the amyloid hypothesis of Alzheimer's disease in a clinical setting.

NMR screening in fragment-based drug design: a practical guide.

PubMed

Kim, Hai-Young; Wyss, Daniel F

2015-01-01

Fragment-based drug design (FBDD) comprises both fragment-based screening (FBS) to find hits and elaboration of these hits to lead compounds. Typical fragment hits have lower molecular weight (<300-350 Da) and lower initial potency but higher ligand efficiency when compared to those from high-throughput screening. NMR spectroscopy has been widely used for FBDD since it identifies and localizes the binding site of weakly interacting hits on the target protein. Here we describe ligand-based NMR methods for hit identification from fragment libraries and for functional cross-validation of primary hits.

Screening of a Novel Fragment Library with Functional Complexity against Mycobacterium tuberculosis InhA.

PubMed

Prati, Federica; Zuccotto, Fabio; Fletcher, Daniel; Convery, Maire A; Fernandez-Menendez, Raquel; Bates, Robert; Encinas, Lourdes; Zeng, Jingkun; Chung, Chun-Wa; De Dios Anton, Paco; Mendoza-Losana, Alfonso; Mackenzie, Claire; Green, Simon R; Huggett, Margaret; Barros, David; Wyatt, Paul G; Ray, Peter C

2018-04-06

Our findings reported herein provide support for the benefits of including functional group complexity (FGC) within fragments when screening against protein targets such as Mycobacterium tuberculosis InhA. We show that InhA fragment actives with FGC maintained their binding pose during elaboration. Furthermore, weak fragment hits with functional group handles also allowed for facile fragment elaboration to afford novel and potent InhA inhibitors with good ligand efficiency metrics for optimization. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Elucidating the druggable interface of protein-protein interactions using fragment docking and coevolutionary analysis.

PubMed

Bai, Fang; Morcos, Faruck; Cheng, Ryan R; Jiang, Hualiang; Onuchic, José N

2016-12-13

Protein-protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein-protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

[Color processing of ultrasonographic images in extracorporeal lithotripsy].

PubMed

Lardennois, B; Ziade, A; Walter, K

1991-02-01

A number of technical difficulties are encountered in the ultrasonographic detection of renal stones which unfortunately limit its performance. The margin of error of firing in extracorporeal shock-wave lithotripsy (ESWL) must be reduced to a minimum. The role of the ultrasonographic monitoring during lithotripsy is also essential: continuous control of the focussing of the short-wave beamand assessment if the quality of fragmentation. The authors propose to improve ultrasonographic imaging in ESWL by means of intraoperative colour processing of the stone. Each shot must be directed to its target with an economy of vision avoiding excessive fatigue. The principle of the technique consists of digitalization of the ultrasound video images using a Macintosh Mac 2 computer. The Graphis Paint II program is interfaced directly with the Quick Capture card and recovers the images on its work surface in real time. The program is then able to attribute to each of these 256 shades of grey any one of the 16.6 million colours of the Macintosh universe with specific intensity and saturation. During fragmentation, using the principle of a palette, the stone changes colour from green to red indicating complete fragmentation. A Color Space card converts the digital image obtained into a video analogue source which is visualized on the monitor. It can be superimposed and/or juxtaposed with the source image by means of a multi-standard mixing table. Colour processing of ultrasonographic images in extracoporeal shockwave lithotripsy allows better visualization of the stones and better follow-up of fragmentation and allows the shockwave treatment to be stopped earlier. It increases the stone-free performance at 6 months. This configuration will eventually be able to integrate into the ultrasound apparatus itself.

Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

PubMed

Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

2018-06-29

The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Electromagnetic Dissociation Cross Sections for High LET Fragments

NASA Technical Reports Server (NTRS)

Norbury, John

2016-01-01

Nuclear interaction cross sections are used in space radiation transport codes to calculate the probability of fragment emission in high energy nucleus-nucleus collisions. Strong interactions usually dominate in these collisions, but electromagnetic (EM) interactions can also sometimes be important. Strong interactions typically occur when the projectile nucleus hits a target nucleus, with a small impact parameter. For impact parameters larger than the sum of the nuclear radii, EM reactions dominate and the process is called electromagnetic dissociation (EMD) if one of the nuclei undergo fragmentation. Previous models of EMD have been used to calculate single proton (p) production, single neutron (n) production or light ion production, where a light ion is defined as an isotope of hydrogen (H) or helium (He), such as a deuteron (2H), a triton (3H), a helion (3He) or an alpha particle (4He). A new model is described which can also account for multiple nucleon production, such as 2p, 2n, 1p1n, 2p1n, 2p2n, etc. in addition to light ion production. Such processes are important to include for the following reasons. Consider, for example, the EMD reaction 56Fe + Al --> 52Cr + X + Al, for a 56Fe projectile impacting Al, which produces the high linear energy transfer (LET) fragment 52Cr. In this reaction, the most probable particles representing X are either 2p2n or 4He. Therefore, production of the high LET fragment 52Cr, must include the multiple nucleon production of 2p2n in addition to the light ion production of 4He. Previous models, such as the NUCFRG3 model, could only account for the 4He production process in this reaction and could not account for 2p2n. The new EMD model presented in this work accounts for both the light ion and multiple nucleon processes, and is therefore able to correctly account for the production of high LET products such as 52Cr. The model will be described and calculations will be presented that show the importance of light ion and multiple nucleon production. The work will also show that EMD reactions contribute most to those fragments with the highest LET.

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

PubMed Central

2017-01-01

Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation. PMID:28633355

Fragment-based screening by protein crystallography: successes and pitfalls.

PubMed

Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J

2012-10-08

Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality.

Fragment-Based Screening by Protein Crystallography: Successes and Pitfalls

PubMed Central

Chilingaryan, Zorik; Yin, Zhou; Oakley, Aaron J.

2012-01-01

Fragment-based drug discovery (FBDD) concerns the screening of low-molecular weight compounds against macromolecular targets of clinical relevance. These compounds act as starting points for the development of drugs. FBDD has evolved and grown in popularity over the past 15 years. In this paper, the rationale and technology behind the use of X-ray crystallography in fragment based screening (FBS) will be described, including fragment library design and use of synchrotron radiation and robotics for high-throughput X-ray data collection. Some recent uses of crystallography in FBS will be described in detail, including interrogation of the drug targets β-secretase, phenylethanolamine N-methyltransferase, phosphodiesterase 4A and Hsp90. These examples provide illustrations of projects where crystallography is straightforward or difficult, and where other screening methods can help overcome the limitations of crystallography necessitated by diffraction quality. PMID:23202926

Design of Potent and Druglike Nonphenolic Inhibitors for Catechol O-Methyltransferase Derived from a Fragment Screening Approach Targeting the S-Adenosyl-l-methionine Pocket.

PubMed

Lerner, Christian; Jakob-Roetne, Roland; Buettelmann, Bernd; Ehler, Andreas; Rudolph, Markus; Rodríguez Sarmiento, Rosa María

2016-11-23

A fragment screening approach designed to target specifically the S-adenosyl-l-methionine pocket of catechol O-methyl transferase allowed the identification of structurally related fragments of high ligand efficiency and with activity on the described orthogonal assays. By use of a reliable enzymatic assay together with X-ray crystallography as guidance, a series of fragment modifications revealed an SAR and, after several expansions, potent lead compounds could be obtained. For the first time nonphenolic and small low nanomolar potent, SAM competitive COMT inhibitors are reported. These compounds represent a novel series of potent COMT inhibitors that might be further optimized to new drugs useful for the treatment of Parkinson's disease, as adjuncts in levodopa based therapy, or for the treatment of schizophrenia.

NMR characterization of weak interactions between RhoGDI2 and fragment screening hits.

PubMed

Liu, Jiuyang; Gao, Jia; Li, Fudong; Ma, Rongsheng; Wei, Qingtao; Wang, Aidong; Wu, Jihui; Ruan, Ke

2017-01-01

The delineation of intrinsically weak interactions between novel targets and fragment screening hits has long limited the pace of hit-to-lead evolution. Rho guanine-nucleotide dissociation inhibitor 2 (RhoGDI2) is a novel target that lacks any chemical probes for the treatment of tumor metastasis. Protein-observed and ligand-observed NMR spectroscopy was used to characterize the weak interactions between RhoGDI2 and fragment screening hits. We identified three hits of RhoGDI2 using streamlined NMR fragment-based screening. The binding site residues were assigned using non-uniformly sampled C α - and H α -based three dimensional NMR spectra. The molecular docking to the proposed geranylgeranyl binding pocket of RhoGDI2 was guided by NMR restraints of chemical shift perturbations and ligand-observed transferred paramagnetic relaxation enhancement. We further validated the weak RhoGDI2-hit interactions using mutagenesis and structure-affinity analysis. Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach. Binders to RhoGDI2 as a potential anti-cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. Our work highlights the powerfulness and the versatility of the integrative NMR techniques to provide valuable structural insight into the intrinsically weak interactions between RhoGDI2 and the fragment screening hits, which could hardly be conceived using other biochemical techniques. Copyright © 2016 Elsevier B.V. All rights reserved.

Target fragmentation in proton-nucleus and16O-nucleus reactions at 60 and 200 GeV/nucleon

NASA Astrophysics Data System (ADS)

Albrecht, R.; Awes, T. C.; Baktash, C.; Beckmann, P.; Claesson, G.; Berger, F.; Bock, R.; Dragon, L.; Ferguson, R. L.; Franz, A.; Garpman, S.; Glasow, R.; Gustafsson, H. Å.; Gutbrod, H. H.; Kampert, K. H.; Kolb, B. W.; Kristiansson, P.; Lee, I. Y.; Löhner, H.; Lund, I.; Obenshain, F. E.; Oskarsson, A.; Otterlund, I.; Peitzmann, T.; Persson, S.; Plasil, F.; Poskanzer, A. M.; Purschke, M.; Ritter, H. G.; Santo, R.; Schmidt, H. R.; Siemiarczuk, T.; Sorensen, S. P.; Stenlund, E.; Young, G. R.

1988-03-01

Target remnants with Z<3 from proton-nucleus and16O-nucleus reactions at 60 and 200 GeV/nucleon were measured in the angular range from 30° to 160° (-1.7<η<1.3) employing the Plastic Ball detector. The excitation energy of the target spectator matter in central oxygen-induced collisions is found to be high enough to allow for complete disintegration of the target nucleus into fragments with Z<3. The average longitudinal momentum transfer per proton to the target in central collisions is considerably higher in the case of16O-induced reactions (≈300 MeV/c) than in proton-induced reactions (≈130 MeV/c). The baryon rapidity distributions are roughly in agreement with one-fluid hydrodynamical calculations at 60 GeV/nucleon16O+Au but are in disagreement at 200 GeV/nucleon, indicating the higher degree of transparency at the higher bombarding energy. Both, the transverse momenta of target spectators and the entropy produced in the target fragmentation region are compared to those attained in head-on collisions of two heavy nuclei at Bevalac energies. They are found to be comparable or do even exceed the values for the participant matter at beam energies of about 1 2 GeV/nucleon.

Epitope mapping of commercial antibodies that detect myocilin.

PubMed

Patterson-Orazem, Athéna C; Hill, Shannon E; Fautsch, Michael P; Lieberman, Raquel L

2018-05-09

The presence of myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial myocilin antibodies, aided by structurally and biochemically characterized myocilin fragments. All four antibodies recognize enriched myocilin secreted from human TM cell media. The detection of myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the myocilin polypeptide chain. A more precise understanding of myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Secondary radiation measurements for particle therapy applications: nuclear fragmentation produced by 4He ion beams in a PMMA target

NASA Astrophysics Data System (ADS)

Marafini, M.; Paramatti, R.; Pinci, D.; Battistoni, G.; Collamati, F.; De Lucia, E.; Faccini, R.; Frallicciardi, P. M.; Mancini-Terracciano, C.; Mattei, I.; Muraro, S.; Piersanti, L.; Rovituso, M.; Rucinski, A.; Russomando, A.; Sarti, A.; Sciubba, A.; Solfaroli Camillocci, E.; Toppi, M.; Traini, G.; Voena, C.; Patera, V.

2017-02-01

Nowadays there is a growing interest in particle therapy treatments exploiting light ion beams against tumors due to their enhanced relative biological effectiveness and high space selectivity. In particular promising results are obtained by the use of 4He projectiles. Unlike the treatments performed using protons, the beam ions can undergo a fragmentation process when interacting with the atomic nuclei in the patient body. In this paper the results of measurements performed at the Heidelberg Ion-Beam Therapy center are reported. For the first time the absolute fluxes and the energy spectra of the fragments—protons, deuterons, and tritons—produced by 4He ion beams of 102, 125 and 145 MeV u-1 energies on a poly-methyl methacrylate target were evaluated at different angles. The obtained results are particularly relevant in view of the necessary optimization and review of the treatment planning software being developed for clinical use of 4He beams in clinical routine and the relative bench-marking of Monte Carlo algorithm predictions.

Using Data Independent Acquisition (DIA) to Model High-responding Peptides for Targeted Proteomics Experiments*

PubMed Central

Searle, Brian C.; Egertson, Jarrett D.; Bollinger, James G.; Stergachis, Andrew B.; MacCoss, Michael J.

2015-01-01

Targeted mass spectrometry is an essential tool for detecting quantitative changes in low abundant proteins throughout the proteome. Although selected reaction monitoring (SRM) is the preferred method for quantifying peptides in complex samples, the process of designing SRM assays is laborious. Peptides have widely varying signal responses dictated by sequence-specific physiochemical properties; one major challenge is in selecting representative peptides to target as a proxy for protein abundance. Here we present PREGO, a software tool that predicts high-responding peptides for SRM experiments. PREGO predicts peptide responses with an artificial neural network trained using 11 minimally redundant, maximally relevant properties. Crucial to its success, PREGO is trained using fragment ion intensities of equimolar synthetic peptides extracted from data independent acquisition experiments. Because of similarities in instrumentation and the nature of data collection, relative peptide responses from data independent acquisition experiments are a suitable substitute for SRM experiments because they both make quantitative measurements from integrated fragment ion chromatograms. Using an SRM experiment containing 12,973 peptides from 724 synthetic proteins, PREGO exhibits a 40–85% improvement over previously published approaches at selecting high-responding peptides. These results also represent a dramatic improvement over the rules-based peptide selection approaches commonly used in the literature. PMID:26100116

Collapse of chain anadiplosis-structured DNA nanowires for highly sensitive colorimetric assay of nucleic acids.

PubMed

Xu, Jianguo; Wu, Zai-Sheng; Chen, Yanru; Zheng, Tingting; Le, Jingqing; Jia, Lee

2017-02-14

In this work, we have proposed a chain anadiplosis-structured DNA nanowire by using two well-defined assembly strands (AS1 and AS2). The presence of a target analyte would drive the single-stranded AS1 dissociate from the pre-formatted nanowire, converting into a fully double-stranded form responsible for extensive accumulation of G-rich cleavage fragment1 (GCF1) because of an autonomously performed polymerization/nicking/displacement process. In turn, the produced GCF1 is able to hybridize with the un-peeled AS2, allowing the replication over AS2 to occur and generate large amounts of G-rich cleavage fragment2 (GCF2) with the ability to hybridize with the un-peeled AS1, thereafter initiating new enzymatic reactions for further collection of GCF1. Because the reactions occur repeatedly, the assembled nanowires gradually dissociated and completely collapsed in the end, achieving the goal of substantial signal amplification for the colorimetric readout of the target analytes. The sensing feasibility is firstly verified by one trigger primer (TP), and then exemplified with the detection of the target, the kras oncogene, with high sensitivity and specificity. As a proof-of-concept strategy, the intelligent signal readout pathway and desired assay ability provide unique insights into the materials research and biological studies.

Targeted microbubbles: a novel application for the treatment of kidney stones.

PubMed

Ramaswamy, Krishna; Marx, Vanessa; Laser, Daniel; Kenny, Thomas; Chi, Thomas; Bailey, Michael; Sorensen, Mathew D; Grubbs, Robert H; Stoller, Marshall L

2015-07-01

Kidney stone disease is endemic. Extracorporeal shockwave lithotripsy was the first major technological breakthrough where focused shockwaves were used to fragment stones in the kidney or ureter. The shockwaves induced the formation of cavitation bubbles, whose collapse released energy at the stone, and the energy fragmented the kidney stones into pieces small enough to be passed spontaneously. Can the concept of microbubbles be used without the bulky machine? The logical progression was to manufacture these powerful microbubbles ex vivo and inject these bubbles directly into the collecting system. An external source can be used to induce cavitation once the microbubbles are at their target; the key is targeting these microbubbles to specifically bind to kidney stones. Two important observations have been established: (i) bisphosphonates attach to hydroxyapatite crystals with high affinity; and (ii) there is substantial hydroxyapatite in most kidney stones. The microbubbles can be equipped with bisphosphonate tags to specifically target kidney stones. These bubbles will preferentially bind to the stone and not surrounding tissue, reducing collateral damage. Ultrasound or another suitable form of energy is then applied causing the microbubbles to induce cavitation and fragment the stones. This can be used as an adjunct to ureteroscopy or percutaneous lithotripsy to aid in fragmentation. Randall's plaques, which also contain hydroxyapatite crystals, can also be targeted to pre-emptively destroy these stone precursors. Additionally, targeted microbubbles can aid in kidney stone diagnostics by virtue of being used as an adjunct to traditional imaging methods, especially useful in high-risk patient populations. This novel application of targeted microbubble technology not only represents the next frontier in minimally invasive stone surgery, but a platform technology for other areas of medicine. © 2014 The Authors BJU International © 2014 BJU International Published by John Wiley & Sons Ltd.

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

Chen, C.; Albergo, S.; Caccia, Z.

A liquid hydrogen target was used to study the nuclear fragmentation of beams of relativistic heavy ions, [sup 22]Ne to [sup 58]Ni, over an energy range 400 to 900 MeV/nucleon. The experiments were carried out at the Lawrence Berkeley Laboratory Bevalac HISS facility, using the charge-velocity-rigidity method to identify the charged fragments. Here we describe the general concept of the experiment and present total charge-changing cross sections obtained from 17 separate runs. These new measured cross sections display an energy dependence which follows semiempirical model predictions. The mass dependence of the cross sections behaves as predicted by optical models, butmore » within the experimental energy range, the optical model parameters display a clear energy dependence. The isospin of the projectile nuclei also appears to be an important factor in the interaction process.« less

NMR-based platform for fragment-based lead discovery used in screening BRD4-targeted compounds

PubMed Central

Yu, Jun-lan; Chen, Tian-tian; Zhou, Chen; Lian, Fu-lin; Tang, Xu-long; Wen, Yi; Shen, Jing-kang; Xu, Ye-chun; Xiong, Bing; Zhang, Nai-xia

2016-01-01

Aim: Fragment-based lead discovery (FBLD) is a complementary approach in drug research and development. In this study, we established an NMR-based FBLD platform that was used to screen novel scaffolds targeting human bromodomain of BRD4, and investigated the binding interactions between hit compounds and the target protein. Methods: 1D NMR techniques were primarily used to generate the fragment library and to screen compounds. The inhibitory activity of hits on the first bromodomain of BRD4 [BRD4(I)] was examined using fluorescence anisotropy binding assay. 2D NMR and X-ray crystallography were applied to characterize the binding interactions between hit compounds and the target protein. Results: An NMR-based fragment library containing 539 compounds was established, which were clustered into 56 groups (8–10 compounds in each group). Eight hits with new scaffolds were found to inhibit BRD4(I). Four out of the 8 hits (compounds 1, 2, 8 and 9) had IC50 values of 100–260 μmol/L, demonstrating their potential for further BRD4-targeted hit-to-lead optimization. Analysis of the binding interactions revealed that compounds 1 and 2 shared a common quinazolin core structure and bound to BRD4(I) in a non-acetylated lysine mimetic mode. Conclusion: An NMR-based platform for FBLD was established and used in discovery of BRD4-targeted compounds. Four potential hit-to-lead optimization candidates have been found, two of them bound to BRD4(I) in a non-acetylated lysine mimetic mode, being selective BRD4(I) inhibitors. PMID:27238211

D meson production asymmetry, unfavored fragmentation, and consequences for prompt atmospheric neutrino production

NASA Astrophysics Data System (ADS)

Maciuła, Rafał; Szczurek, Antoni

2018-04-01

We consider unfavored light quark/antiquark to D meson fragmentation. We discuss nonperturbative effects for small transverse momenta. The asymmetry for D+ and D- production measured by the LHCb collaboration provides natural constraints on the parton (quark/antiquark) fragmentation functions. We find that already a fraction of q /q ¯→D fragmentation probability is sufficient to account for the measured asymmetry. We make predictions for similar asymmetry for neutral D mesons. Large D -meson production asymmetries are found for large xF which is related to dominance of light quark/antiquark q /q ¯→D fragmentation over the standard c →D fragmentation. As a consequence, prompt atmospheric neutrino flux at high neutrino energies can be much larger than for the conventional c →D fragmentation. The latter can constitute a sizeable background for the cosmic neutrinos claimed to be observed recently by the IceCube Observatory. Large rapidity-dependent D+/D- and D0/D¯0 asymmetries are predicted for low (√{s }=20 - 100 GeV ) energies. The q /q ¯→D fragmentation leads to enhanced production of D mesons at low energies. At √{s }=20 GeV the enhancement factor with respect to the conventional contribution is larger than a factor of five. In the considered picture the large-xF D mesons are produced dominantly via fragmentation of light quarks/antiquarks. Predictions for fixed target p + 4He collisions relevant for a fixed target LHCb experiment are presented.

Fragment informatics and computational fragment-based drug design: an overview and update.

PubMed

Sheng, Chunquan; Zhang, Wannian

2013-05-01

Fragment-based drug design (FBDD) is a promising approach for the discovery and optimization of lead compounds. Despite its successes, FBDD also faces some internal limitations and challenges. FBDD requires a high quality of target protein and good solubility of fragments. Biophysical techniques for fragment screening necessitate expensive detection equipment and the strategies for evolving fragment hits to leads remain to be improved. Regardless, FBDD is necessary for investigating larger chemical space and can be applied to challenging biological targets. In this scenario, cheminformatics and computational chemistry can be used as alternative approaches that can significantly improve the efficiency and success rate of lead discovery and optimization. Cheminformatics and computational tools assist FBDD in a very flexible manner. Computational FBDD can be used independently or in parallel with experimental FBDD for efficiently generating and optimizing leads. Computational FBDD can also be integrated into each step of experimental FBDD and help to play a synergistic role by maximizing its performance. This review will provide critical analysis of the complementarity between computational and experimental FBDD and highlight recent advances in new algorithms and successful examples of their applications. In particular, fragment-based cheminformatics tools, high-throughput fragment docking, and fragment-based de novo drug design will provide the focus of this review. We will also discuss the advantages and limitations of different methods and the trends in new developments that should inspire future research. © 2012 Wiley Periodicals, Inc.

A machine learning approach to computer-aided molecular design

NASA Astrophysics Data System (ADS)

Bolis, Giorgio; Di Pace, Luigi; Fabrocini, Filippo

1991-12-01

Preliminary results of a machine learning application concerning computer-aided molecular design applied to drug discovery are presented. The artificial intelligence techniques of machine learning use a sample of active and inactive compounds, which is viewed as a set of positive and negative examples, to allow the induction of a molecular model characterizing the interaction between the compounds and a target molecule. The algorithm is based on a twofold phase. In the first one — the specialization step — the program identifies a number of active/inactive pairs of compounds which appear to be the most useful in order to make the learning process as effective as possible and generates a dictionary of molecular fragments, deemed to be responsible for the activity of the compounds. In the second phase — the generalization step — the fragments thus generated are combined and generalized in order to select the most plausible hypothesis with respect to the sample of compounds. A knowledge base concerning physical and chemical properties is utilized during the inductive process.

Fast and Efficient Fragment-Based Lead Generation by Fully Automated Processing and Analysis of Ligand-Observed NMR Binding Data.

PubMed

Peng, Chen; Frommlet, Alexandra; Perez, Manuel; Cobas, Carlos; Blechschmidt, Anke; Dominguez, Santiago; Lingel, Andreas

2016-04-14

NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.

From small to powerful: the fragments universe and its "chem-appeal".

PubMed

Sancineto, Luca; Massari, Serena; Iraci, Nunzio; Tabarrini, Oriana

2013-01-01

While increasing expertise in molecular biology and proteomics is markedly speeding up the target elucidation process, various strategies have been proposed that improve the chances of identifying active molecules. Among them, the Fragment Based Drug Design (FBDD) is surely worth noting. The FBDD entails the screening of a small number of low molecular weight compounds in the hopes of finding even low affine but high ligand efficient fragments that have high probability to became drug candidates. Since 1996, when the first paper on FBDD was reported, the potentialities of this strategy became progressively more apparent as testified by the growing number of publications. Many drug discovery projects started with the identification of fragments which after the optimization gave many molecules close to the approval and one marketed drug Vemurafenib, approved in 2011. A preamble that highlights the advantages of dealing with simple and "very small" molecules over conventional drug-like compounds will be herein given prior to discussing the canonical FBDD stages, from fragment library design, to the different screening methods concluding with the various optimization strategies, in an attempt to illustrate the whole FBDD workflow while discussing the most recent and successful applications. While this review is a tribute to the success achieved by the researchers in this field, it is particularly addressed to scientists who want to become aware of the versatility and potentiality of FBDD.

Fragmentation of Structural Energetic Materials: Implications for Performance

NASA Astrophysics Data System (ADS)

Aydelotte, Brady; Braithwaite, Christopher; Thadhani, Naresh

2013-06-01

Fragmentation results for structural energetic materials based on intermetallic forming mixtures are reviewed and the implications of the fragment populations are discussed. Cold Sprayed Ni+Al and explosively compacted mixtures of Ni+Al+W and Ni+Al+W+Zr powders were fabricated into ring shaped samples and subjected to fragmentation tests. Ring velocity was monitored and fragments were soft captured in order to study the fragmentation process. It was determined that the fragments produced by these structural energetic materials are much smaller than those typically produced by ductile metals such as steel or aluminum. This has implications for combustion processes that may occur subsequent to the fragmentation process. ONR/MURI grant No. N00014-07-1-0740 Dr. Cliff Bedford PM.

Growth and regeneration in cultivated fragments of the boreal deep water sponge Geodia barretti Bowerbank, 1858 (Geodiidae, Tetractinellida, Demospongiae).

PubMed

Hoffmann, Friederike; Rapp, Hans Tore; Zöller, Tobias; Reitner, Joachim

2003-01-23

A cultivation method has been developed for the boreal deep-water sponge Geodia barretti (Demospongiae, Geodiidae), a species which is common in the deep Norwegian fjords. The species is known to contain secondary metabolites which are biologically active. Choanosomal fragments of 2-4 cm(3) (approximately 3-7 g) were kept in half-open systems. Cicatrisation and regeneration processes were surveyed by histological examination during 8 months of cultivation. During the first weeks, the weight of the fragments decreased. However, after about 6 weeks the weight equalled the original weight, and after 1 year the weight had increased by about 40% compared to the original weight. The initial decrease was due to complex healing processes and the regeneration of the cortex, a sterrastral layer typical for the family of the Geodiidae. We document, for the first time, the complete cortex reconstruction in an adult G. barretti, as well as the development of egg cells during cultivation. Our study represents the first attempt at biotechnological production of boreal sponge tissue. For successful farming of G. barretti and other boreal and arctic sponges, however, further investigation is needed on factors stimulating growth and secondary metabolite production in the target species.

Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: A case report.

PubMed

Cavalcanti, Amanda; Lobo, Rogério; Cupolillo, Elisa; Bustamante, Fábio; Porrozzi, Renato

2012-12-01

Visceral leishmaniasis is an anthropozoonosis caused by a protozoan Leishmania infantum (syn. Leishmania chagasi). Here, we report a typical case of canine cutaneous leishmaniasis due to L. infantum infection without any other systemic symptom in one dog in the city of Rio de Janeiro, Brazil. A mongrel female dog was admitted in a veterinary clinic with reports of chronic wounds in the body. Physical examination revealed erosive lesions in the limbs, nasal ulcers, presence of ectoparasites and seborrheic dermatitis. Blood samples and fragments of healthy and injured skin were collected. The complete hemogram revealed aregenerative normocytic normochromic anemia and erythrocyte rouleaux, and biochemical analysis revealed normal renal and hepatic functions. Cytology of the muzzle and skin lesions suggested pyogranulomatous inflammatory process. The histopathology of a skin fragment was performed and revealed suspicion of protozoa accompanied by necrotizing dermatitis. The diagnosis of leishmaniasis was accomplished by positive serology, isolation of Leishmania from the skin lesion, and also by molecular test (PCR targeting the conserved region of Leishmania kDNA). Culture was positive for damaged skin samples. PCR targeting a fragment of Leishmania hsp70 gene was performed employing DNA extracted from damaged skin. RFLP of the amplified hsp70 fragment identified the parasite as L. infantum, instead of Leishmania braziliensis, the main agent of cutaneous leishmaniasis in Rio de Janeiro. Characterization of isolated promastigotes by five different enzymatic systems confirmed the species identification of the etiological agent. Serology was positive by ELISA and rapid test. This case warns to the suspicion of viscerotropic Leishmania in cases of chronic skin lesions and brings the discussion of the mechanisms involved in the parasite tissue tropism. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Fragmentation cross sections of medium-energy {sup 35}Cl, {sup 40}Ar, and {sup 48}Ti beams on elemental targets

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

Zeitlin, C.; Guetersloh, S.; Heilbronn, L.

Charge-changing and fragment production cross sections at 0 deg. have been obtained for interactions of 290, 400, and 650 MeV/nucleon {sup 40}Ar beams, 650 and 1000 MeV/nucleon {sup 35}Cl beams, and a 1000 MeV/nucleon {sup 48}Ti beam. Targets of C, CH{sub 2}, Al, Cu, Sn, and Pb were used. Using standard analysis methods, we obtained fragment cross sections for charges as low as 8 for Cl and Ar beams and as low as 10 for the Ti beam. Using data obtained with small-acceptance detectors, we report fragment production cross sections for charges as low as 5, corrected for acceptance usingmore » a simple model of fragment angular distributions. With the lower-charged fragment cross sections, we can compare the data to predictions from several models (including NUCFRG2, EPAX2, and PHITS) in a region largely unexplored in earlier work. As found in earlier work with other beams, NUCFRG2 and PHITS predictions agree reasonably well with the data for charge-changing cross sections, but these models do not accurately predict the fragment production cross sections. The cross sections for the lightest fragments demonstrate the inadequacy of several models in which the cross sections fall monotonically with the charge of the fragment. PHITS, despite its not agreeing particularly well with the fragment production cross sections on average, nonetheless qualitatively reproduces some significant features of the data that are missing from the other models.« less

Fragment screening for drug leads by weak affinity chromatography (WAC-MS).

PubMed

Ohlson, Sten; Duong-Thi, Minh-Dao

2018-02-23

Fragment-based drug discovery is an important tool for design of small molecule hit-to-lead compounds against various biological targets. Several approved drugs have been derived from an initial fragment screen and many such candidates are in various stages of clinical trials. Finding fragment hits, that are suitable for optimisation by medicinal chemists, is still a challenge as the binding between the small fragment and its target is weak in the range of mM to µM of K d and irrelevant non-specific interactions are abundant in this area of transient interactions. Fortunately, there are methods that can study weak interactions quite efficiently of which NMR, surface plasmon resonance (SPR) and X-ray crystallography are the most prominent. Now, a new technology based on zonal affinity chromatography, weak affinity chromatography (WAC), has been introduced which has remedied many of the problems with other technologies. By combining WAC with mass spectrometry (WAC-MS), it is a powerful tool to identify binders quantitatively in terms of affinity and kinetics either from fragment libraries or from complex mixtures of biological extracts. As WAC-MS can be multiplexed by analysing mixtures of fragments (20-100 fragments) in one sample, this approach yields high throughput, where a whole library of e.g. >2000 fragments can be analysed quantitatively within a day. WAC-MS is easy to perform, where the robustness and quality of HPLC is fully utilized. This review will highlight the rationale behind the application of WAC-MS for fragment screening in drug discovery. Copyright © 2018 Elsevier Inc. All rights reserved.

Despacito: the slow evolutionary changes in plant microRNAs.

PubMed

Baldrich, Patricia; Beric, Aleksandra; Meyers, Blake C

2018-02-12

MicroRNAs (miRNAs) are key regulators of gene expression. A handful of miRNAs are broadly conserved in land plants, while the majority are lineage specific; this review describes the processes by which new miRNAs are hypothesized to have emerged. Two major models describe miRNA origins, firstly, de novo emergence via inverted duplication of target gene fragments, and secondly, the expansion and neofunctionalization of existing miRNA families. The occasional acquisition of target sites by previously un-targeted genes adds further dynamism to the process by which miRNAs may shift roles during evolution. Additional factors guiding miRNA evolution include functional constraints on their length and the importance of precursor conservation that is observed in regions above or below the mature miRNA duplex; these regions represent recognition sites for components of biogenesis machinery and direct precursor processing. Insights into the mechanisms of miRNA emergence and divergence are important for understanding plant genome evolution and the impact of miRNA regulatory networks. Copyright © 2018 Elsevier Ltd. All rights reserved.

Aligning physics and physiology: Engineering antibodies for radionuclide delivery.

PubMed

Tsai, Wen-Ting K; Wu, Anna M

2018-03-14

The exquisite specificity of antibodies and antibody fragments renders them excellent agents for targeted delivery of radionuclides. Radiolabeled antibodies and fragments have been successfully used for molecular imaging and radioimmunotherapy (RIT) of cell surface targets in oncology and immunology. Protein engineering has been used for antibody humanization essential for clinical applications, as well as optimization of important characteristics including pharmacokinetics, biodistribution, and clearance. Although intact antibodies have high potential as imaging and therapeutic agents, challenges include long circulation time in blood, which leads to later imaging time points post-injection and higher blood absorbed dose that may be disadvantageous for RIT. Using engineered fragments may address these challenges, as size reduction and removal of Fc function decreases serum half-life. Radiolabeled fragments and pretargeting strategies can result in high contrast images within hours to days, and a reduction of RIT toxicity in normal tissues. Additionally, fragments can be engineered to direct hepatic or renal clearance, which may be chosen based on the application and disease setting. This review discusses aligning the physical properties of radionuclides (positron, gamma, beta, alpha, and Auger emitters) with antibodies and fragments and highlights recent advances of engineered antibodies and fragments in preclinical and clinical development for imaging and therapy. Copyright © 2018 John Wiley & Sons, Ltd.

Fragment-based drug discovery and its application to challenging drug targets.

PubMed

Price, Amanda J; Howard, Steven; Cons, Benjamin D

2017-11-08

Fragment-based drug discovery (FBDD) is a technique for identifying low molecular weight chemical starting points for drug discovery. Since its inception 20 years ago, FBDD has grown in popularity to the point where it is now an established technique in industry and academia. The approach involves the biophysical screening of proteins against collections of low molecular weight compounds (fragments). Although fragments bind to proteins with relatively low affinity, they form efficient, high quality binding interactions with the protein architecture as they have to overcome a significant entropy barrier to bind. Of the biophysical methods available for fragment screening, X-ray protein crystallography is one of the most sensitive and least prone to false positives. It also provides detailed structural information of the protein-fragment complex at the atomic level. Fragment-based screening using X-ray crystallography is therefore an efficient method for identifying binding hotspots on proteins, which can then be exploited by chemists and biologists for the discovery of new drugs. The use of FBDD is illustrated here with a recently published case study of a drug discovery programme targeting the challenging protein-protein interaction Kelch-like ECH-associated protein 1:nuclear factor erythroid 2-related factor 2. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The new double energy-velocity spectrometer VERDI

NASA Astrophysics Data System (ADS)

Jansson, Kaj; Frégeau, Marc Olivier; Al-Adili, Ali; Göök, Alf; Gustavsson, Cecilia; Hambsch, Franz-Josef; Oberstedt, Stephan; Pomp, Stephan

2017-09-01

VERDI (VElocity foR Direct particle Identification) is a fission-fragment spectrometer recently put into operation at JRC-Geel. It allows measuring the kinetic energy and velocity of both fission fragments simultaneously. The velocity provides information about the pre-neutron mass of each fission fragment when isotropic prompt-neutron emission from the fragments is assumed. The kinetic energy, in combination with the velocity, provides the post-neutron mass. From the difference between pre- and post-neutron masses, the number of neutrons emitted by each fragment can be determined. Multiplicity as a function of fragment mass and total kinetic energy is one important ingredient, essential for understanding the sharing of excitation energy between fission fragments at scission, and may be used to benchmark nuclear de-excitation models. The VERDI spectrometer design is a compromise between geometrical efficiency and mass resolution. The spectrometer consists of an electron detector located close to the target and two arrays of silicon detectors, each located 50 cm away from the target. In the present configuration pre-neutron and post-neutron mass distributions are in good agreement with reference data were obtained. Our latest measurements performed with spontaneously fissioning 252Cf is presented along with the developed calibration procedure to obtain pulse height defect and plasma delay time corrections.

Engineering Archeal Surrogate Systems for the Development of Protein-Protein Interaction Inhibitors against Human RAD51.

PubMed

Moschetti, Tommaso; Sharpe, Timothy; Fischer, Gerhard; Marsh, May E; Ng, Hong Kin; Morgan, Matthew; Scott, Duncan E; Blundell, Tom L; R Venkitaraman, Ashok; Skidmore, John; Abell, Chris; Hyvönen, Marko

2016-11-20

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Model Cortical Association Fields Account for the Time Course and Dependence on Target Complexity of Human Contour Perception

PubMed Central

Gintautas, Vadas; Ham, Michael I.; Kunsberg, Benjamin; Barr, Shawn; Brumby, Steven P.; Rasmussen, Craig; George, John S.; Nemenman, Ilya; Bettencourt, Luís M. A.; Kenyon, Garret T.

2011-01-01

Can lateral connectivity in the primary visual cortex account for the time dependence and intrinsic task difficulty of human contour detection? To answer this question, we created a synthetic image set that prevents sole reliance on either low-level visual features or high-level context for the detection of target objects. Rendered images consist of smoothly varying, globally aligned contour fragments (amoebas) distributed among groups of randomly rotated fragments (clutter). The time course and accuracy of amoeba detection by humans was measured using a two-alternative forced choice protocol with self-reported confidence and variable image presentation time (20-200 ms), followed by an image mask optimized so as to interrupt visual processing. Measured psychometric functions were well fit by sigmoidal functions with exponential time constants of 30-91 ms, depending on amoeba complexity. Key aspects of the psychophysical experiments were accounted for by a computational network model, in which simulated responses across retinotopic arrays of orientation-selective elements were modulated by cortical association fields, represented as multiplicative kernels computed from the differences in pairwise edge statistics between target and distractor images. Comparing the experimental and the computational results suggests that each iteration of the lateral interactions takes at least ms of cortical processing time. Our results provide evidence that cortical association fields between orientation selective elements in early visual areas can account for important temporal and task-dependent aspects of the psychometric curves characterizing human contour perception, with the remaining discrepancies postulated to arise from the influence of higher cortical areas. PMID:21998562

Successful de-fragmentation of woodland by planting in an agricultural landscape? An assessment based on landscape indicators.

PubMed

Quine, C P; Watts, K

2009-01-01

Habitat fragmentation is the focus of much conservation concern and associated research. In some countries, such as Britain, the main phase of fragmentation occurred centuries ago and the focus of conservation management is now on restoration and recovery. Scenario studies have suggested that spatial targeting is preferable if landscape scale restoration is to be achieved, and that this should bring greater benefits than site-focussed activities but this has rarely been tested in practice. In Britain, woodland expansion has been encouraged through a number of financial incentives, which have evolved from instruments that encouraged almost any addition to the potential woodland resource, to grant schemes that have set out to restore connectivity to remnant ancient woodland. This study assessed the degree of de-fragmentation achieved by woodland expansion on the Isle of Wight and in particular the success of spatial targeting of new woodland planting implemented through grant aid in the JIGSAW (Joining and Increasing Grant Scheme for Ancient Woodland) scheme. Five steps in the re-development of broad-leaved woodland were tested using eight indicators - six commonly used landscape metrics, and two ecologically scaled indicators derived from application of least-cost network evaluation. Only half of the measures indicated de-fragmentation over the whole sequence of five steps. However, the spatial targeting did appear successful, when compared to equivalent untargeted grant-aided woodland expansion, and resulted in positive change to six of the eight indicators. We discuss the utility of the indicators and ways in which future targeting could be supported by their application.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment.

PubMed

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition "modules" to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo . Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads.

Coherent dissociation of relativistic {sup 9}C nuclei in nuclear track emulsion

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

Krivenkov, D. O.; Artemenkov, D. A.; Bradnova, V.

2010-04-30

For the first time nuclear track emulsion is exposed to relativistic {sup 9}C nuclei. A systematic pattern of the distributions of charge combinations of fragments in the peripheral interactions of {sup 9}C nuclei in a nuclear track emulsion has been obtained. The main conclusion is that the contribution of the channel {sup 9}C->{sup 8}B+p and {sup 9}C->{sup 7}Be+2p is most important in events that do not involve the production of target-nucleus fragments or mesons (coherent dissociation). It can be concluded that in the peripheral {sup 9}C dissociation the picture hitherto obtained for {sup 8}B and {sup 7}Be with the additionmore » of one or two protons, respectively, is reproduced. Three coherent dissociation events {sup 9}C->3{sup 3}He accompanied by neither target fragments of the nucleus target nor charged mesons are identified.« less

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

Avakian, Harut; Pisano, Silvia

The Deep Inelastic Scattering (DIS) proved to be a great tool in testing of the theory of strong in- teractions. Semi-Inclusive DIS (SIDIS), with detection of an additional hadron allowed first stud- ies of 3D structure of the nucleon, moving the main focus from testing the QCD to understanding of strong interactions and quark gluon dynamics to address a number of puzzles accumulated in recent years. Detection of two hadrons in SIDIS, which is even more complicated, provides ac- cess to details of quark gluon interactions inaccessible in single-hadron SIDIS, providing a new avenue to study the complex nucleon structure.more » Large acceptance of the CLAS detector at Jef- ferson Lab, allowing detection of two hadrons, produced back-to-back (b2b) in the current and target fragmentation regions, provides a unique possibility to study the nucleon structure in target fragmentation region, and correlations of target and current fragmentation regions« less

Ultra high molecular weight polyethylene (UHMWPE) fiber epoxy composite hybridized with Gadolinium and Boron nanoparticles for radiation shielding

NASA Astrophysics Data System (ADS)

Mani, Venkat; Prasad, Narasimha S.; Kelkar, Ajit

2016-09-01

Deep space radiations pose a major threat to the astronauts and their spacecraft during long duration space exploration missions. The two sources of radiation that are of concern are the galactic cosmic radiation (GCR) and the short lived secondary neutron radiations that are generated as a result of fragmentation that occurs when GCR strikes target nuclei in a spacecraft. Energy loss, during the interaction of GCR and the shielding material, increases with the charge to mass ratio of the shielding material. Hydrogen with no neutron in its nucleus has the highest charge to mass ratio and is the element which is the most effective shield against GCR. Some of the polymers because of their higher hydrogen content also serve as radiation shield materials. Ultra High Molecular Weight Polyethylene (UHMWPE) fibers, apart from possessing radiation shielding properties by the virtue of the high hydrogen content, are known for extraordinary properties. An effective radiation shielding material is the one that will offer protection from GCR and impede the secondary neutron radiations resulting from the fragmentation process. Neutrons, which result from fragmentation, do not respond to the Coulombic interaction that shield against GCR. To prevent the deleterious effects of secondary neutrons, targets such as Gadolinium are required. In this paper, the radiation shielding studies that were carried out on the fabricated sandwich panels by vacuum-assisted resin transfer molding (VARTM) process are presented. VARTM is a manufacturing process used for making large composite structures by infusing resin into base materials formed with woven fabric or fiber using vacuum pressure. Using the VARTM process, the hybridization of Epoxy/UHMWPE composites with Gadolinium nanoparticles, Boron, and Boron carbide nanoparticles in the form of sandwich panels were successfully carried out. The preliminary results from neutron radiation tests show that greater than 99% shielding performance was achieved with these sandwich panels. Moreover, the mechanical testing and thermo-physical analysis performed show that core materials can preserve their thermo-physical and mechanical integrity after radiation.

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

PubMed Central

Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

2016-01-01

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

Recombinant human antibody fragment against tetanus toxoid produced by phage display

PubMed Central

Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

2014-01-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

Binding-Site Assessment by Virtual Fragment Screening

PubMed Central

Huang, Niu; Jacobson, Matthew P.

2010-01-01

The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules) would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock ∼11000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors. PMID:20404926

Experimental determination of particle range and dose distribution in thick targets through fragmentation reactions of stable heavy ions.

PubMed

Inaniwa, Taku; Kohno, Toshiyuki; Tomitani, Takehiro; Urakabe, Eriko; Sato, Shinji; Kanazawa, Mitsutaka; Kanai, Tatsuaki

2006-09-07

In radiation therapy with highly energetic heavy ions, the conformal irradiation of a tumour can be achieved by using their advantageous features such as the good dose localization and the high relative biological effectiveness around their mean range. For effective utilization of such properties, it is necessary to evaluate the range of incident ions and the deposited dose distribution in a patient's body. Several methods have been proposed to derive such physical quantities; one of them uses positron emitters generated through projectile fragmentation reactions of incident ions with target nuclei. We have proposed the application of the maximum likelihood estimation (MLE) method to a detected annihilation gamma-ray distribution for determination of the range of incident ions in a target and we have demonstrated the effectiveness of the method with computer simulations. In this paper, a water, a polyethylene and a polymethyl methacrylate target were each irradiated with stable (12)C, (14)N, (16)O and (20)Ne beams. Except for a few combinations of incident beams and targets, the MLE method could determine the range of incident ions R(MLE) with a difference between R(MLE) and the experimental range of less than 2.0 mm under the circumstance that the measurement of annihilation gamma rays was started just after the irradiation of 61.4 s and lasted for 500 s. In the process of evaluating the range of incident ions with the MLE method, we must calculate many physical quantities such as the fluence and the energy of both primary ions and fragments as a function of depth in a target. Consequently, by using them we can obtain the dose distribution. Thus, when the mean range of incident ions is determined with the MLE method, the annihilation gamma-ray distribution and the deposited dose distribution can be derived simultaneously. The derived dose distributions in water for the mono-energetic heavy-ion beams of four species were compared with those measured with an ionization chamber. The good agreement between the derived and the measured distributions implies that the deposited dose distribution in a target can be estimated from the detected annihilation gamma-ray distribution with a positron camera.

Antipodal fragment velocities for porous and weak targets at catastrophic impacts

NASA Technical Reports Server (NTRS)

Yanagisawa, M.; Itoi, T.

1993-01-01

Mortar, porous alumina, and sand targets, which were spherical in shape and from 11 to 15 cm in diameter, were impacted normally by plastic (polycarbonate) projectiles of nearly 1 g in mass at velocities about 6 km/s. Fragment velocity at the antipole of impact site (antipodal velocity, V(sub a)), for each experiment, was obtained from two Flash X-ray images recorded prior to and at predetermined delayed time after impact event. It has been revealed that the velocities for the same E/M(sub t) (impact energy divided by target mass) depend strongly on target material, and differ about an order of magnitude between the sand and basalt.

Antipodal fragment velocities for porous and weak targets at catastrophic impacts

NASA Astrophysics Data System (ADS)

Yanagisawa, M.; Itoi, T.

1993-03-01

Mortar, porous alumina, and sand targets, which were spherical in shape and from 11 to 15 cm in diameter, were impacted normally by plastic (polycarbonate) projectiles of nearly 1 g in mass at velocities about 6 km/s. Fragment velocity at the antipole of impact site (antipodal velocity, V(sub a)), for each experiment, was obtained from two Flash X-ray images recorded prior to and at predetermined delayed time after impact event. It has been revealed that the velocities for the same E/M(sub t) (impact energy divided by target mass) depend strongly on target material, and differ about an order of magnitude between the sand and basalt.

Incorporation of the statistical multi-fragmentation model in PHITS and its application for simulation of fragmentation by heavy ions and protons

NASA Astrophysics Data System (ADS)

Ogawa, Tatsuhiko; Sato, Tatsuhiko; Hashimoto, Shintaro; Niita, Koji

2014-06-01

The fragmentation reactions of relativistic-energy nucleus-nucleus and proton-nucleus collisions were simulated using the Statistical Multi-fragmentation Model (SMM) incorporated with the Particle and Heavy Ion Transport code System (PHITS). The comparisons of calculated cross-sections with literature data showed that PHITS-SMM predicts the fragmentation cross-sections of heavy nuclei up to two orders of magnitude more accurately than PHITS for heavy-ion-induced reactions. For proton-induced reactions, noticeable improvements are observed for interactions of the heavy target with protons at an energy greater than 1 GeV. Therefore, consideration for multi-fragmentation reactions is necessary for the accurate simulation of energetic fragmentation reactions of heavy nuclei.

Development and Application of a Virtual Screening Protocol for the Identification of Multitarget Fragments.

PubMed

Bottegoni, Giovanni; Veronesi, Marina; Bisignano, Paola; Kacker, Puneet; Favia, Angelo D; Cavalli, Andrea

2016-06-20

In this study, we report on a virtual ligand screening protocol optimized to identify fragments endowed with activity at multiple targets. Thanks to this protocol, we were able to identify a fragment that displays activity in the low-micromolar range at both β-secretase 1 (BACE-1) and glycogen synthase kinase 3β (GSK-3β). These two structurally and physiologically unrelated enzymes likely contribute, through different pathways, to the onset of Alzheimer's disease (AD). Therefore, their simultaneous inhibition holds great potential in exerting a profound effect on AD. In perspective, the strategy outlined herein can be adapted to other target combinations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Experiences in fragment-based drug discovery.

PubMed

Murray, Christopher W; Verdonk, Marcel L; Rees, David C

2012-05-01

Fragment-based drug discovery (FBDD) has become established in both industry and academia as an alternative approach to high-throughput screening for the generation of chemical leads for drug targets. In FBDD, specialised detection methods are used to identify small chemical compounds (fragments) that bind to the drug target, and structural biology is usually employed to establish their binding mode and to facilitate their optimisation. In this article, we present three recent and successful case histories in FBDD. We then re-examine the key concepts and challenges of FBDD with particular emphasis on recent literature and our own experience from a substantial number of FBDD applications. Our opinion is that careful application of FBDD is living up to its promise of delivering high quality leads with good physical properties and that in future many drug molecules will be derived from fragment-based approaches. Copyright © 2012 Elsevier Ltd. All rights reserved.

Heavy fragment production cross sections from 1.05 GeV/nucleon 56Fe in C, Al, Cu, Pb, and CH2 targets

NASA Technical Reports Server (NTRS)

Zeitlin, C.; Heilbronn, L.; Miller, J.; Rademacher, S. E.; Borak, T.; Carter, T. R.; Frankel, K. A.; Schimmerling, W.; Stronach, C. E.; Chatterjee, A. (Principal Investigator)

1997-01-01

We have obtained charge-changing cross sections and partial cross sections for fragmentation of 1.05 GeV/nucleon Fe projectiles incident on H, C, Al, Cu, and Pb nuclei. The energy region covered by this experiment is critical for an understanding of galactic cosmic ray propagation and space radiation biophysics. Surviving primary beam particles and fragments with charges from 12 to 25 produced within a forward cone of half-angle 61 mrad were detected using a silicon detector telescope to identify their charge and the cross sections were calculated after correction of the measured yields for finite target thickness effects. The cross sections are compared to model calculations and to previous measurements. Cross sections for the production of fragments with even-numbered nuclear charges are seen to be enhanced in almost all cases.

The fragmentation of 510 MeV/nucleon iron-56 in polyethylene. I. Fragment fluence spectra

NASA Technical Reports Server (NTRS)

Zeitlin, C.; Miller, J.; Heilbronn, L.; Frankel, K.; Gong, W.; Schimmerling, W.

1996-01-01

The fragmentation of 510 MeV/nucleon iron ions in several thicknesses of polyethylene has been measured. Non-interacting primary beam particles and fragments have been identified and their LETs calculated by measuring ionization energy loss in a stack of silicon detectors. Fluences, normalized to the incident beam intensity and corrected for detector effects, are presented for each fragment charge and target. Histograms of fluence as a function of LET are also presented. Some implications of these data for measurements of the biological effects of heavy ions are discussed.

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

PubMed Central

Zavašnik-Bergant, Tina; Bergant Marušič, Martina

2016-01-01

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii. PMID:26960148

In situ magnetic separation of antibody fragments from Escherichia coli in complex media

PubMed Central

2013-01-01

Background In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies (“D1.3”) produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used. Results Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments. Conclusions We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps. PMID:23688064

Characterization of single chain antibody targets through yeast two hybrid

PubMed Central

2010-01-01

Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear epitopes, confirmation of non-linear epitopes for conformational sensors, and detection of secondary binding partners. This approach may thus prove to be an elegant and rapid method for the target characterization of newly obtained scFv antibodies. It may be considered prior to any research application and particularly before any use of such recombinant antibodies in clinical medicine. PMID:20727208

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei

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

Begley, Darren W.; Hartley, Robert C.; Davies, Douglas R.

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification ofmore » several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.« less

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

PubMed Central

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

2015-01-01

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

A SPR strategy for high-throughput ligand screenings based on synthetic peptides mimicking a selected subdomain of the target protein: a proof of concept on HER2 receptor.

PubMed

Monfregola, Luca; Vitale, Rosa Maria; Amodeo, Pietro; De Luca, Stefania

2009-10-01

The discovery of pharmaceutical agents is a complex, lengthy and costly process, critically depending on the availability of rapid and efficient screening methods. In particular, when targets are large, multidomain proteins, their complexity may affect unfavorably technical feasibility, costs and unambiguity of binding test interpretation. A possible strategy to overcome these problems relies on molecular design of receptor fragments that are: sensible targets for ligand screenings, conformationally stable also as standalone domains, easily synthesized and immobilized on chip for Biacore experiments. An additional desirable feature for new ligands is the ability of selectively targeting alternative conformational states typical of many proteins. To test the feasibility of such approach on a case with potential applicative interest, we developed a surface plasmon resonance (SPR)-based screening method for drug candidates toward HER2, a Tyr-kinase receptor targeted in anticancer therapies. HER2 was mimicked by HER2-DIVMP, a modified fragment of it immobilized onto the sensor surface specifically modeling HER2 domain IV in its bounded form, designed by structural comparison of HER2 alone and in complex with Herceptin, a monoclonal therapeutic anti-HER2 antibody. This design and its implementation in SPR devices was validated by investigating Herceptin- HER2-DIVMP affinity, measuring its dissociation constant (K(D)=19.2 nM). An efficient synthetic procedure to prepare the HER2-DIVMP peptide was also developed. The HER2-DIVMP conformational stability suggested by experimental and computational results, makes it also a valuable candidate as a mold to design new molecules selectively targeting domain IV of HER2.

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection.

PubMed

Melendez, Johan H; Santaus, Tonya M; Brinsley, Gregory; Kiang, Daniel; Mali, Buddha; Hardick, Justin; Gaydos, Charlotte A; Geddes, Chris D

2016-10-01

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections. Copyright © 2016 Elsevier Inc. All rights reserved.

Fragment-based discovery of a potent NAMPT inhibitor.

PubMed

Korepanova, Alla; Longenecker, Kenton L; Pratt, Steve D; Panchal, Sanjay C; Clark, Richard F; Lake, Marc; Gopalakrishnan, Sujatha M; Raich, Diana; Sun, Chaohong; Petros, Andrew M

2017-12-12

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Physics of Protoplanetesimal Dust Agglomerates. V. Multiple Impacts of Dusty Agglomerates at Velocities Above the Fragmentation Threshold

NASA Astrophysics Data System (ADS)

Kothe, Stefan; Güttler, Carsten; Blum, Jürgen

2010-12-01

In recent years, a number of new experiments have advanced our knowledge on the early growth phases of protoplanetary dust aggregates. Some of these experiments have shown that collisions between porous and compacted agglomerates at velocities above the fragmentation threshold velocity can lead to growth of the compact body, when the porous collision partner fragments upon impact and transfers mass to the compact agglomerate. To obtain a deeper understanding of this potentially important growth process, we performed laboratory and drop tower experiments to study multiple impacts of small, highly porous dust-aggregate projectiles onto sintered dust targets. The projectile and target consisted of 1.5 μm monodisperse, spherical SiO2 monomers with volume filling factors of 0.15 ± 0.01 and 0.45 ± 0.05, respectively. The fragile projectiles were accelerated by a solenoid magnet and combined with a projectile magazine with which 25 impacts onto the same spot on the target could be performed in vacuum. We measured the mass-accretion efficiency and the volume filling factor for different impact velocities between 1.5 and 6.0 m s^{-1}. The experiments at the lowest impact speeds were performed in the Bremen drop tower under microgravity conditions to allow partial mass transfer also for the lowest adhesion case. Within this velocity range, we found a linear increase of the accretion efficiency with increasing velocity. In the laboratory experiments, the accretion efficiency increases from 0.12 to 0.21 in units of the projectile mass. The recorded images of the impacts showed that the mass transfer from the projectile to the target leads to the growth of a conical structure on the target after less than 100 impacts. From the images, we also measured the volume filling factors of the grown structures, which ranged from 0.15 (uncompacted) to 0.40 (significantly compacted) with increasing impact speed. The velocity dependency of the mass-transfer efficiency and the packing density of the resulting aggregates augment our knowledge of the aggregate growth in protoplanetary disks and should be taken into account for future models of protoplanetary dust growth.

Towards novel therapeutics for HIV through fragment-based screening and drug design.

PubMed

Tiefendbrunn, Theresa; Stout, C David

2014-01-01

Fragment-based drug discovery has been applied with varying levels of success to a number of proteins involved in the HIV (Human Immunodeficiency Virus) life cycle. Fragment-based approaches have led to the discovery of novel binding sites within protease, reverse transcriptase, integrase, and gp41. Novel compounds that bind to known pockets within CCR5 have also been identified via fragment screening, and a fragment-based approach to target the TAR-Tat interaction was explored. In the context of HIV-1 reverse transcriptase (RT), fragment-based approaches have yielded fragment hits with mid-μM activity in an in vitro activity assay, as well as fragment hits that are active against drug-resistant variants of RT. Fragment-based drug discovery is a powerful method to elucidate novel binding sites within proteins, and the method has had significant success in the context of HIV proteins.

Proton and antiproton production in deep inelastic muon-nucleon scattering at 280 GeV

NASA Astrophysics Data System (ADS)

Arneodo, M.; Arvidson, A.; Aubert, J. J.; Badelek, B.; Beaufays, J.; Bee, C. P.; Benchouk, C.; Berghoff, G.; Bird, I.; Blum, D.; Böhm, E.; de Bouard, X.; Brasse, F. W.; Braun, H.; Broll, C.; Brown, S.; Brück, H.; Calen, H.; Chima, J. S.; Ciborowski, J.; Clifft, R.; Coignet, G.; Combley, F.; Coughlan, J.; D'Agostini, G.; Dahlgren, S.; Dengler, F.; Derado, I.; Dreyer, T.; Drees, J.; Düren, M.; Eckardt, V.; Edwards, A.; Edwards, M.; Ernst, T.; Eszes, G.; Favier, J.; Ferrero, M. I.; Figiel, J.; Flauger, W.; Foster, J.; Gabathuler, E.; Gajewski, J.; Gamet, R.; Gayler, J.; Geddes, N.; Grafström, P.; Grard, F.; Haas, J.; Hagberg, E.; Hasert, F. J.; Hayman, P.; Heusse, P.; Jaffré, M.; Jacholkowska, A.; Janata, F.; Jansco, G.; Johnson, A. S.; Kabuss, E. M.; Kellner, G.; Korbel, V.; Krüger, A.; Krüger, J.; Kullander, S.; Landgraf, U.; Lanske, D.; Loken, J.; Long, K.; Maire, M.; Malecki, P.; Manz, A.; Maselli, S.; Mohr, W.; Montanet, F.; Montgomery, H. E.; Nagy, E.; Nassalski, J.; Norton, P. R.; Oakham, F. G.; Osborne, A. M.; Pascaud, C.; Pawlik, B.; Payre, P.; Peroni, C.; Peschel, H.; Pessard, H.; Pettingale, J.; Pietrzyk, B.; Poensgen, B.; Pötsch, M.; Renton, P.; Ribarics, P.; Rith, K.; Rondio, E.; Sandacz, A.; Scheer, M.; Schlagböhmer, A.; Schiemann, H.; Schmitz, N.; Schneegans, M.; Scholz, M.; Schouten, M.; Schröder, T.; Schultze, K.; Sloan, T.; Stier, H. E.; Studt, M.; Taylor, G. N.; Thénard, J. M.; Thompson, J. C.; de La Torre, A.; Toth, J.; Urban, L.; Wallucks, W.; Whalley, M.; Wheeler, S.; Williams, W. S. C.; Wimpenny, S. J.; Windmolders, R.; Wolf, G.

1987-12-01

New results on proton and antiproton production in the target and current fragmentation regions of high energy muon-nucleon scattering are presented. Proton and antiproton production is investigated as a function of Feynman x and rapidity. No significant difference is observed between production on hydrogen and deuterium targets. Correlations between pp,pbar p andbar pbar p pairs are analysed and the results are compared with the predictions of the Lund fragmentation model.

Method and apparatus for biological sequence comparison

DOEpatents

Marr, T.G.; Chang, W.I.

1997-12-23

A method and apparatus are disclosed for comparing biological sequences from a known source of sequences, with a subject (query) sequence. The apparatus takes as input a set of target similarity levels (such as evolutionary distances in units of PAM), and finds all fragments of known sequences that are similar to the subject sequence at each target similarity level, and are long enough to be statistically significant. The invention device filters out fragments from the known sequences that are too short, or have a lower average similarity to the subject sequence than is required by each target similarity level. The subject sequence is then compared only to the remaining known sequences to find the best matches. The filtering member divides the subject sequence into overlapping blocks, each block being sufficiently large to contain a minimum-length alignment from a known sequence. For each block, the filter member compares the block with every possible short fragment in the known sequences and determines a best match for each comparison. The determined set of short fragment best matches for the block provide an upper threshold on alignment values. Regions of a certain length from the known sequences that have a mean alignment value upper threshold greater than a target unit score are concatenated to form a union. The current block is compared to the union and provides an indication of best local alignment with the subject sequence. 5 figs.

Method and apparatus for biological sequence comparison

DOEpatents

Marr, Thomas G.; Chang, William I-Wei

1997-01-01

A method and apparatus for comparing biological sequences from a known source of sequences, with a subject (query) sequence. The apparatus takes as input a set of target similarity levels (such as evolutionary distances in units of PAM), and finds all fragments of known sequences that are similar to the subject sequence at each target similarity level, and are long enough to be statistically significant. The invention device filters out fragments from the known sequences that are too short, or have a lower average similarity to the subject sequence than is required by each target similarity level. The subject sequence is then compared only to the remaining known sequences to find the best matches. The filtering member divides the subject sequence into overlapping blocks, each block being sufficiently large to contain a minimum-length alignment from a known sequence. For each block, the filter member compares the block with every possible short fragment in the known sequences and determines a best match for each comparison. The determined set of short fragment best matches for the block provide an upper threshold on alignment values. Regions of a certain length from the known sequences that have a mean alignment value upper threshold greater than a target unit score are concatenated to form a union. The current block is compared to the union and provides an indication of best local alignment with the subject sequence.

Fragment library design: using cheminformatics and expert chemists to fill gaps in existing fragment libraries.

PubMed

Kutchukian, Peter S; So, Sung-Sau; Fischer, Christian; Waller, Chris L

2015-01-01

Fragment based screening (FBS) has emerged as a mainstream lead discovery strategy in academia, biotechnology start-ups, and large pharma. As a prerequisite of FBS, a structurally diverse library of fragments is desirable in order to identify chemical matter that will interact with the range of diverse target classes that are prosecuted in contemporary screening campaigns. In addition, it is also desirable to offer synthetically amenable starting points to increase the probability of a successful fragment evolution through medicinal chemistry. Herein we describe a method to identify biologically relevant chemical substructures that are missing from an existing fragment library (chemical gaps), and organize these chemical gaps hierarchically so that medicinal chemists can efficiently navigate the prioritized chemical space and subsequently select purchasable fragments for inclusion in an enhanced fragment library.

Fragment-based design of symmetrical bis-benzimidazoles as selective inhibitors of the trimethoprim-resistant, type II R67 dihydrofolate reductase.

PubMed

Bastien, Dominic; Ebert, Maximilian C C J C; Forge, Delphine; Toulouse, Jacynthe; Kadnikova, Natalia; Perron, Florent; Mayence, Annie; Huang, Tien L; Vanden Eynde, Jean Jacques; Pelletier, Joelle N

2012-04-12

The continuously increasing use of trimethoprim as a common antibiotic for medical use and for prophylactic application in terrestrial and aquatic animal farming has increased its prevalence in the environment. This has been accompanied by increased drug resistance, generally in the form of alterations in the drug target, dihydrofolate reductase (DHFR). The most highly resistant variants of DHFR are known as type II DHFR, among which R67 DHFR is the most broadly studied variant. We report the first attempt at designing specific inhibitors to this emerging drug target by fragment-based design. The detection of inhibition in R67 DHFR was accompanied by parallel monitoring of the human DHFR, as an assessment of compound selectivity. By those means, small aromatic molecules of 150-250 g/mol (fragments) inhibiting R67 DHFR selectively in the low millimolar range were identified. More complex, symmetrical bis-benzimidazoles and a bis-carboxyphenyl were then assayed as fragment-based leads, which procured selective inhibition of the target in the low micromolar range (K(i) = 2-4 μM). The putative mode of inhibition is discussed according to molecular modeling supported by in vitro tests. © 2012 American Chemical Society

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

PubMed

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-02-17

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

PubMed Central

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-01-01

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins. PMID:26883295

Fragment virtual screening based on Bayesian categorization for discovering novel VEGFR-2 scaffolds.

PubMed

Zhang, Yanmin; Jiao, Yu; Xiong, Xiao; Liu, Haichun; Ran, Ting; Xu, Jinxing; Lu, Shuai; Xu, Anyang; Pan, Jing; Qiao, Xin; Shi, Zhihao; Lu, Tao; Chen, Yadong

2015-11-01

The discovery of novel scaffolds against a specific target has long been one of the most significant but challengeable goals in discovering lead compounds. A scaffold that binds in important regions of the active pocket is more favorable as a starting point because scaffolds generally possess greater optimization possibilities. However, due to the lack of sufficient chemical space diversity of the databases and the ineffectiveness of the screening methods, it still remains a great challenge to discover novel active scaffolds. Since the strengths and weaknesses of both fragment-based drug design and traditional virtual screening (VS), we proposed a fragment VS concept based on Bayesian categorization for the discovery of novel scaffolds. This work investigated the proposal through an application on VEGFR-2 target. Firstly, scaffold and structural diversity of chemical space for 10 compound databases were explicitly evaluated. Simultaneously, a robust Bayesian classification model was constructed for screening not only compound databases but also their corresponding fragment databases. Although analysis of the scaffold diversity demonstrated a very unevenly distribution of scaffolds over molecules, results showed that our Bayesian model behaved better in screening fragments than molecules. Through a literature retrospective research, several generated fragments with relatively high Bayesian scores indeed exhibit VEGFR-2 biological activity, which strongly proved the effectiveness of fragment VS based on Bayesian categorization models. This investigation of Bayesian-based fragment VS can further emphasize the necessity for enrichment of compound databases employed in lead discovery by amplifying the diversity of databases with novel structures.

Dereplication of plant phenolics using a mass-spectrometry database independent method.

PubMed

Borges, Ricardo M; Taujale, Rahil; de Souza, Juliana Santana; de Andrade Bezerra, Thaís; Silva, Eder Lana E; Herzog, Ronny; Ponce, Francesca V; Wolfender, Jean-Luc; Edison, Arthur S

2018-05-29

Dereplication, an approach to sidestep the efforts involved in the isolation of known compounds, is generally accepted as being the first stage of novel discoveries in natural product research. It is based on metabolite profiling analysis of complex natural extracts. To present the application of LipidXplorer for automatic targeted dereplication of phenolics in plant crude extracts based on direct infusion high-resolution tandem mass spectrometry data. LipidXplorer uses a user-defined molecular fragmentation query language (MFQL) to search for specific characteristic fragmentation patterns in large data sets and highlight the corresponding metabolites. To this end, MFQL files were written to dereplicate common phenolics occurring in plant extracts. Complementary MFQL files were used for validation purposes. New MFQL files with molecular formula restrictions for common classes of phenolic natural products were generated for the metabolite profiling of different representative crude plant extracts. This method was evaluated against an open-source software for mass-spectrometry data processing (MZMine®) and against manual annotation based on published data. The targeted LipidXplorer method implemented using common phenolic fragmentation patterns, was found to be able to annotate more phenolics than MZMine® that is based on automated queries on the available databases. Additionally, screening for ascarosides, natural products with unrelated structures to plant phenolics collected from the nematode Caenorhabditis elegans, demonstrated the specificity of this method by cross-testing both groups of chemicals in both plants and nematodes. Copyright © 2018 John Wiley & Sons, Ltd.

What Hinders Electron Transfer Dissociation (ETD) of DNA Cations?

NASA Astrophysics Data System (ADS)

Hari, Yvonne; Leumann, Christian J.; Schürch, Stefan

2017-12-01

Radical activation methods, such as electron transfer dissociation (ETD), produce structural information complementary to collision-induced dissociation. Herein, electron transfer dissociation of 3-fold protonated DNA hexamers was studied to gain insight into the fragmentation mechanism. The fragmentation patterns of a large set of DNA hexamers confirm cytosine as the primary target of electron transfer. The reported data reveal backbone cleavage by internal electron transfer from the nucleobase to the phosphate linker leading either to a•/ w or d/ z• ion pairs. This reaction pathway contrasts with previous findings on the dissociation processes after electron capture by DNA cations, suggesting multiple, parallel dissociation channels. However, all these channels merely result in partial fragmentation of the precursor ion because the charge-reduced DNA radical cations are quite stable. Two hypotheses are put forward to explain the low dissociation yield of DNA radical cations: it is either attributed to non-covalent interactions between complementary fragments or to the stabilization of the unpaired electron in stacked nucleobases. MS3 experiments suggest that the charge-reduced species is the intact oligonucleotide. Moreover, introducing abasic sites significantly increases the dissociation yield of DNA cations. Consequently, the stabilization of the unpaired electron by π-π-stacking provides an appropriate rationale for the high intensity of DNA radical cations after electron transfer. [Figure not available: see fulltext.

Gas gun driven dynamic fracture and fragmentation of Ti-6Al-4V cylinders

NASA Astrophysics Data System (ADS)

Jones, D. R.; Chapman, D. J.; Eakins, D. E.

2014-05-01

The dynamic fracture and fragmentation of a material is a complex late stage phenomenon occurring in many shock loading scenarios. Improving our predictive capability depends upon exercising our current failure models against new loading schemes and data. We present axially-symmetric high strain rate (104 s-1) expansion of Ti-6Al-4V cylinders using a single stage light gas gun technique. A steel ogive insert was located inside the target cylinder, into which a polycarbonate rod was launched. Deformation of this rod around the insert drives the cylinder into rapid expansion. This technique we have developed facilitates repeatable loading, independent of the temperature of the sample cylinder, with straightforward adjustment of the radial strain rate. Expansion velocity was measured with multiple channels of photon Doppler velocimetry. High speed imaging was used to track the overall expansion process and record strain to failure and crack growth. Results from a cylinder at a temperature of 150 K are compared with work at room temperature, examining the deformation, failure mechanisms and differences in fragmentation.

Fragmentation Cross Sections of Medium-Energy 35Cl, 40Ar, and 48TiBeams on Elemental Targets

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

Zeitlin, C.; Guetersloh, S.; Heilbronn, L.

Charge-changing and fragment production cross sections at 0degrees have been obtained for interactions of 290, 400, and 650MeV/nucleon 40Ar beams, 650 and 1000 MeV/nucleon 35Cl beams, and a 1000MeV/nucleon 48Ti beam. Targets of C, CH2, Al, Cu, Sn, and Pb were used.Using standard analysis methods, we obtain fragment cross sections forcharges as low as 8 for Cl and Ar beams, and as low as 10 for the Tibeam. Using data obtained with small-acceptance detectors, we reportfragment production cross sections for charges as low as 5, corrected foracceptance using a simple model of fragment angular distributions. Withthe lower-charged fragment cross sections,more » we cancompare the data topredictions from several models (including NUCFRG2, EPAX2, and PHITS) ina region largely unexplored in earlier work. As found in earlier workwith other beams, NUCFRG2 and PHITS predictions agree reasonably wellwith the data for charge-changing cross sections, but do not accuratelypredict the fragment production cross sections. The cross sections forthe lightest fragments demonstrate the inadequacy of several models inwhich the cross sections fall monotonically with the charge of thefragment. PHITS, despite not agreeing particularly well with the fragmentproduction cross sections on average, nonetheless qualitativelyreproduces somesignificant features of the data that are missing from theother models.« less

Interactions in hydrogen of relativistic neon to nickel projectiles: Total charge-changing cross sections

NASA Astrophysics Data System (ADS)

Chen, C.-X.; Albergo, S.; Caccia, Z.; Costa, S.; Crawford, H. J.; Cronqvist, M.; Engelage, J.; Ferrando, P.; Fonte, R.; Greiner, L.; Guzik, T. G.; Insolia, A.; Jones, F. C.; Knott, C. N.; Lindstrom, P. J.; Mitchell, J. W.; Potenza, R.; Romanski, J.; Russo, G. V.; Soutoul, A.; Testard, O.; Tull, C. E.; Tuvé, C.; Waddington, C. J.; Webber, W. R.; Wefel, J. P.; Zhang, X.

1994-06-01

A liquid hydrogen target was used to study the nuclear fragmentation of beams of relativistic heavy ions, 22Ne to 58Ni, over an energy range 400 to 900 MeV/nucleon. The experiments were carried out at the Lawrence Berkeley Laboratory Bevalac HISS facility, using the charge-velocity-rigidity method to identify the charged fragments. Here we describe the general concept of the experiment and present total charge-changing cross sections obtained from 17 separate runs. These new measured cross sections display an energy dependence which follows semiempirical model predictions. The mass dependence of the cross sections behaves as predicted by optical models, but within the experimental energy range, the optical model parameters display a clear energy dependence. The isospin of the projectile nuclei also appears to be an important factor in the interaction process.

Repetition priming with Japanese Kana scripts in word-fragment completion.

PubMed

Komatsu, S; Naito, M

1992-03-01

Manipulating two types of Japanese Kana script, Katakana and Hiragana, we examined the effects of a script change between study and test on later word-fragment completion. Throughout three experiments, materials were composed of foreign loan nouns normally written in Katakana, but not in Hiragana, according to approved usage in Japanese. Experiment 1 demonstrated the reliable size of cross-script priming between Katakana and Hiragana. In Experiment 2, cross-modal priming was substantial when modality of presentation was changed from auditory to visual. In Experiment 3, generating a target word from its definition induced priming comparable in size to that in the prior reading condition. These results have been confirmed in the Hiragana test, as well as in the Katakana test, thereby suggesting that some conceptual and modality-independent processes may also mediate repetition priming.

Single-Domain Antibodies and the Promise of Modular Targeting in Cancer Imaging and Treatment

PubMed Central

Iezzi, María Elena; Policastro, Lucía; Werbajh, Santiago; Podhajcer, Osvaldo; Canziani, Gabriela Alicia

2018-01-01

Monoclonal antibodies and their fragments have significantly changed the outcome of cancer in the clinic, effectively inhibiting tumor cell proliferation, triggering antibody-dependent immune effector cell activation and complement mediated cell death. Along with a continued expansion in number, diversity, and complexity of validated tumor targets there is an increasing focus on engineering recombinant antibody fragments for lead development. Single-domain antibodies (sdAbs), in particular those engineered from the variable heavy-chain fragment (VHH gene) found in Camelidae heavy-chain antibodies (or IgG2 and IgG3), are the smallest fragments that retain the full antigen-binding capacity of the antibody with advantageous properties as drugs. For similar reasons, growing attention is being paid to the yet smaller variable heavy chain new antigen receptor (VNAR) fragments found in Squalidae. sdAbs have been selected, mostly from immune VHH libraries, to inhibit or modulate enzyme activity, bind soluble factors, internalize cell membrane receptors, or block cytoplasmic targets. This succinct review is a compilation of recent data documenting the application of engineered, recombinant sdAb in the clinic as epitope recognition “modules” to build monomeric, dimeric and multimeric ligands that target, tag and stall solid tumor growth in vivo. Size, affinity, specificity, and the development profile of sdAbs drugs are seemingly consistent with desirable clinical efficacy and safety requirements. But the hepatotoxicity of the tetrameric anti-DR5-VHH drug in patients with pre-existing anti-drug antibodies halted the phase I clinical trial and called for a thorough pre-screening of the immune and poly-specific reactivities of the sdAb leads. PMID:29520274

Unravelling the structural and mechanistic basis of CRISPR-Cas systems.

PubMed

van der Oost, John; Westra, Edze R; Jackson, Ryan N; Wiedenheft, Blake

2014-07-01

Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR-Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.

Creation of a Novel Class of Potent and Selective MutT Homologue 1 (MTH1) Inhibitors Using Fragment-Based Screening and Structure-Based Drug Design.

PubMed

Rahm, Fredrik; Viklund, Jenny; Trésaugues, Lionel; Ellermann, Manuel; Giese, Anja; Ericsson, Ulrika; Forsblom, Rickard; Ginman, Tobias; Günther, Judith; Hallberg, Kenth; Lindström, Johan; Persson, Lars Boukharta; Silvander, Camilla; Talagas, Antoine; Díaz-Sáez, Laura; Fedorov, Oleg; Huber, Kilian V M; Panagakou, Ioanna; Siejka, Paulina; Gorjánácz, Mátyás; Bauser, Marcus; Andersson, Martin

2018-03-22

Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo. (1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment-based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.

Ejection of spalled layers from laser shock-loaded metals

NASA Astrophysics Data System (ADS)

Lescoute, E.; De Rességuier, T.; Chevalier, J.-M.; Loison, D.; Cuq-Lelandais, J.-P.; Boustie, M.; Breil, J.; Maire, P.-H.; Schurtz, G.

2010-11-01

Dynamic fragmentation of shock-loaded metals is an issue of considerable importance for both basic science and a variety of technological applications, such as inertial confinement fusion, which involves high energy laser irradiation of thin metallic shells. In this context, we present an experimental and numerical study of debris ejection in laser shock-loaded metallic targets (aluminum, gold, and iron) where fragmentation is mainly governed by spall fracture occurring upon tensile loading due to wave interactions inside the sample. Experimental results consist of time-resolved velocity measurements, transverse optical shadowgraphy of ejected debris, and postshock observations of targets and fragments recovered within a transparent gel of low density. They are compared to numerical computations performed with a hydrodynamic code. A correct overall consistency is obtained.

A modular solid state detector for measuring high energy heavy ion fragmentation near the beam axis

NASA Technical Reports Server (NTRS)

Zeitlin, C. J.; Frankel, K. A.; Gong, W.; Heilbronn, L.; Lampo, E. J.; Leres, R.; Miller, J.; Schimmerling, W.

1994-01-01

A multi-element solid state detector has been designed to measure fluences of fragments produced near the beam axis by high energy heavy ion beams in thick targets. The detector is compact and modular, so as to be readily reconfigured according to the range of fragment charges and energies to be measured. Preamplifier gain settings and detector calibrations are adjustable remotely under computer control. We describe the central detector, its associated detectors and electronics, triggering scheme, data acquisition and particle identification techniques, illustrated by data taken with 600 MeV/u 56Fe beams and thick polyethylene targets at the LBL Bevalac. The applications of this work to space radiation protection are discussed.

TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery.

PubMed

Vanwetswinkel, Sophie; Heetebrij, Robert J; van Duynhoven, John; Hollander, Johan G; Filippov, Dmitri V; Hajduk, Philip J; Siegal, Gregg

2005-02-01

We propose a ligand screening method, called TINS (target immobilized NMR screening), which reduces the amount of target required for the fragment-based approach to drug discovery. Binding is detected by comparing 1D NMR spectra of compound mixtures in the presence of a target immobilized on a solid support to a control sample. The method has been validated by the detection of a variety of ligands for protein and nucleic acid targets (K(D) from 60 to 5000 muM). The ligand binding capacity of a protein was undiminished after 2000 different compounds had been applied, indicating the potential to apply the assay for screening typical fragment libraries. TINS can be used in competition mode, allowing rapid characterization of the ligand binding site. TINS may allow screening of targets that are difficult to produce or that are insoluble, such as membrane proteins.

Two-proton correlations in the target fragmentation region of nuclear collisions at 200 A GeV

NASA Astrophysics Data System (ADS)

Awes, T. C.; Barlag, C.; Berger, F.; Bloomer, M. A.; Blume, C.; Bock, D.; Bock, R.; Bohne, E.-M.; Bucher, D.; Claussen, A.; Clewing, G.; Dragon, L.; Eklund, A.; Garpman, S.; Glasow, R.; Gustafsson, H.; Gutbrod, H. H.; Hölker, G.; Idh, J.; Jacobs, P.; Kampert, K. H.; Kolb, B. W.; Löhner, H.; Lund, I.; Obenshain, F. E.; Oskarsson, A.; Otterlund, I.; Peitzmann, T.; Plasil, F.; Poskanzer, A. M.; Purschke, M.; Roters, B.; Saini, S.; Santo, R.; Schmidt, H. R.; Sørensen, S. P.; Steffens, K.; Steinhaeuser, P.; Stenlund, E.; Stüken, D.; Young, G. R.

1995-06-01

Correlations between protons are studied in the target fragmentation region of reactions of protons and16O with C, Cu, Ag, Au and of32S with Al and Au at 200 A GeV. The emitted protons were measured with the Plastic Ball detector in the WA80 experiment at the CERN SPS. The comparison of the correlation function with calculations, assuming a spherical, gaussian shaped source with a lifetime τ=0 fm/ c, allows the extraction of radius parameters. The values are very close to those expected from the geometry of the target nuclei and increase with the target mass as α A {Target/1/3}. Even in proton induced reactions the whole target nucleus is involved. The dependence of the radii on centrality, polar angle θ lab, and energy, and their relation to measured proton yields are presented.

On collisional disruption - Experimental results and scaling laws

NASA Technical Reports Server (NTRS)

Davis, Donald R.; Ryan, Eileen V.

1990-01-01

Both homogeneous and inhomogeneous targets have been addressed by the present experimental consideration of the impact strengths, fragment sizes, and fragment velocities generated by cement mortar targets whose crushing strengths vary by an order of magnitude, upon impact of projectiles in the velocity range of 50-5700 m/sec. When combined with additional published data, dynamic impact strength is found to correlate with quasi-static material strengths for materials ranging in character from basalt to ice; two materials not following this trend, however, are weak mortar and clay targets. Values consistent with experimental results are obtainable with a simple scaling algorithm based on impact energy, material properties, and collisional strain rate.

Interaction of 160-GeV muon with emulsion nuclei

NASA Astrophysics Data System (ADS)

Othman, S. M.; Ghoneim, M. T.; Hussein, M. T.; El-Samman, H.; Hussein, A.

In this work we present some results of the interaction of high-energy muons with emulsion nuclei. The interaction results in emission of a number of fragments as a consequence of electromagnetic dissociation of the excited target nuclei. This excitation is attributed to absorption of photons by the target nuclei due to the intense electric field of the very fast incident muon particles. The interactions take place at impact parameters that allow ultra-peripheral collisions to take place, leading to giant resonances and hence multifragmentation of emulsion targets. Charge identification, range, energy spectra, angular distribution and topological cross-section of the produced fragments are measured and evaluated.

A Computational Study of Laminate Transparent Armor Impacted by FSP

DTIC Science & Technology

2009-06-01

of Hsieh et al [1], on targets consisting of 3mm PC-12mm PMMA-3mm PC impacted by 17-gr, 0.22 caliber fragment simulating projectile (FSP) at impact...results from the experiments of Hsieh et al [1], on targets consisting of 3mm PC-12mm PMMA-3mm PC impacted by 17-gr, 0.22 caliber fragment simulating...investigate several different analysis techniques to qualitatively determine their accuracy when compared with experiments of Hsieh et al [1]. The

AKT1, LKB1, and YAP1 revealed as MYC interactors with NanoLuc-based protein-fragment complementation assay. | Office of Cancer Genomics

Cancer.gov

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. Here we report the development of a NanoLuc®-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells.

Electrochemistry Combined with LC-HRMS: Elucidating Transformation Products of the Recalcitrant Pharmaceutical Compound Carbamazepine Generated by the White-Rot Fungus Pleurotus ostreatus.

PubMed

Seiwert, Bettina; Golan-Rozen, Naama; Weidauer, Cindy; Riemenschneider, Christina; Chefetz, Benny; Hadar, Yitzhak; Reemtsma, Thorsten

2015-10-20

Transformation products (TPs) of environmental pollutants must be identified to understand biodegradation processes and reaction mechanisms and to assess the efficiency of treatment processes. The combination of oxidation by an electrochemical cell (EC) with analysis by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is a rapid approach for the determination and identification of TPs generated by natural microbial processes. Electrochemically generated TPs of the recalcitrant pharmaceutical carbamazepine (CBZ) were used for a target screening for TPs formed by the white-rot fungus Pleurotus ostreatus. EC with LC-HRMS facilitates detection and identification of TPs because the product spectrum is not superimposed with biogenic metabolites and elevated substrate concentrations can be used. A group of 10 TPs formed in the microbial process were detected by target screening for molecular ions, and another 4 were detected by screening on the basis of characteristic fragment ions. Three of these TPs have never been reported before. For CBZ, EC with LC-HRMS was found to be more effective than software tools in defining targets for the screening and faster than nontarget screening alone in TP identification. EC with LC-HRMS may be used to feed MS databases with spectra of possible TPs of larger numbers of environmental contaminants for an efficient target screening.

Fragment-Based Optimization of Small Molecule CXCL12 Inhibitors for Antagonizing the CXCL12/CXCR4 Interaction

PubMed Central

Ziarek, Joshua J.; Liu, Yan; Smith, Emmanuel; Zhang, Guolin; Peterson, Francis C.; Chen, Jun; Yu, Yongping; Chen, Yu; Volkman, Brian F.; Li, Rongshi

2013-01-01

The chemokine CXCL12 and its G protein-coupled receptor (GPCR) CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines interact with two distinct sites to bind and activate their receptors, both the GPCRs and chemokines are potential targets for small molecule inhibition. A number of chemokines have been validated as targets for drug development, but virtually all drug discovery efforts focus on the GPCRs. However, all CXCR4 receptor antagonists with the exception of MSX-122 have failed in clinical trials due to unmanageable toxicities, emphasizing the need for alternative strategies to interfere with CXCL12/CXCR4-guided metastatic homing. Although targeting the relatively featureless surface of CXCL12 was presumed to be challenging, focusing efforts at the sulfotyrosine (sY) binding pockets proved successful for procuring initial hits. Using a hybrid structure-based in silico/NMR screening strategy, we recently identified a ligand that occludes the receptor recognition site. From this initial hit, we designed a small fragment library containing only nine tetrazole derivatives using a fragment-based and bioisostere approach to target the sY binding sites of CXCL12. Compound binding modes and affinities were studied by 2D NMR spectroscopy, X-ray crystallography, molecular docking and cell-based functional assays. Our results demonstrate that the sY binding sites are conducive to the development of high affinity inhibitors with better ligand efficiency (LE) than typical protein-protein interaction inhibitors (LE ≤ 0.24). Our novel tetrazole-based fragment 18 was identified to bind the sY21 site with a Kd of 24 μM (LE = 0.30). Optimization of 18 yielded compound 25 which specifically inhibits CXCL12-induced migration with an improvement in potency over the initial hit 9. The fragment from this library that exhibited the highest affinity and ligand efficiency (11: Kd = 13 μM, LE = 0.33) may serve as a starting point for development of inhibitors targeting the sY12 site. PMID:23368099

First results from the new double velocity-double energy spectrometer VERDI

NASA Astrophysics Data System (ADS)

Frégeau, M. O.; Oberstedt, S.; Gamboni, Th.; Geerts, W.; Hambsch, F.-J.; Vidali, M.

2016-05-01

The VERDI spectrometer (VElocity foR Direct mass Identification) is a two arm time-of-flight spectrometer built at the European Commission Joint Research Centre IRMM. It determines fragment masses and kinetic energy distributions produced in nuclear fission by means of the double velocity and double energy (2v-2E) method. The simultaneous measurement of pre- and post neutron fragment characteristics allows studying the share of excitation energy between the two fragments. In particular, the evolution of fission modes and neutron multiplicity may be studied as a function of the available excitation energy. Both topics are of great importance for the development of models used in the evaluation of nuclear data, and also have important implications for the fundamental understanding of the fission process. The development of VERDI focus on maximum geometrical efficiency while striving for highest possible mass resolution. An innovative transmission start detector, using electrons ejected from the target itself, was developed. Stop signal and kinetic energy of both fragments are provided by two arrays of silicon detectors. The present design provides about 200 times higher geometrical efficiency than that of the famous COSI FAN TUTTE spectrometer [Nuclear Instruments and Methods in Physics Research 219 (1984) 569]. We report about a commissioning experiment of the VERDI spectrometer, present first results from a 2v-2E measurement of 252Cf spontaneous fission and discuss the potential of this instrument to contribute to the investigation prompt fission neutron characteristics as a function of fission fragment properties.

Momentum peak shift and width of longitudinal momentum distribution of projectilelike fragments produced at E =290 MeV /nucleon

NASA Astrophysics Data System (ADS)

Momota, S.; Kanazawa, M.; Kitagawa, A.; Sato, S.

2018-04-01

Longitudinal momentum (PL) distributions of projectilelike fragments produced at E =290 MeV /nucleon are investigated. PL distributions of fragments produced by Ar and Kr beams with a wide variety of targets (C, Al, Nb, Tb, and Au) were measured using the fragment separator at HIMAC. PL distributions observed for fragments with a wide range of mass losses Δ A (1-30 for Ar beam and 1-64 for Kr beam), show a slightly, but definitely asymmetric nature. The peak shift and width were obtained from the observed PL distributions. No significant target dependence was found in either the peak shift or width. For the practical application, the variation in momentum peak shift with fragment mass (AF) was represented by a parabolic function. The width on the high-PL side (σHigh) is well reproduced by the Goldhaber formula, which is obtained from the contribution of the Fermi momentum. The behavior of the reduced width, σ0, obtained from σHigh via the Goldhaber formulation, is consistent with the mass-dependent Fermi momentum of a nucleon. The width on the low-PL side (σLow) is markedly larger than σHigh and exhibits a clear AF dependence.

Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1991-01-01

A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

Forest fragmentation in the Pacific Northwest: quantification and correlations

Treesearch

Brett J. Butler; Jennifer J. Swenson; Ralph J. Alig

2004-01-01

A forest fragmentation index was produced for western Oregon and western Washington that combined measures of forested area, percentage edge, and interspersion. While natural, human land-cover, and human land-use processes contribute to forest fragmentation in the region, the drivers of these processes are categorically different. Here we examine forest fragmentation...

Primary and secondary fragmentation of crystal-bearing intermediate magma

NASA Astrophysics Data System (ADS)

Jones, Thomas J.; McNamara, Keri; Eychenne, Julia; Rust, Alison C.; Cashman, Katharine V.; Scheu, Bettina; Edwards, Robyn

2016-11-01

Crystal-rich intermediate magmas are subjected to both primary and secondary fragmentation processes, each of which may produce texturally distinct tephra. Of particular interest for volcanic hazards is the extent to which each process contributes ash to volcanic plumes. One way to address this question is by fragmenting pyroclasts under controlled conditions. We fragmented pumice samples from Soufriere Hills Volcano (SHV), Montserrat, by three methods: rapid decompression in a shock tube-like apparatus, impact by a falling piston, and milling in a ball mill. Grain size distributions of the products reveal that all three mechanisms produce fractal breakage patterns, and that the fractal dimension increases from a minimum of 2.1 for decompression fragmentation (primary fragmentation) to a maximum of 2.7 by repeated impact (secondary fragmentation). To assess the details of the fragmentation process, we quantified the shape, texture and components of constituent ash particles. Ash shape analysis shows that the axial ratio increases during milling and that particle convexity increases with repeated impacts. We also quantify the extent to which the matrix is separated from the crystals, which shows that secondary processes efficiently remove adhering matrix from crystals, particularly during milling (abrasion). Furthermore, measurements of crystal size distributions before (using x-ray computed tomography) and after (by componentry of individual grain size classes) decompression-driven fragmentation show not only that crystals influence particular size fractions across the total grain size distribution, but also that free crystals are smaller in the fragmented material than in the original pumice clast. Taken together, our results confirm previous work showing both the control of initial texture on the primary fragmentation process and the contributions of secondary processes to ash formation. Critically, however, our extension of previous analyses to characterisation of shape, texture and componentry provides new analytical tools that can be used to assess contributions of secondary processes to ash deposits of uncertain or mixed origin. We illustrate this application with examples from SHV deposits.

Characterizing DebriSat Fragments: So Many Fragments, So Much Data, and So Little Time

NASA Technical Reports Server (NTRS)

Shiotani, B.; Rivero, M.; Carrasquilla, M.; Allen, S.; Fitz-Coy, N.; Liou, J.-C.; Huynh, T.; Sorge, M.; Cowardin, H.; Opiela, J.;

Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies

NASA Astrophysics Data System (ADS)

Manoharan, Prabu; Vijayan, R. S. K.; Ghoshal, Nanda

2010-10-01

The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables ( X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

Rationalizing fragment based drug discovery for BACE1: insights from FB-QSAR, FB-QSSR, multi objective (MO-QSPR) and MIF studies.

PubMed

Manoharan, Prabu; Vijayan, R S K; Ghoshal, Nanda

2010-10-01

The ability to identify fragments that interact with a biological target is a key step in FBDD. To date, the concept of fragment based drug design (FBDD) is increasingly driven by bio-physical methods. To expand the boundaries of QSAR paradigm, and to rationalize FBDD using In silico approach, we propose a fragment based QSAR methodology referred here in as FB-QSAR. The FB-QSAR methodology was validated on a dataset consisting of 52 Hydroxy ethylamine (HEA) inhibitors, disclosed by GlaxoSmithKline Pharmaceuticals as potential anti-Alzheimer agents. To address the issue of target selectivity, a major confounding factor in the development of selective BACE1 inhibitors, FB-QSSR models were developed using the reported off target activity values. A heat map constructed, based on the activity and selectivity profile of the individual R-group fragments, and was in turn used to identify superior R-group fragments. Further, simultaneous optimization of multiple properties, an issue encountered in real-world drug discovery scenario, and often overlooked in QSAR approaches, was addressed using a Multi Objective (MO-QSPR) method that balances properties, based on the defined objectives. MO-QSPR was implemented using Derringer and Suich desirability algorithm to identify the optimal level of independent variables (X) that could confer a trade-off between selectivity and activity. The results obtained from FB-QSAR were further substantiated using MIF (Molecular Interaction Fields) studies. To exemplify the potentials of FB-QSAR and MO-QSPR in a pragmatic fashion, the insights gleaned from the MO-QSPR study was reverse engineered using Inverse-QSAR in a combinatorial fashion to enumerate some prospective novel, potent and selective BACE1 inhibitors.

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

PubMed

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

2015-12-15

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

miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor.

PubMed

Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung

2014-11-28

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.

miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor

PubMed Central

Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung

2014-01-01

Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3′-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF. PMID:25431021

Studies of the nucleon structure in back-to-back SIDIS

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

Avakian, Harut

2016-03-01

The Deep Inelastic Scattering (DIS) proved to be a great tool in testing of the theory of strong interactions, which was a major focus in last decades. Semi-Inclusive DIS (SIDIS), with detection of an additional hadron allowed first studies of 3D structure of the nucleon, moving the main focus from testing the QCD to understanding of strong interactions and quark gluon dynamics to address a number of puzzles accumulated in recent years. Detection of two hadrons in SIDIS, which is even more complicated, provides access to details of quark gluon interactions inaccessible in single-hadron SIDIS, providing a new avenue tomore » study the complex nucleon structure. Large acceptance of the Electron Ion Collider, allowing detection of two hadrons, produced back-to-back in the current and target fragmentation regions, combined with clear separation of two regions, would provide a unique possibility to study the nucleon structure in target fragmentation region, and correlations of target and current fragmentation regions.« less

Synthesis and profiling of a 3-aminopyridin-2-one-based kinase targeted fragment library: Identification of 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one scaffold for monopolar spindle 1 (MPS1) and Aurora kinases inhibition.

PubMed

Fearon, Daren; Westwood, Isaac M; van Montfort, Rob L M; Bayliss, Richard; Jones, Keith; Bavetsias, Vassilios

2018-07-15

Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin.

PubMed

Teixeira, Pedro F; Masuyer, Geoffrey; Pinho, Catarina M; Branca, Rui M M; Kmiec, Beata; Wallin, Cecilia; Wärmländer, Sebastian K T S; Berntsson, Ronnie P-A; Ankarcrona, Maria; Gräslund, Astrid; Lehtiö, Janne; Stenmark, Pål; Glaser, Elzbieta

2018-02-02

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN E475Q in complex with the products of neurotensin cleavage at 2.7Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-β peptide, Aβ1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria. Copyright © 2017 Elsevier Ltd. All rights reserved.

Shape Distribution of Fragments from Microsatellite Impact Tests

NASA Technical Reports Server (NTRS)

Liou, J.C.; Hanada, T.

2009-01-01

Fragment shape is an important factor for conducting reliable orbital debris damage assessments for critical space assets, such as the International Space Station. To date, seven microsatellite impact tests have been completed as part of an ongoing collaboration between Kyushu University and the NASA Orbital Debris Program Office. The target satellites ranged in size from 15 cm 15 cm 15 cm to 20 cm 20 cm 20 cm. Each target satellite was equipped with fully functional electronics, including circuits, battery, and transmitter. Solar panels and multi-layer insulation (MLI) were added to the target satellites of the last two tests. The impact tests were carried out with projectiles of different sizes and impact speeds. All fragments down to about 2 mm in size were collected and analyzed based on their three orthogonal dimensions, x, y, and z, where x is the longest dimension, y is the longest dimension in the plane perpendicular to x, and z is the longest dimension perpendicular to both x and y. Each fragment was also photographed and classified by shape and material composition. This data set serves as the basis of our effort to develop a fragment shape distribution. Two distinct groups can be observed in the x/y versus y/z distribution of the fragments. Objects in the first group typically have large x/y values. Many of them are needle-like objects originating from the fragmentation of carbon fiber reinforced plastic materials used to construct the satellites. Objects in the second group tend to have small x/y values, and many of them are box-like or plate-like objects, depending on their y/z values. Each group forms the corresponding peak in the x/y distribution. However, only one peak can be observed in the y/z distribution. These distributions and how they vary with size, material type, and impact parameters will be described in detail within the paper.

Gene disruptions indicate an essential function for the LmmCRK1 cdc2-related kinase of Leishmania mexicana.

PubMed

Mottram, J C; McCready, B P; Brown, K G; Grant, K M

1996-11-01

The generation of homozygous null mutants for the crk1 Cdc2-Related Kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg-targeting fragment, but attempts to create null mutants by second-round transfections with a bie-targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild-type crk1 alleles as well as hyg and ble integrations. DNA-content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L mexicana transfected with a Trypanosoma brucel homologue, tbcrk1, was shown to be viable in an immcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six-histidine tag at the C-terminus of the kinase, allowing purification of the active complex by affinity selection on Nl(2+)-nitriloacetic acid (NTA) agarose.

The rise of fragment-based drug discovery.

PubMed

Murray, Christopher W; Rees, David C

2009-06-01

The search for new drugs is plagued by high attrition rates at all stages in research and development. Chemists have an opportunity to tackle this problem because attrition can be traced back, in part, to the quality of the chemical leads. Fragment-based drug discovery (FBDD) is a new approach, increasingly used in the pharmaceutical industry, for reducing attrition and providing leads for previously intractable biological targets. FBDD identifies low-molecular-weight ligands (∼150 Da) that bind to biologically important macromolecules. The three-dimensional experimental binding mode of these fragments is determined using X-ray crystallography or NMR spectroscopy, and is used to facilitate their optimization into potent molecules with drug-like properties. Compared with high-throughput-screening, the fragment approach requires fewer compounds to be screened, and, despite the lower initial potency of the screening hits, offers more efficient and fruitful optimization campaigns. Here, we review the rise of FBDD, including its application to discovering clinical candidates against targets for which other chemistry approaches have struggled.

Targeting YAP/TAZ-TEAD protein-protein interactions using fragment-based and computational modeling approaches

PubMed Central

Verma, Chandra

2017-01-01

The Hippo signaling pathway, which is implicated in the regulation of organ size, has emerged as a potential target for the development of cancer therapeutics. YAP, TAZ (transcription co-activators) and TEAD (transcription factor) are the downstream transcriptional machinery and effectors of the pathway. Formation of the YAP/TAZ-TEAD complex leads to transcription of growth-promoting genes. Conversely, disrupting the interactions of the complex decreases cell proliferation. Herein, we screened a 1000-member fragment library using Thermal Shift Assay and identified a hit fragment. We confirmed its binding at the YAP/TAZ-TEAD interface by X-ray crystallography, and showed that it occupies the same hydrophobic pocket as a conserved phenylalanine of YAP/TAZ. This hit fragment serves as a scaffold for the development of compounds that have the potential to disrupt YAP/TAZ-TEAD interactions. Structure-activity relationship studies and computational modeling were also carried out to identify more potent compounds that may bind at this validated druggable binding site. PMID:28570566

RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana.

PubMed

Zhang, Zhonghui; Hu, Fuqu; Sung, Min Woo; Shu, Chang; Castillo-González, Claudia; Koiwa, Hisashi; Tang, Guiliang; Dickman, Martin; Li, Pingwei; Zhang, Xiuren

2017-05-02

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5' products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

Large Genomic Fragment Deletions and Insertions in Mouse Using CRISPR/Cas9

PubMed Central

Satheka, Achim Cchitvsanzwhoh; Togo, Jacques; An, Yao; Humphrey, Mabwi; Ban, Luying; Ji, Yan; Jin, Honghong; Feng, Xuechao; Zheng, Yaowu

2015-01-01

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies. PMID:25803037

Asteroid collisions: Target size effects and resultant velocity distributions

NASA Technical Reports Server (NTRS)

Ryan, Eileen V.

1993-01-01

To study the dynamic fragmentation of rock to simulate asteroid collisions, we use a 2-D, continuum damage numerical hydrocode which models two-body impacts. This hydrocode monitors stress wave propagation and interaction within the target body, and includes a physical model for the formation and growth of cracks in rock. With this algorithm we have successfully reproduced fragment size distributions and mean ejecta speeds from laboratory impact experiments using basalt, and weak and strong mortar as target materials. Using the hydrocode, we have determined that the energy needed to fracture a body has a much stronger dependence on target size than predicted from most scaling theories. In addition, velocity distributions obtained indicate that mean ejecta speeds resulting from large-body collisions do not exceed escape velocities.

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

PubMed Central

Margalit, Y; Yarus, S; Shapira, E; Gruenbaum, Y; Fainsod, A

1993-01-01

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture. Images PMID:7909943

Fragmentation of cosmic-string loops

NASA Technical Reports Server (NTRS)

York, Thomas

1989-01-01

The fragmentation of cosmic string loops is discussed, and the results of a simulation of this process are presented. The simulation can evolve any of a large class of loops essentially exactly, including allowing fragments that collide to join together. Such reconnection enhances the production of small fragments, but not drastically. With or without reconnections, the fragmentation process produces a collection of nonself-intersecting loops whose typical length is on the order of the persistence length of the initial loop.

An Imaging System for Automated Characteristic Length Measurement of Debrisat Fragments

NASA Technical Reports Server (NTRS)

Moraguez, Mathew; Patankar, Kunal; Fitz-Coy, Norman; Liou, J.-C.; Sorge, Marlon; Cowardin, Heather; Opiela, John; Krisko, Paula H.

2015-01-01

The debris fragments generated by DebriSat's hypervelocity impact test are currently being processed and characterized through an effort of NASA and USAF. The debris characteristics will be used to update satellite breakup models. In particular, the physical dimensions of the debris fragments must be measured to provide characteristic lengths for use in these models. Calipers and commercial 3D scanners were considered as measurement options, but an automated imaging system was ultimately developed to measure debris fragments. By automating the entire process, the measurement results are made repeatable and the human factor associated with calipers and 3D scanning is eliminated. Unlike using calipers to measure, the imaging system obtains non-contact measurements to avoid damaging delicate fragments. Furthermore, this fully automated measurement system minimizes fragment handling, which reduces the potential for fragment damage during the characterization process. In addition, the imaging system reduces the time required to determine the characteristic length of the debris fragment. In this way, the imaging system can measure the tens of thousands of DebriSat fragments at a rate of about six minutes per fragment, compared to hours per fragment in NASA's current 3D scanning measurement approach. The imaging system utilizes a space carving algorithm to generate a 3D point cloud of the article being measured and a custom developed algorithm then extracts the characteristic length from the point cloud. This paper describes the measurement process, results, challenges, and future work of the imaging system used for automated characteristic length measurement of DebriSat fragments.

OPTIMIZATION OF EXPERIMENTAL DESIGNS BY INCORPORATING NIF FACILITY IMPACTS

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

Eder, D C; Whitman, P K; Koniges, A E

2005-08-31

For experimental campaigns on the National Ignition Facility (NIF) to be successful, they must obtain useful data without causing unacceptable impact on the facility. Of particular concern is excessive damage to optics and diagnostic components. There are 192 fused silica main debris shields (MDS) exposed to the potentially hostile target chamber environment on each shot. Damage in these optics results either from the interaction of laser light with contamination and pre-existing imperfections on the optic surface or from the impact of shrapnel fragments. Mitigation of this second damage source is possible by identifying shrapnel sources and shielding optics from them.more » It was recently demonstrated that the addition of 1.1-mm thick borosilicate disposable debris shields (DDS) block the majority of debris and shrapnel fragments from reaching the relatively expensive MDS's. However, DDS's cannot stop large, faster moving fragments. We have experimentally demonstrated one shrapnel mitigation technique showing that it is possible to direct fast moving fragments by changing the source orientation, in this case a Ta pinhole array. Another mitigation method is to change the source material to one that produces smaller fragments. Simulations and validating experiments are necessary to determine which fragments can penetrate or break 1-3 mm thick DDS's. Three-dimensional modeling of complex target-diagnostic configurations is necessary to predict the size, velocity, and spatial distribution of shrapnel fragments. The tools we are developing will be used to set the allowed level of debris and shrapnel generation for all NIF experimental campaigns.« less

Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth.

PubMed

Battersby, J E; Snedecor, B; Chen, C; Champion, K M; Riddle, L; Vanderlaan, M

2001-08-24

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-related protein species found in crude cell extracts of recombinant origin is described. Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-tissue factor (anti-TF), from various stages throughout a biopharmaceutical production process to describe the assay details. The targeted proteins were captured on an affinity column containing an anti-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column was placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by eluting the reversed-phase column with a shallow acetonitrile gradient. Characterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chain dimer and multiple forms of Fab'. Analysis of full-length antibody preparations also resolved these fragments as well as a completely assembled form. Co-eluting with the full-length antibody were high-molecular-mass variants that were missing one or both light chains. Resolved components were quantified by comparison with peak areas of similarly treated standards. By comparing the two-dimensional polyacrylamide gel electrophoresis patterns of an Escherichia coli blank run, a production run and the material affinity captured (AME5) from a production run, it was determined that the AME5 antibody captured isoforms of light chain, light chain covalently attached to heavy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments.

Mass spectrometry for fragment screening.

PubMed

Chan, Daniel Shiu-Hin; Whitehouse, Andrew J; Coyne, Anthony G; Abell, Chris

2017-11-08

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Forward-Backward Emission of Target Evaporated Fragments at High Energy Nucleus-Nucleus Collisions

NASA Astrophysics Data System (ADS)

Zhang, Zhi; Ma, Tian-Li; Zhang, Dong-Hai

The multiplicity distribution, multiplicity moments, scaled variance and entropy of target evaporated fragment emitted in forward and backward hemispheres in relativistic heavy ions induced emulsion heavy targets (AgBr) interactions are investigated. It is found that the multiplicity distribution can be fitted by the Gaussian distribution, and the fitting parameters are different between two hemispheres for all the interactions. The multiplicity moment increases with the order of the moment q, and second-order multiplicity moment is energy independent over the entire energy for all the interactions. The scaled variance is close to one for all the interactions. The entropy in forward hemisphere is greater than that in backward hemisphere for all the interactions.

Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

DOEpatents

Gray, J.W.; Pinkel, D.

1991-07-02

A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro.

PubMed

Kang, H W; Weissleder, R; Bogdanov, A

2002-01-01

Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab')(2). We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1 beta inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab')(2), or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20-30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1 beta.

Captured metagenomics: large-scale targeting of genes based on ‘sequence capture’ reveals functional diversity in soils

PubMed Central

Manoharan, Lokeshwaran; Kushwaha, Sandeep K.; Hedlund, Katarina; Ahrén, Dag

2015-01-01

Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances. PMID:26490729

Investigation of Shapes and Spins of Reaccumulated Remnants from Asteroid Disruption Simulations

NASA Astrophysics Data System (ADS)

Michel, Patrick; Ballouz, R.; Richardson, D. C.; Schwartz, S. R.

2012-10-01

Evidence that asteroids larger than a few hundred meters diameter can be gravitational aggregates of smaller, cohesive pieces comes, for instance, from images returned by the Hayabusa spacecraft of asteroid 25143 Itokawa (Fujiwara et al., 2006, Science 312, 1330). These images show an irregular 500-meter-long body with a boulder-strewn surface, as might be expected from reaccumulation following catastrophic disruption of a larger parent asteroid (Michel et al., 2001, Science 294, 1696). However, numerical simulations of this process to date essentially focus on the size/mass and velocity distributions of reaccumulated fragments, matching asteroid families. Reaccumulation was simplified by merging the objects into growing spheres. However, understanding shapes, spins and surface properties of gravitational aggregates formed by reaccumulation is required to interpret information from ground-based observations and space missions. E.g., do boulders on Itokawa originate from reaccumulation of material ejected from a catastrophic impact or from other processes (such as the Brazil-nut effect)? How does reaccumulation affect the observed shapes? A model was developed (Richardson et al., 2009, Planet. Space Sci. 57, 183) to preserve shape and spin information of reaccumulated bodies in simulations of asteroid disruption, by allowing fragments to stick on contact (and optionally bounce or fragment further, depending on user-selectable parameters). Such treatments are computationally expensive, and we could only recently start to explore the parameter space. Preliminary results will be presented, showing that some observed surface and shape features may be explained by how fragments produced by a disruption reaccumulate. Simulations of rubble pile collisions without particle cohesion, and an investigation of the influence of initial target rotation on the outcome will also be shown. We acknowledge the National Science Foundation (AST1009579) and NASA (NNX08AM39G).

Study on the isospin equilibration phenomenon in nuclear reactions 40Ca + 40Ca , 40Ca + 46Ti , 40Ca + 48Ca , 48Ca + 48Ca at 25 MeV/nucleon by using the CHIMERA multidetector

NASA Astrophysics Data System (ADS)

Martorana, N. S.; Auditore, L.; Berceanu, I.; Cardella, G.; Chatterjee, M. B.; De Luca, S.; De Filippo, E.; Dell'Aquila, D.; Gnoffo, B.; Lanzalone, G.; Lombardo, I.; Maiolino, C.; Norella, S.; Pagano, A.; Pagano, E. V.; Papa, M.; Pirrone, S.; Politi, G.; Porto, F.; Quattrocchi, L.; Rizzo, F.; Russotto, P.; Trifirò, A.; Trimarchi, M.; Verde, G.; Vigilante, M.

2017-11-01

We report on the results obtained by studying nuclear reactions between isotopes of Ca and Ti at 25 MeV/nucleon. We used the multidetector CHIMERA to detect charged reaction products. In particular, we studied two main effects: the isospin diffusion and the isospin drift. In order to study these processes we performed a moving-source analysis on kinetic energy spectra of the isobar nuclei ^{3H} and ^{3He} . This method allows to isolate the emission from the typical sources produced in reactions at Fermi energy: projectile like fragment (PLF), target like fragment (TLF), and mid-velocity (MV) emission. The obtained results are compared to previous experimental investigations and to simulations obtained with CoMD-II model.

Crystallographic Fragment Based Drug Discovery: Use of a Brominated Fragment Library Targeting HIV Protease

PubMed Central

Tiefenbrunn, Theresa; Forli, Stefano; Happer, Meaghan; Gonzalez, Ana; Tsai, Yingssu; Soltis, Michael; Elder, John H.; Olson, Arthur J.; Stout, C. David

2013-01-01

A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV-1 protease in order to probe surface sites in soaking experiments. Often fragments are weak binders with partial occupancy, resulting in weak, difficult-to-fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3-bromo-2,6-dimethoxybenzoic acid (Br6) in the flap site, and 1-bromo-2-naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br-fragments provides further insight into the nature of these surface pockets. PMID:23998903

Multiple lesion track structure model

NASA Technical Reports Server (NTRS)

Wilson, John W.; Cucinotta, Francis A.; Shinn, Judy L.

1992-01-01

A multilesion cell kinetic model is derived, and radiation kinetic coefficients are related to the Katz track structure model. The repair-related coefficients are determined from the delayed plating experiments of Yang et al. for the C3H10T1/2 cell system. The model agrees well with the x ray and heavy ion experiments of Yang et al. for the immediate plating, delaying plating, and fractionated exposure protocols employed by Yang. A study is made of the effects of target fragments in energetic proton exposures and of the repair-deficient target-fragment-induced lesions.

η-Secretase processing of APP inhibits neuronal activity in the hippocampus.

PubMed

Willem, Michael; Tahirovic, Sabina; Busche, Marc Aurel; Ovsepian, Saak V; Chafai, Magda; Kootar, Scherazad; Hornburg, Daniel; Evans, Lewis D B; Moore, Steven; Daria, Anna; Hampel, Heike; Müller, Veronika; Giudici, Camilla; Nuscher, Brigitte; Wenninger-Weinzierl, Andrea; Kremmer, Elisabeth; Heneka, Michael T; Thal, Dietmar R; Giedraitis, Vilmantas; Lannfelt, Lars; Müller, Ulrike; Livesey, Frederick J; Meissner, Felix; Herms, Jochen; Konnerth, Arthur; Marie, Hélène; Haass, Christian

2015-10-15

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-β peptide. Two principal physiological pathways either prevent or promote amyloid-β generation from its precursor, β-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-β fragments generated by the α- and β-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (β-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504-505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-β). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

Unravelling the structural and mechanistic basis of CRISPR–Cas systems

PubMed Central

van der Oost, John; Westra, Edze R.; Jackson, Ryan N.; Wiedenheft, Blake

2014-01-01

Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR–Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea. PMID:24909109

Simulations of Collisional Disruption at the Catastrophic Impact Energy Threshold: Effect of the Target's Internal Structure and Diameter

NASA Astrophysics Data System (ADS)

Michel, P.; Benz, W.; Richardson, D. C.

2005-08-01

Recent simulations of asteroid break-ups, including both the fragmentation of the parent body and the gravitational interactions of the fragments, have allowed to reproduced successfully the main properties of asteroid families formed in different regimes of impact energy. Here, using the same kind of simulations, we concentrate on a single regime of impact energy, the so-called catastrophic threshold usually designated by Qcrit, which results in the escape of half of the target's mass. Considering a wide range of diameter values and two kinds of internal structures of the parent body, monolithic and pre-shattered, we analyse their potential influences on the value of Qcrit and on the collisional outcome limited here to the fragment size and ejection speed distributions, which are the main outcome properties used by collisional models to study the evolutions of the different populations of small bodies. For all the considered diameters and the two internal structures of the parent body, we confirm that the process of gravitational reaccumulation is at the origin of the largest remnant's mass. We then find that, for a given diameter of the parent body, the impact energy corresponding to the catastrophic disruption threshold is highly dependent on the internal structure of the parent body. In particular, a pre-shattered parent body containing only damaged zones but no macroscopic voids is easier to disrupt than a monolithic parent body. Other kinds of internal properties that can also characterize small bodies in real populations will be investigated in a future work.

The Development of Novel Chemical Fragment-Based Descriptors Using Frequent Common Subgraph Mining Approach and Their Application in QSAR Modeling.

PubMed

Khashan, Raed; Zheng, Weifan; Tropsha, Alexander

2014-03-01

We present a novel approach to generating fragment-based molecular descriptors. The molecules are represented by labeled undirected chemical graph. Fast Frequent Subgraph Mining (FFSM) is used to find chemical-fragments (subgraphs) that occur in at least a subset of all molecules in a dataset. The collection of frequent subgraphs (FSG) forms a dataset-specific descriptors whose values for each molecule are defined by the number of times each frequent fragment occurs in this molecule. We have employed the FSG descriptors to develop variable selection k Nearest Neighbor (kNN) QSAR models of several datasets with binary target property including Maximum Recommended Therapeutic Dose (MRTD), Salmonella Mutagenicity (Ames Genotoxicity), and P-Glycoprotein (PGP) data. Each dataset was divided into training, test, and validation sets to establish the statistical figures of merit reflecting the model validated predictive power. The classification accuracies of models for both training and test sets for all datasets exceeded 75 %, and the accuracy for the external validation sets exceeded 72 %. The model accuracies were comparable or better than those reported earlier in the literature for the same datasets. Furthermore, the use of fragment-based descriptors affords mechanistic interpretation of validated QSAR models in terms of essential chemical fragments responsible for the compounds' target property. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distribution of rock fragments and their effects on hillslope soil erosion in purple soil, China

NASA Astrophysics Data System (ADS)

Wang, Xiaoyan

2017-04-01

Purple soil is widely distributed in Sichuan Basin and Three Gorges Reservoir Area. Purple soil region is abundant in soil fertility and hydrothermal resources, playing an important role in the agricultural development of China. Soil erosion has long been recognized as a major environmental problem in the purple soil region where the population is large and slope farming is commonly practiced, and rainstorm is numerous. The existence of rock fragments is one of the most important characteristics of purple soil. Rock fragments at the soil surface or in the soil layer affect soil erosion processes by water in various direct and indirect ways, thus the erosion processes of soil containing rock fragments have unique features. Against the severe soil degradation by erosion of purple soil slope, carrying out the research about the characteristics of purple soil containing rock fragments and understanding the influence of rock fragments on soil erosion processes have important significance, which would promote the rational utilization of purple soil slope land resources and accurate prediction of purple soil loss. Therefore, the aims of this study were to investigate the distribution of rock fragments in purple soil slope and the impact of rock fragment content on soil physical properties and soil erosion. First, field sampling methods were used to survey the spatial variability of rock fragments in soil profiles and along slope and the physical properties of soils containing rock fragments. Secondly, indoor simulated rainfall experiments were used to exam the effect of rock fragments in the soil layer on soil erosion processes and the relationships between rainfall infiltration, change of surface flow velocity, surface runoff volume and sediment on one hand, and rock fragment content (Rv, 0% 30%, which was determined according the results of field investigation for rock fragment distribution) on the other were investigated. Thirdly, systematic analysis about the influence of rock fragment cover on purple soil slope erosion process were carried on, under different conditions with two kind of rock fragment positions (resting on soil surface and embedded into top soil layer), varied rock fragment coverage (Rc, 0% 40%), two kind of soils with textural porosity or structural porosity, and three kind of rainfall intensities (I, 1 mm/min, 1.5 mm/min and 2 mm/min). Simulated rainfall experiments in situ plots in the field, combined with simulated rainfall experiments in soil pans indoor, were used. The main conclusions of this dissertation are as following: 1. The spatial distribution characteristics of rock fragments in purple soil slope and its effects on the soil physical properties were clarified basically. 2. The mechanism of influence of rock fragments within top soil layer on soil erosion processes was understood and a threshold of rock fragment content on the infiltration was figured out. 3. The relationships between surface rock fragment cover and hillslope soil erosion in purple soil under different conditions with varied rock fragment positions, soil structures and rainfall intensities were obtained and the soil and water conservation function of surface rock fragment cover on reducing soil loss was affirmed.

New multiplex PCR methods for rapid screening of genetically modified organisms in foods

PubMed Central

Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

2015-01-01

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products. PMID:26257724

New multiplex PCR methods for rapid screening of genetically modified organisms in foods.

PubMed

Datukishvili, Nelly; Kutateladze, Tamara; Gabriadze, Inga; Bitskinashvili, Kakha; Vishnepolsky, Boris

2015-01-01

We present novel multiplex PCR methods for rapid and reliable screening of genetically modified organisms (GMOs). New designed PCR primers targeting four frequently used GMO specific sequences permitted identification of new DNA markers, in particular 141 bp fragment of cauliflower mosaic virus (CaMV) 35S promoter, 224 bp fragment of Agrobacterium tumefaciens nopaline synthase (NOS) terminator, 256 bp fragment of 5-enolppyruvylshikimate-phosphate synthase (epsps) gene and 258 bp fragment of Cry1Ab delta-endotoxin (cry1Ab) gene for GMO screening. The certified reference materials containing Roundup Ready soybean (RRS) and maize MON 810 were applied for the development and optimization of uniplex and multiplex PCR systems. Evaluation of amplification products by agarose gel electrophoresis using negative and positive controls confirmed high specificity and sensitivity at 0.1% GMO for both RRS and MON 810. The fourplex PCR was developed and optimized that allows simultaneous detection of three common transgenic elements, such as: CaMV 35S promoter, NOS terminator, epsps gene together with soybean-specific lectin gene. The triplex PCR developed enables simultaneous identification of transgenic elements, such as: 35S promoter and cry1Ab gene together with maize zein gene. The analysis of different processed foods demonstrated that multiplex PCR methods developed in this study are useful for accurate and fast screening of GM food products.

Identification of chemogenomic features from drug–target interaction networks using interpretable classifiers

PubMed Central

Tabei, Yasuo; Pauwels, Edouard; Stoven, Véronique; Takemoto, Kazuhiro; Yamanishi, Yoshihiro

2012-01-01

Motivation: Drug effects are mainly caused by the interactions between drug molecules and their target proteins including primary targets and off-targets. Identification of the molecular mechanisms behind overall drug–target interactions is crucial in the drug design process. Results: We develop a classifier-based approach to identify chemogenomic features (the underlying associations between drug chemical substructures and protein domains) that are involved in drug–target interaction networks. We propose a novel algorithm for extracting informative chemogenomic features by using L1 regularized classifiers over the tensor product space of possible drug–target pairs. It is shown that the proposed method can extract a very limited number of chemogenomic features without loosing the performance of predicting drug–target interactions and the extracted features are biologically meaningful. The extracted substructure–domain association network enables us to suggest ligand chemical fragments specific for each protein domain and ligand core substructures important for a wide range of protein families. Availability: Softwares are available at the supplemental website. Contact: yamanishi@bioreg.kyushu-u.ac.jp Supplementary Information: Datasets and all results are available at http://cbio.ensmp.fr/~yyamanishi/l1binary/ . PMID:22962471

Investigating the mixture and subdivision of perceptual and conceptual processing in Japanese memory tests.

PubMed

Gabeza, R

1995-03-01

The dual nature of the Japanese writing system was used to investigate two assumptions of the processing view of memory transfer: (1) that both perceptual and conceptual processing can contribute to the same memory test (mixture assumption) and (2) that both can be broken into more specific processes (subdivision assumption). Supporting the mixture assumption, a word fragment completion test based on ideographic kanji characters (kanji fragment completion test) was affected by both perceptual (hiragana/kanji script shift) and conceptual (levels-of-processing) study manipulations kanji fragments, because it did not occur with the use of meaningless hiragana fragments. The mixture assumption is also supported by an effect of study script on an implicit conceptual test (sentence completion), and the subdivision assumption is supported by a crossover dissociation between hiragana and kanji fragment completion as a function of study script.

Virtual fragment preparation for computational fragment-based drug design.

PubMed

Ludington, Jennifer L

2015-01-01

Fragment-based drug design (FBDD) has become an important component of the drug discovery process. The use of fragments can accelerate both the search for a hit molecule and the development of that hit into a lead molecule for clinical testing. In addition to experimental methodologies for FBDD such as NMR and X-ray Crystallography screens, computational techniques are playing an increasingly important role. The success of the computational simulations is due in large part to how the database of virtual fragments is prepared. In order to prepare the fragments appropriately it is necessary to understand how FBDD differs from other approaches and the issues inherent in building up molecules from smaller fragment pieces. The ultimate goal of these calculations is to link two or more simulated fragments into a molecule that has an experimental binding affinity consistent with the additive predicted binding affinities of the virtual fragments. Computationally predicting binding affinities is a complex process, with many opportunities for introducing error. Therefore, care should be taken with the fragment preparation procedure to avoid introducing additional inaccuracies.This chapter is focused on the preparation process used to create a virtual fragment database. Several key issues of fragment preparation which affect the accuracy of binding affinity predictions are discussed. The first issue is the selection of the two-dimensional atomic structure of the virtual fragment. Although the particular usage of the fragment can affect this choice (i.e., whether the fragment will be used for calibration, binding site characterization, hit identification, or lead optimization), general factors such as synthetic accessibility, size, and flexibility are major considerations in selecting the 2D structure. Other aspects of preparing the virtual fragments for simulation are the generation of three-dimensional conformations and the assignment of the associated atomic point charges.

DebriSat Fragment Characterization System and Processing Status

NASA Technical Reports Server (NTRS)

Rivero, M.; Shiotani, B.; M. Carrasquilla; Fitz-Coy, N.; Liou, J. C.; Sorge, M.; Huynh, T.; Opiela, J.; Krisko, P.; Cowardin, H.

2016-01-01

The DebriSat project is a continuing effort sponsored by NASA and DoD to update existing break-up models using data obtained from hypervelocity impact tests performed to simulate on-orbit collisions. After the impact tests, a team at the University of Florida has been working to characterize the fragments in terms of their mass, size, shape, color and material content. The focus of the post-impact effort has been the collection of 2 mm and larger fragments resulting from the hypervelocity impact test. To date, in excess of 125K fragments have been recovered which is approximately 40K more than the 85K fragments predicted by the existing models. While the fragment collection activities continue, there has been a transition to the characterization of the recovered fragments. Since the start of the characterization effort, the focus has been on the use of automation to (i) expedite the fragment characterization process and (ii) minimize the effects of human subjectivity on the results; e.g., automated data entry processes were developed and implemented to minimize errors during transcription of the measurement data. At all steps of the process, however, there is human oversight to ensure the integrity of the data. Additionally, repeatability and reproducibility tests have been developed and implemented to ensure that the instrumentations used in the characterization process are accurate and properly calibrated.

The Evolution of Grain Size Distribution in Explosive Rock Fragmentation - Sequential Fragmentation Theory Revisited

NASA Astrophysics Data System (ADS)

Scheu, B.; Fowler, A. C.

2015-12-01

Fragmentation is a ubiquitous phenomenon in many natural and engineering systems. It is the process by which an initially competent medium, solid or liquid, is broken up into a population of constituents. Examples occur in collisions and impacts of asteroids/meteorites, explosion driven fragmentation of munitions on a battlefield, as well as of magma in a volcanic conduit causing explosive volcanic eruptions and break-up of liquid drops. Besides the mechanism of fragmentation the resulting frequency-size distribution of the generated constituents is of central interest. Initially their distributions were fitted empirically using lognormal, Rosin-Rammler and Weibull distributions (e.g. Brown & Wohletz 1995). The sequential fragmentation theory (Brown 1989, Wohletz at al. 1989, Wohletz & Brown 1995) and the application of fractal theory to fragmentation products (Turcotte 1986, Perfect 1997, Perugini & Kueppers 2012) attempt to overcome this shortcoming by providing a more physical basis for the applied distribution. Both rely on an at least partially scale-invariant and thus self-similar random fragmentation process. Here we provide a stochastic model for the evolution of grain size distribution during the explosion process. Our model is based on laboratory experiments in which volcanic rock samples explode naturally when rapidly depressurized from initial pressures of several MPa to ambient conditions. The physics governing this fragmentation process has been successfully modelled and the observed fragmentation pattern could be numerically reproduced (Fowler et al. 2010). The fragmentation of these natural rocks leads to grain size distributions which vary depending on the experimental starting conditions. Our model provides a theoretical description of these different grain size distributions. Our model combines a sequential model of the type outlined by Turcotte (1986), but generalized to cater for the explosive process appropriate here, in particular by including in the description of the fracturing events in which the rock fragments, with a recipe for the production of fines, as observed in the experiments. To our knowledge, this implementation of a deterministic fracturing process into a stochastic (sequential) model is unique, further it provides the model with some forecasting power.

Causes of fragmented crystals in ignimbrites: a case study of the Cardones ignimbrite, Northern Chile

NASA Astrophysics Data System (ADS)

van Zalinge, M. E.; Cashman, K. V.; Sparks, R. S. J.

2018-03-01

Broken crystals have been documented in many large-volume caldera-forming ignimbrites and can help to understand the role of crystal fragmentation in both eruption and compaction processes, the latter generally overlooked in the literature. This study investigates the origin of fragmented crystals in the > 1260 km3, crystal-rich Cardones ignimbrites located in the Central Andes. Observations of fragmented crystals in non-welded pumice clasts indicate that primary fragmentation includes extensive crystal breakage and an associated ca. 5 vol% expansion of individual crystals while preserving their original shapes. These observations are consistent with the hypothesis that crystals fragment in a brittle response to rapid decompression associated with the eruption. Additionally, we observe that the extent of crystal fragmentation increases with increasing stratigraphic depth in the ignimbrite, recording secondary crystal fragmentation during welding and compaction. Secondary crystal fragmentation aids welding and compaction in two ways. First, enhanced crystal fragmentation at crystal-crystal contacts accommodates compaction along the principal axis of stress. Second, rotation and displacement of individual crystal fragments enhances lateral flow in the direction(s) of least principal stress. This process increases crystal aspect ratios and forms textures that resemble mantled porphyroclasts in shear zones, indicating lateral flow adds to processes of compaction and welding alongside bubble collapse. In the Cardones ignimbrite, secondary fragmentation commences at depths of 175-250 m (lithostatic pressures 4-6 MPa), and is modulated by both the overlying crystal load and the time spent above the glass transition temperature. Under these conditions, the existence of force-chains can produce stresses at crystal-crystal contacts of a few times the lithostatic pressure. We suggest that documenting crystal textures, in addition to conventional welding parameters, can provide useful information about welding processes in thick crystal-rich ignimbrites.

Fragmentation targeted at preferred discontinuities: A new concept in endolithotripsy with Holmium laser:YAG.

PubMed

Sánchez-Martín, F M; Emiliani, E; Pueyo-Morer, E; Angerri-Feu, O; Sanguedolce, F; Millán, F; Villavicencio, H

2018-04-17

There are currently 3holmium laser, YAG (Ho:YAG) endolithotripsy procedures that are considered basic (fragmentation, pulverisation, "pop-corn" technique). We present the technique of fragmentation targeted at preferred discontinuities (FTPD), a new concept of endolithotripsy by Ho:YAG laser. The FTPD technique is based on the selective application of energy (targeting a specific preselected point) to an area that is visually prone to the formation of a fracture line or preferred discontinuity (conditioned by the anisotropy of the urolithiasis). The ideal energy regimen (setting) is a high range of working energy (2-3J) with a very low frequency range (5-8Hz) and short pulse width. Between January 2015 to February 2017, the FTPD technique was used in 37 procedures (7 NLP, 16 RIRS, 12 URS, 2 cystolithotomies), with a Ho:YAG laser (Lumenis Pulse 120H ® , Tel-Aviv, Israel). Maximum power used: 24W (3J/8Hz) with fibres of 365μ and 273μ (URS, RIRS), and 32W (4J/8Hz) with fibres of 550μ (NLP, cystolithotomy). Strategic improvement was achieved in all cases using the TFPD technique to continue the endolithotripsy or remove fragments. No complications were recorded after the use of this method. FTPD can be considered a complementary option in combination with the basic methods of fragmentation and pulverisation. In our experience, it constitutes significant progress in optimising the performance of Ho:YAG laser endolithotripsy. Copyright © 2018 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

Modularity of Protein Folds as a Tool for Template-Free Modeling of Structures.

PubMed

Vallat, Brinda; Madrid-Aliste, Carlos; Fiser, Andras

2015-08-01

Predicting the three-dimensional structure of proteins from their amino acid sequences remains a challenging problem in molecular biology. While the current structural coverage of proteins is almost exclusively provided by template-based techniques, the modeling of the rest of the protein sequences increasingly require template-free methods. However, template-free modeling methods are much less reliable and are usually applicable for smaller proteins, leaving much space for improvement. We present here a novel computational method that uses a library of supersecondary structure fragments, known as Smotifs, to model protein structures. The library of Smotifs has saturated over time, providing a theoretical foundation for efficient modeling. The method relies on weak sequence signals from remotely related protein structures to create a library of Smotif fragments specific to the target protein sequence. This Smotif library is exploited in a fragment assembly protocol to sample decoys, which are assessed by a composite scoring function. Since the Smotif fragments are larger in size compared to the ones used in other fragment-based methods, the proposed modeling algorithm, SmotifTF, can employ an exhaustive sampling during decoy assembly. SmotifTF successfully predicts the overall fold of the target proteins in about 50% of the test cases and performs competitively when compared to other state of the art prediction methods, especially when sequence signal to remote homologs is diminishing. Smotif-based modeling is complementary to current prediction methods and provides a promising direction in addressing the structure prediction problem, especially when targeting larger proteins for modeling.

Salt Weathering on Mars

NASA Astrophysics Data System (ADS)

Jagoutz, E.

2006-12-01

Large well rounded boulders and angular rock fragments characterizes the Martian landscape as seen on the recent excellent quality photos. Analyzing the different rock-shapes indicates a time sequence of emplacement, fragmentation and transport of different rocks on Mars, which might give interesting insight into transport and weathering processes. Larger commonly well rounded boulders were emplaced onto gravel plains. After emplacement, these rocks were fragmented and disassembled. Nests of angular rock fragments are marking the locations of preexisting larger rocks. Frequently it is possible to reconstruct larger rounded rocks from smaller angular fragments. In other cases transport after fragmentation obscured the relationship of the fragments. However, a strewn field of fragments is still reminiscent of the preexisting rock. Mechanical salt weathering could be a plausible explanation for the insitu fragmentation of larger rounded blocks into angular fragments. Impact or secondary air fall induced fragmentation produces very different patterns, as observed around impact crates on Earth. Salt weathering of rocks is a common process in terrestrial environments. Salt crystallization in capillaries causes fragmentation of rocks, irrespective of the process of salt transportation and concentration. On Earth significant salt weathering can be observed in different climatic environments: in the transition zone of alluvial aprons and salt playas in desserts and in dry valleys of Antarctica. In terrestrial semi-arid areas the salt is transported by salt solution, which is progressively concentrated by evaporation. In Antarctic dry valleys freeze-thaw cycles causes salt transportation and crystallization resulting in rock fragmentation. This salt induced process can lead to complete destruction of rocks and converts rocks to fine sand. The efficient breakdown of rocks is dominating the landscape in some dry valleys of the Earth but possibly also on Mars. (Malin, 1974). However, irrespectively of the climatic environment a liquid brine is a necessity for salt induced fragmentation of rocks.M. C. Malin (1974) JGR Vol 79,26 p 3888-3894

A MATHEMATICAL ANALYSIS OF SELEX

PubMed Central

Levine, Howard A.; Nilsen-Hamilton, Marit

2007-01-01

SELEX (Systematic Evolution of Ligands by Exponential Enrichment) is a procedure by which a mixture of nucleic acids can be separated into pure components with the goal of isolating those with specific biochemical activities. The basic idea is to combine the mixture with a specific target molecule and then separate the target-NA complex from the resulting reaction. The target-NA complex is then separated by mechanical means (for example by nitrocellulose filtration), the NA is then eluted from the complex, amplified by PCR (polymerase chain reaction) and the process repeated. After several rounds, one should be left with a pool of [NA]that consists mostly of the species in the original pool that best binds to the target. In Irvine et al. (1991) a mathematical analysis of this process was given. In this paper we revisit Irvine et al. (1991). By rewriting the equations for the SELEX process, we considerably reduce the labor of computing the round to round distribution of nucleic acid fractions. We also establish necessary and sufficient conditions for the SELEX process to converge to a pool consisting solely of the best binding nucleic acid to a fixed target in a manner that maximizes the percentage of bound target. The assumption is that there is a single nucleic acid binding site on the target that permits occupation by no more than one nucleic acid. We analyze the case for which there is no background loss, (no support losses and no free [NA] left on the support.) We then examine the case in which such there are such losses. The significance of the analysis is that it suggests an experimental approach for the SELEX process as defined in Irvine et al. (1991) to converge to a pool consisting of a single best binding nucleic acid without recourse to any a-priori information about the nature of the binding constants or the distribution of the individual nucleic acid fragments. PMID:17218151

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment.

PubMed

Zdobnova, Tatiana A; Dorofeev, Sergey G; Tananaev, Piter N; Vasiliev, Roman B; Balandin, Taras G; Edelweiss, Eveline F; Stremovskiy, Oleg A; Balalaeva, Irina V; Turchin, Ilya V; Lebedenko, Ekaterina N; Zlomanov, Vladimir P; Deyev, Sergey M

2009-01-01

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Smart linkers in polymer-drug conjugates for tumor-targeted delivery.

PubMed

Chang, Minglu; Zhang, Fang; Wei, Ting; Zuo, Tiantian; Guan, Yuanyuan; Lin, Guimei; Shao, Wei

2016-01-01

To achieve effective chemotherapy, many types of drug delivery systems have been developed for the specific environments in tumor tissues. Polymer-drug conjugates are increasingly used in tumor therapy due to several significant advantages over traditional delivery systems. In the fabrication of polymer-drug conjugates, a smart linker is an important component that joins two fragments or molecules together and can be cleared by a specific stimulus, which results in targeted drug delivery and controlled release. By regulating the conjugation between the drug and the nanocarriers, stimulus-sensitive systems based on smart linkers can offer high payloads, certified stability, controlled release and targeted delivery. In this review, we summarize the current state of smart linkers (e.g. disulfide, hydrazone, peptide, azo) used recently in various polymer-drug conjugate-based delivery systems with a primary focus on their sophisticated design principles and drug delivery mechanisms as well as in vivo processes.

Holographic studies of the vapor explosion of vaporizing water-in-fuel emulsion droplets

NASA Technical Reports Server (NTRS)

Sheffield, S. A.; Hess, C. F.; Trolinger, J. D.

1982-01-01

Holographic studies were performed which examined the fragmentation process during vapor explosion of a water-in-fuel (hexadecane/water) emulsion droplet. Holograms were taken at 700 to 1000 microseconds after the vapor explosion. Photographs of the reconstructed holograms reveal a wide range of fragment droplet sizes created during the explosion process. Fragment droplet diameters range from below 10 microns to over 100 microns. It is estimated that between ten thousand and a million fragment droplets can result from this extremely violent vapor explosion process. This enhanced atomization is thus expected to have a pronounced effect on vaporization processes which are present during combustion of emulsified fuels.

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors.

PubMed

Austin, Carol; Pettit, Simon N; Magnolo, Sharon K; Sanvoisin, Jonathan; Chen, Wenjie; Wood, Stephen P; Freeman, Lauren D; Pengelly, Reuben J; Hughes, Dallas E

2012-08-01

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 µM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.

Evolutions in fragment-based drug design: the deconstruction–reconstruction approach

PubMed Central

Chen, Haijun; Zhou, Xiaobin; Wang, Ailan; Zheng, Yunquan; Gao, Yu; Zhou, Jia

2014-01-01

Recent advances in the understanding of molecular recognition and protein–ligand interactions have facilitated rapid development of potent and selective ligands for therapeutically relevant targets. Over the past two decades, a variety of useful approaches and emerging techniques have been developed to promote the identification and optimization of leads that have high potential for generating new therapeutic agents. Intriguingly, the innovation of a fragment-based drug design (FBDD) approach has enabled rapid and efficient progress in drug discovery. In this critical review, we focus on the construction of fragment libraries and the advantages and disadvantages of various fragment-based screening (FBS) for constructing such libraries. We also highlight the deconstruction–reconstruction strategy by utilizing privileged fragments of reported ligands. PMID:25263697

The DINGO dataset: a comprehensive set of data for the SAMPL challenge

NASA Astrophysics Data System (ADS)

Newman, Janet; Dolezal, Olan; Fazio, Vincent; Caradoc-Davies, Tom; Peat, Thomas S.

2012-05-01

Part of the latest SAMPL challenge was to predict how a small fragment library of 500 commercially available compounds would bind to a protein target. In order to assess the modellers' work, a reasonably comprehensive set of data was collected using a number of techniques. These included surface plasmon resonance, isothermal titration calorimetry, protein crystallization and protein crystallography. Using these techniques we could determine the kinetics of fragment binding, the energy of binding, how this affects the ability of the target to crystallize, and when the fragment did bind, the pose or orientation of binding. Both the final data set and all of the raw images have been made available to the community for scrutiny and further work. This overview sets out to give the parameters of the experiments done and what might be done differently for future studies.

On the influence of particle morphology on the post-impact ballistic response of ceramic armour materials

NASA Astrophysics Data System (ADS)

Hameed, Amer; Appleby-Thomas, Gareth; Wood, David; Jaansalu, Kevin

2015-06-01

Recent studies have shown evidence that the ballistic-resistance of fragmented (comminuted) ceramics is independent of the original strength of the material. In particular, experimental investigations into the ballistic behaviour of such fragmented ceramics have indicated that this response is correlated to shattered ceramic morphology. This suggests that careful control of ceramic microstructure - and therefore failure paths - might provide a route to optimise post-impact ballistic performance, thereby enhancing multi-hit capability. In this study, building on previous in-house work, ballistic tests were conducted using pre-formed `fragmented-ceramic' analogues based around three morphologically differing (but chemically identical) alumina feedstock materials compacted into target `pucks. In an evolution of previous work, variation of target thickness provided additional insight into an apparent morphology-based contribution to ballistic response.

Development of a Double Allylboration Reagent Targeting 1,5-syn-(E)-Diols: Application to the Synthesis of the C(23)-C(40) Fragment of Tetrafibricin

PubMed Central

Nuhant, Philippe; Kister, Jeremy; Lira, Ricardo; Sorg, Achim; Roush, William R.

2011-01-01

Interest in the synthesis of the C(23)-C(40) fragment 2 of tetrafibricin prompted us to develop a new method for the synthesis of 1,5-syn-(E)-diols. Toward this end, the kinetically controlled hydroboration of allenes 6, 33, ent-39, 42 and 45 with the Soderquist borane 25R were studied. Tetrabutylammonium allenyltrifluoroborate 45 gave superior results and was utilized in a double allylboration sequence with two different aldehydes to provide the targeted 1,5-syn-(E)-diols in generally high yields (72–98%), and with high enantioselectivity (>95% e.e.), diastereoselectivity (d.r. >20:1), and (E)/(Z) selectivity (>20:1). This new method was applied to the synthesis of the C(23)-C(40) fragment 2 of tetrafibricin. PMID:21857752

Current perspectives in fragment-based lead discovery (FBLD)

PubMed Central

Lamoree, Bas; Hubbard, Roderick E.

2017-01-01

It is over 20 years since the first fragment-based discovery projects were disclosed. The methods are now mature for most ‘conventional’ targets in drug discovery such as enzymes (kinases and proteases) but there has also been growing success on more challenging targets, such as disruption of protein–protein interactions. The main application is to identify tractable chemical startpoints that non-covalently modulate the activity of a biological molecule. In this essay, we overview current practice in the methods and discuss how they have had an impact in lead discovery – generating a large number of fragment-derived compounds that are in clinical trials and two medicines treating patients. In addition, we discuss some of the more recent applications of the methods in chemical biology – providing chemical tools to investigate biological molecules, mechanisms and systems. PMID:29118093

Prompt-delayed $$\\gamma$$-ray spectroscopy with AGATA, EXOGAM and VAMOS++

DOE PAGES

Kim, Y. H.; Lemasson, A.; Rejmund, M.; ...

2017-08-10

Here, a new experimental setup to measure prompt-delayed γ-ray coincidences from isotopically identified fission fragments, over a wide time range of 100ns-200μ s, is presented. The fission fragments were isotopically identified, on an event-by-event basis, using the VAMOS++ large acceptance spectrometer. The prompt γ rays emitted at the target position and corresponding delayed γ rays emitted at the focal plane of the spectrometer were detected using, respectively, thirty two crystals of the AGATA γ-ray tracking array and seven EXOGAM HPGe Clover detectors. Finally, fission fragments produced in fusion and transfer-induced fission reactions, using a 238U beam at an energy ofmore » 6.2 MeV/u impinging on a 9Be target, were used to characterize and qualify the performance of the detection system.« less

Modelling fragmentations of aminoacids after resonant electron attachment: quantum evidence of possible direct -OH detachment

NASA Astrophysics Data System (ADS)

Panosetti, C.; Baccarelli, I.; Sebastianelli, F.; Gianturco, F. A.

2010-10-01

We investigate some aspects of the radiation damage mechanisms in biomolecules, focusing on the modelling of resonant fragmentation caused by the attachment of low-energy electrons (LEEs) initially ejected by biological tissues when exposed to ionizing radiation. Scattering equations are formulated within a symmetry-adapted, single-center expansion of both continuum and bound electrons, and the interaction forces are obtained from a combination of ab initio calculations and a nonempirical model of exchange and correlation effects developped in our group. We present total elastic scattering cross-sections and resonance features obtained for the equilibrium geometries of glycine, alanine, proline and valine. Our results at those geometries of the target molecules are briefly shown to qualitatively explain some of the fragmentation patterns obtained in experiments. We further carry out a one-dimensional (1D) modeling for the dynamics of intramolecular energy transfers mediated by the vibrational activation of selected bonds: our calculations indicate that resonant electron attachment to glycine can trigger direct, dissociative evolution of the complex into (Gly-OH)- and -OH losses, while they also find that the same process does not occur via a direct, 1D dissociative path in the larger aminoacids of the present study.

Structure Of Neutron-Rich Nuclei In A˜100 Region Observed In Fusion-Fission Reactions

NASA Astrophysics Data System (ADS)

Wu, C. Y.; Hua, H.; Cline, D.; Hayes, A. B.; Teng, R.; Clark, R. M.; Fallon, P.; Görgen, A.; Macchiavelli, A. O.; Vetter, K.

2003-03-01

Neutron-rich nuclei around A˜100 were populated as fission fragments produced by the 238U(α,f) fusion-fission reaction. The deexcitation γ rays were detected by Gammasphere in coincidence with the detection of both fission fragments by the Rochester 4π heavy-ion detector array, CHICO. This technique allows Doppler-shift corrections to be applied for the observed γ rays on an event-by-event basis thus establishing the origin of γ rays from either fission fragment. In addition, it allows observation of γ-ray transitions from states with short lifetimes and offers the opportunity to study nuclear species beyond the reach of the spontaneous fission process. With these advantages, one can extend the spectroscopic study to higher spins than those derived using the thick-target technique, and to more neutron-rich nuclei than those derived from spontaneous fissions. Among the new and interesting phenomena identified in this rapid shape-changing region, the most distinct result is the evidence for a prolate-to-oblate shape transition occurring at 116Pd, which may have important implications to our understanding of the shell structure for neutron-rich nuclei.

A mutation within the transmembrane domain of melanosomal protein Silver (Pmel17) changes lumenal fragment interactions

PubMed Central

Kuliawat, Regina; Santambrogio, Laura

2009-01-01

Melanocytes synthesize and store melanin within tissue-specific organelles, the melanosomes. Melanin deposition takes place along fibrils found within these organelles and fibril formation is known to depend on trafficking of the membrane glycoprotein Silver/Pmel17. However, correctly targeted, full-length Silver/Pmel17 cannot form fibers. Proteolytic processing in endosomal compartments and the generation of a lumenal Mα fragment that is incorporated into amyloid-like structures is also essential. Dominant White (DWhite), a mutant form of Silver/Pmel17 first described in chicken, causes disorganized fibers and severe hypopigmentation due to melanocyte death. Surprisingly, the DWhite mutation is an insertion of three amino acids into the transmembrane domain; the DWhite-Mα fragment is unaffected. To determine the functional importance of the transmembrane domain in organized fibril assembly, we investigated membrane trafficking and multimerization of Silver/Pmel17/DWhite proteins. We demonstrate that the DWhite mutation changes lipid interactions and disulfide bond-mediated associations of lumenal domains. Thus, partitioning into membrane microdomains and effects on conformation explain how the transmembrane region may contribute to the structural integrity of Silver/Pmel17 oligomers or influence toxic, amyloidogenic properties. PMID:19679373

A spatially explicit decision support model for restoration of forest bird habitat

USGS Publications Warehouse

Twedt, D.J.; Uihlein, W.B.; Elliott, A.B.

2006-01-01

The historical area of bottomland hardwood forest in the Mississippi Alluvial Valley has been reduced by >75%. Agricultural production was the primary motivator for deforestation; hence, clearing deliberately targeted higher and drier sites. Remaining forests are highly fragmented and hydrologically altered, with larger forest fragments subject to greater inundation, which has negatively affected many forest bird populations. We developed a spatially explicit decision support model, based on a Partners in Flight plan for forest bird conservation, that prioritizes forest restoration to reduce forest fragmentation and increase the area of forest core (interior forest >1 km from 'hostile' edge). Our primary objective was to increase the number of forest patches that harbor >2000 ha of forest core, but we also sought to increase the number and area of forest cores >5000 ha. Concurrently, we targeted restoration within local (320 km2) landscapes to achieve >60% forest cover. Finally, we emphasized restoration of higher-elevation bottomland hardwood forests in areas where restoration would not increase forest fragmentation. Reforestation of 10% of restorable land in the Mississippi Alluvial Valley (approximately 880,000 ha) targeted at priorities established by this decision support model resulted in approximately 824,000 ha of new forest core. This is more than 32 times the amount of core forest added through reforestation of randomly located fields (approximately 25,000 ha). The total area of forest core (1.6 million ha) that resulted from targeted restoration exceeded habitat objectives identified in the Partners in Flight Bird Conservation Plan and approached the area of forest core present in the 1950s.

Optical model calculations of 14.6A GeV silicon fragmentation cross sections

NASA Technical Reports Server (NTRS)

Townsend, Lawrence W.; Khan, Ferdous; Tripathi, Ram K.

1993-01-01

An optical potential abrasion-ablation collision model is used to calculate hadronic dissociation cross sections for a 14.6 A GeV(exp 28) Si beam fragmenting in aluminum, tin, and lead targets. The frictional-spectator-interaction (FSI) contributions are computed with two different formalisms for the energy-dependent mean free path. These estimates are compared with experimental data and with estimates obtained from semi-empirical fragmentation models commonly used in galactic cosmic ray transport studies.

Comparisons of cross-section predictions for relativistic iron and argon beams with semiempirical fragmentation models

NASA Technical Reports Server (NTRS)

Townsend, Lawrence W.; Tripathi, Ram K.; Khan, Ferdous

1993-01-01

Cross-section predictions with semi-empirical nuclear fragmentation models from the Langley Research Center and the Naval Research Laboratory are compared with experimental data for the breakup of relativistic iron and argon projectile nuclei in various targets. Both these models are commonly used to provide fragmentation cross-section inputs into galactic cosmic ray transport codes for shielding and exposure analyses. Overall, the Langley model appears to yield better agreement with the experimental data.

Comprehensive analytical strategy for biomonitoring of pesticides in urine by liquid chromatography–orbitrap high resolution masss pectrometry.

PubMed

Roca, M; Leon, N; Pastor, A; Yusà, V

2014-12-29

In this study we propose an analytical strategy that combines a target approach for the quantitative analysis of contemporary pesticide metabolites with a comprehensive post-target screening for the identification of biomarkers of exposure to environmental contaminants in urine using liquid chromatography coupled to high-resolution mass spectrometry (LC–HRMS). The quantitative method for the target analysis of 29 urinary metabolites of organophosphate (OP) insecticides, synthetic pyrethroids, herbicides and fungicides was validated after a previous statistical optimization of the main factors governing the ion source ionization and a fragmentation study using the high energy collision dissociation (HCD) cell. The full scan accurate mass data were acquired with a resolving power of 50,000 FWHM (scan speed, 2 Hz), in both ESI+ and ESI− modes, and with and without HCD-fragmentation. The method – LOQ was lower than 3.2 μg L−1 for the majority of the analytes. For post-target screening a customized theoretical database was built, for the identification of 60 metabolites including pesticides, PAHs, phenols, and other metabolites of environmental pollutants. For identification purposes, accurate exact mass with less than 5 ppm, and diagnostic ions including isotopes and/or fragments were used. The analytical strategy was applied to 20 urine sample collected from children living in Valencia Region. Eleven target metabolites were detected with concentrations ranging from 1.18 to 131 μg L−1. Likewise, several compounds were tentatively identified in the post-target analysis belonging to the families of phthalates, phenols and parabenes. The proposed strategy is suitable for the determination of target pesticide biomarkers in urine in the framework of biomonitoring studies, and appropriate for the identification of other non-target metabolites.

Target analysis of primary aromatic amines combined with a comprehensive screening of migrating substances in kitchen utensils by liquid chromatography-high resolution mass spectrometry.

PubMed

Sanchis, Yovana; Coscollà, Clara; Roca, Marta; Yusà, Vicent

2015-06-01

An analytical strategy including both the quantitative target analysis of 8 regulated primary aromatic amines (PAAs), as well as a comprehensive post-run target screening of 77 migrating substances, was developed for nylon utensils, using liquid chromatography-orbitrap-high resolution mass spectrometry (LC-HRMS) operating in full scan mode. The accurate mass data were acquired with a resolving power of 50,000 FWHM (scan speed, 2 Hz), and by alternating two acquisition events, ESI+ with and without fragmentation. The target method was validated after statistical optimization of the main ionization and fragmentation parameters. The quantitative method presented appropriate performance to be used in official monitoring with recoveries ranging from 78% to 112%, precision in terms of Relative Standard Deviation (RSD) was less than 15%, and the limits of quantification were between 2 and 2.5 µg kg(-1). For post-target screening, a customized theoretical database was built for food contact material migrants, including bisphenols, phthalates, and other amines. For identification purposes, accurate exact mass (<5 ppm) and some diagnostic ions including fragments were used. The strategy was applied to 10 real samples collected from different retailers in the Valencian Region (Spain) during 2014. Six out of eight target PAAs were detected in at least one sample in the target analysis. The most frequently detected compounds were 4,4'-methylenedianiline and aniline, with concentrations ranging from 2.4 to 19,715 µg kg(-1) and 2.5 to 283 µg kg(-1), respectively. Two phthalates were identified and confirmed in the post-run target screening analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Status of Reconstruction of Fragmented Diamond-on-Silicon Collector From Genesis Spacecraft Solar Wind Concentrator

NASA Technical Reports Server (NTRS)

Rodriquez, Melissa C.; Calaway, M. C.; McNamara, K. M.; Hittle, J. D.

2009-01-01

In addition to passive solar wind collector surfaces, the Genesis Discovery Mission science canister had on board an electrostatic concave mirror for concentrating the solar wind ions, known as the concentrator . The 30-mm-radius collector focal point (the target) was comprised of 4 quadrants: two of single crystal SiC, one of polycrystalline 13C diamond and one of diamond-like-carbon (DLC) on a silicon substrate. [DLC-on-silicon is also sometimes referenced as Diamond-on-silicon, DOS.] Three of target quadrants survived the hard landing intact, but the DLC-on-silicon quadrant fractured into numerous pieces (Fig. 1). This abstract reports the status of identifying the DLC target fragments and reconstructing their original orientation.

A note on the self-similar solutions to the spontaneous fragmentation equation

NASA Astrophysics Data System (ADS)

Breschi, Giancarlo; Fontelos, Marco A.

2017-05-01

We provide a method to compute self-similar solutions for various fragmentation equations and use it to compute their asymptotic behaviours. Our procedure is applied to specific cases: (i) the case of mitosis, where fragmentation results into two identical fragments, (ii) fragmentation limited to the formation of sufficiently large fragments, and (iii) processes with fragmentation kernel presenting a power-like behaviour.

Onset of damping in energetic heavy-ion interactions

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

Kowalski, L.; Haustein, P.E.; Cumming, J.B.

1983-08-22

Measurements of longitudinal momenta transferred to mass-identified products of the fragmentation of Cu by /sup 12/C ions give clear evidence for a change in reaction mechanism between 22 and 84 MeV/u. Results at 84 MeV/u are generally consistent with peripheral interactions. However, at 22 MeV/u large momentum transfers observed for near-target products suggest that strongly damped processes have become important. Limits to momentum transfer of the type reported by Galin et al. are shown to arise in a natural way from this transition.

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

Highly efficient CRISPR/HDR-mediated knock-in for mouse embryonic stem cells and zygotes.

PubMed

Wang, Bangmei; Li, Kunyu; Wang, Amy; Reiser, Michelle; Saunders, Thom; Lockey, Richard F; Wang, Jia-Wang

2015-10-01

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Efficient search of multiple types of targets

NASA Astrophysics Data System (ADS)

Wosniack, M. E.; Raposo, E. P.; Viswanathan, G. M.; da Luz, M. G. E.

2015-12-01

Random searches often take place in fragmented landscapes. Also, in many instances like animal foraging, significant benefits to the searcher arise from visits to a large diversity of patches with a well-balanced distribution of targets found. Up to date, such aspects have been widely ignored in the usual single-objective analysis of search efficiency, in which one seeks to maximize just the number of targets found per distance traversed. Here we address the problem of determining the best strategies for the random search when these multiple-objective factors play a key role in the process. We consider a figure of merit (efficiency function), which properly "scores" the mentioned tasks. By considering random walk searchers with a power-law asymptotic Lévy distribution of step lengths, p (ℓ ) ˜ℓ-μ , with 1 <μ ≤3 , we show that the standard optimal strategy with μopt≈2 no longer holds universally. Instead, optimal searches with enhanced superdiffusivity emerge, including values as low as μopt≈1.3 (i.e., tending to the ballistic limit). For the general theory of random search optimization, our findings emphasize the necessity to correctly characterize the multitude of aims in any concrete metric to compare among possible candidates to efficient strategies. In the context of animal foraging, our results might explain some empirical data pointing to stronger superdiffusion (μ <2 ) in the search behavior of different animal species, conceivably associated to multiple goals to be achieved in fragmented landscapes.

RISC-interacting clearing 3’- 5’ exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana

PubMed Central

Zhang, Zhonghui; Hu, Fuqu; Sung, Min Woo; Shu, Chang; Castillo-González, Claudia; Koiwa, Hisashi; Tang, Guiliang; Dickman, Martin; Li, Pingwei; Zhang, Xiuren

2017-01-01

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC. DOI: http://dx.doi.org/10.7554/eLife.24466.001 PMID:28463111

Targeting α-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies.

PubMed

Moussaud, Simon; Malany, Siobhan; Mehta, Alka; Vasile, Stefan; Smith, Layton H; McLean, Pamela J

2015-05-01

Reducing the burden of α-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target α-synuclein oligomers has been a limitation to drug discovery programs. Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein-synuclein interactions. We have developed two robust models to monitor α-synuclein oligomerization by generating novel stable cell lines expressing α-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system. A pilot screen was performed resulting in the identification of two potential hits, a p38 MAPK inhibitor and a casein kinase 2 inhibitor, thereby demonstrating the suitability of our protein-fragment complementation assay for the measurement of α-synuclein oligomerization in living cells at high throughput. The application of the strategy described herein to monitor α-synuclein oligomer formation in living cells with high throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

Anthropogenic Matrices Favor Homogenization of Tree Reproductive Functions in a Highly Fragmented Landscape.

PubMed

Carneiro, Magda Silva; Campos, Caroline Cambraia Furtado; Beijo, Luiz Alberto; Ramos, Flavio Nunes

2016-01-01

Species homogenization or floristic differentiation are two possible consequences of the fragmentation process in plant communities. Despite the few studies, it seems clear that fragments with low forest cover inserted in anthropogenic matrices are more likely to experience floristic homogenization. However, the homogenization process has two other components, genetic and functional, which have not been investigated. The purpose of this study was to verify whether there was homogenization of tree reproductive functions in a fragmented landscape and, if found, to determine how the process was influenced by landscape composition. The study was conducted in eight fragments in southwest Brazil. The study was conducted in eight fragments in southwestern Brazil. In each fragment, all individual trees were sampled that had a diameter at breast height ≥3 cm, in ten plots (0.2 ha) and, classified within 26 reproductive functional types (RFTs). The process of functional homogenization was evaluated using additive partitioning of diversity. Additionally, the effect of landscape composition on functional diversity and on the number of individuals within each RFT was evaluated using a generalized linear mixed model. appeared to be in a process of functional homogenization (dominance of RFTs, alpha diversity lower than expected by chance and and low beta diversity). More than 50% of the RFTs and the functional diversity were affected by the landscape parameters. In general, the percentage of forest cover has a positive effect on RFTs while the percentage of coffee matrix has a negative one. The process of functional homogenization has serious consequences for biodiversity conservation because some functions may disappear that, in the long term, would threaten the fragments. This study contributes to a better understanding of how landscape changes affect the functional diversity, abundance of individuals in RFTs and the process of functional homogenization, as well as how to manage fragmented landscapes.

Anthropogenic Matrices Favor Homogenization of Tree Reproductive Functions in a Highly Fragmented Landscape

PubMed Central

2016-01-01

Species homogenization or floristic differentiation are two possible consequences of the fragmentation process in plant communities. Despite the few studies, it seems clear that fragments with low forest cover inserted in anthropogenic matrices are more likely to experience floristic homogenization. However, the homogenization process has two other components, genetic and functional, which have not been investigated. The purpose of this study was to verify whether there was homogenization of tree reproductive functions in a fragmented landscape and, if found, to determine how the process was influenced by landscape composition. The study was conducted in eight fragments in southwest Brazil. The study was conducted in eight fragments in southwestern Brazil. In each fragment, all individual trees were sampled that had a diameter at breast height ≥3 cm, in ten plots (0.2 ha) and, classified within 26 reproductive functional types (RFTs). The process of functional homogenization was evaluated using additive partitioning of diversity. Additionally, the effect of landscape composition on functional diversity and on the number of individuals within each RFT was evaluated using a generalized linear mixed model. appeared to be in a process of functional homogenization (dominance of RFTs, alpha diversity lower than expected by chance and and low beta diversity). More than 50% of the RFTs and the functional diversity were affected by the landscape parameters. In general, the percentage of forest cover has a positive effect on RFTs while the percentage of coffee matrix has a negative one. The process of functional homogenization has serious consequences for biodiversity conservation because some functions may disappear that, in the long term, would threaten the fragments. This study contributes to a better understanding of how landscape changes affect the functional diversity, abundance of individuals in RFTs and the process of functional homogenization, as well as how to manage fragmented landscapes. PMID:27760218

Active Debris Removal of Multiple Priority Targets

NASA Astrophysics Data System (ADS)

Braun, Vitali; Flegel, Sven Kevin; Vörsmann, Peter; Wiedemann, Carsten; Gelhaus, Johannes; Moeckel, Marek; Kebschull, Christopher

2012-07-01

Today's space debris environment shows major concentrations of objects within distinct orbital regions for nearly all size regimes. The most critical region is found at orbital altitudes near 800 kilometers with high declinations. Within this region many satellites are operated in so called sun-synchronous orbits (SSO). Among those, there are Earth observation, communication and weather satellites. Due to the orbital geometry, head-on encounters with relative velocities of about 15 km/s are most probable and would thus result in highly energetic collisions, which are often referred to as catastrophic collisions, leading to the complete fragmentation of the participating objects. So called feedback collisions can then be triggered by the newly generated fragments, thus leading to a further population increase in the affected orbital region. This effect is known as the Kessler syndrome. Current studies show that catastrophic collisions are not a major problem today, but will become the main process for debris generation within the SSO region in the near future, even without any future launches. In order to avoid this effect, objects with a major impact on collisional cascading have to be actively removed from the critical region after their end of life. Not having the capability to perform an end-of-life maneuver in order to transfer to a graveyard orbit or to de-orbit, many satellites and rocket bodies would have to be de-orbited within a dedicated mission. In such a mission, a service satellite would perform a de-orbit maneuver, after having docked to a specific target. In this paper several systems, e.g. chemical and electrical engines are analysed with the main focus on removing multiple targets within one single mission. The service satellite has to undock from the previously de-orbited target in order to start the rendezvous and docking phase for a subsequent target. The targets are chosen from a previously defined priority list in order to enhance the mission efficiency. Total mission time and system mass shall enable the evaluation of the different concepts.

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

PubMed

Upadhyay, Anup K; Judge, Russell A; Li, Leiming; Pithawalla, Ron; Simanis, Justin; Bodelle, Pierre M; Marin, Violeta L; Henry, Rodger F; Petros, Andrew M; Sun, Chaohong

2018-06-01

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain. Copyright © 2018 Elsevier Ltd. All rights reserved.

The soluble extracellular fragment of neuroligin-1 targets Aβ oligomers to the postsynaptic region of excitatory synapses.

PubMed

Dinamarca, Margarita C; Di Luca, Monica; Godoy, Juan A; Inestrosa, Nibaldo C

2015-10-09

Amyloid-β oligomers (Aβo) play a major role in the synaptic dysfunction of Alzheimer's disease (AD). Neuroligins are postsynaptic cell-adhesion molecules, that share an extracellular domain with high degree of similarity to acetylcholinesterase (AChE), one of the first putative Aβo receptors. We recently found that Aβo interact with the soluble N-terminal fragment of neuroligin-1 (NL-1). We report here that Aβo associate with NL-1 at excitatory hippocampal synapses, whereas almost no association was observed with neuroligin-2, an isoform present at inhibitory synapses. Studies using purified hippocampal postsynaptic densities indicate that NL-1 interacts with Aβo in a complex with GluN2B-containing NMDA receptors. Additionally, the soluble fragment of NL-1 was used as a scavenger for Aβo. Field excitatory postsynaptic potentials indicate that fragments of NL-1 protect hippocampal neurons from the impairment induced by Aβo. To our knowledge, this is the first report of the interaction between this extracellular fragment of NL-1 and Aβo, strongly suggest that NL-1 facilitates the targeting of Aβo to the postsynaptic regions of excitatory synapses. Copyright © 2015 Elsevier Inc. All rights reserved.

Forest fragmentation and selective logging have inconsistent effects on multiple animal-mediated ecosystem processes in a tropical forest.

PubMed

Schleuning, Matthias; Farwig, Nina; Peters, Marcell K; Bergsdorf, Thomas; Bleher, Bärbel; Brandl, Roland; Dalitz, Helmut; Fischer, Georg; Freund, Wolfram; Gikungu, Mary W; Hagen, Melanie; Garcia, Francisco Hita; Kagezi, Godfrey H; Kaib, Manfred; Kraemer, Manfred; Lung, Tobias; Naumann, Clas M; Schaab, Gertrud; Templin, Mathias; Uster, Dana; Wägele, J Wolfgang; Böhning-Gaese, Katrin

2011-01-01

Forest fragmentation and selective logging are two main drivers of global environmental change and modify biodiversity and environmental conditions in many tropical forests. The consequences of these changes for the functioning of tropical forest ecosystems have rarely been explored in a comprehensive approach. In a Kenyan rainforest, we studied six animal-mediated ecosystem processes and recorded species richness and community composition of all animal taxa involved in these processes. We used linear models and a formal meta-analysis to test whether forest fragmentation and selective logging affected ecosystem processes and biodiversity and used structural equation models to disentangle direct from biodiversity-related indirect effects of human disturbance on multiple ecosystem processes. Fragmentation increased decomposition and reduced antbird predation, while selective logging consistently increased pollination, seed dispersal and army-ant raiding. Fragmentation modified species richness or community composition of five taxa, whereas selective logging did not affect any component of biodiversity. Changes in the abundance of functionally important species were related to lower predation by antbirds and higher decomposition rates in small forest fragments. The positive effects of selective logging on bee pollination, bird seed dispersal and army-ant raiding were direct, i.e. not related to changes in biodiversity, and were probably due to behavioural changes of these highly mobile animal taxa. We conclude that animal-mediated ecosystem processes respond in distinct ways to different types of human disturbance in Kakamega Forest. Our findings suggest that forest fragmentation affects ecosystem processes indirectly by changes in biodiversity, whereas selective logging influences processes directly by modifying local environmental conditions and resource distributions. The positive to neutral effects of selective logging on ecosystem processes show that the functionality of tropical forests can be maintained in moderately disturbed forest fragments. Conservation concepts for tropical forests should thus include not only remaining pristine forests but also functionally viable forest remnants.

Forest Fragmentation and Selective Logging Have Inconsistent Effects on Multiple Animal-Mediated Ecosystem Processes in a Tropical Forest

PubMed Central

Schleuning, Matthias; Farwig, Nina; Peters, Marcell K.; Bergsdorf, Thomas; Bleher, Bärbel; Brandl, Roland; Dalitz, Helmut; Fischer, Georg; Freund, Wolfram; Gikungu, Mary W.; Hagen, Melanie; Garcia, Francisco Hita; Kagezi, Godfrey H.; Kaib, Manfred; Kraemer, Manfred; Lung, Tobias; Schaab, Gertrud; Templin, Mathias; Uster, Dana; Wägele, J. Wolfgang; Böhning-Gaese, Katrin

2011-01-01

Forest fragmentation and selective logging are two main drivers of global environmental change and modify biodiversity and environmental conditions in many tropical forests. The consequences of these changes for the functioning of tropical forest ecosystems have rarely been explored in a comprehensive approach. In a Kenyan rainforest, we studied six animal-mediated ecosystem processes and recorded species richness and community composition of all animal taxa involved in these processes. We used linear models and a formal meta-analysis to test whether forest fragmentation and selective logging affected ecosystem processes and biodiversity and used structural equation models to disentangle direct from biodiversity-related indirect effects of human disturbance on multiple ecosystem processes. Fragmentation increased decomposition and reduced antbird predation, while selective logging consistently increased pollination, seed dispersal and army-ant raiding. Fragmentation modified species richness or community composition of five taxa, whereas selective logging did not affect any component of biodiversity. Changes in the abundance of functionally important species were related to lower predation by antbirds and higher decomposition rates in small forest fragments. The positive effects of selective logging on bee pollination, bird seed dispersal and army-ant raiding were direct, i.e. not related to changes in biodiversity, and were probably due to behavioural changes of these highly mobile animal taxa. We conclude that animal-mediated ecosystem processes respond in distinct ways to different types of human disturbance in Kakamega Forest. Our findings suggest that forest fragmentation affects ecosystem processes indirectly by changes in biodiversity, whereas selective logging influences processes directly by modifying local environmental conditions and resource distributions. The positive to neutral effects of selective logging on ecosystem processes show that the functionality of tropical forests can be maintained in moderately disturbed forest fragments. Conservation concepts for tropical forests should thus include not only remaining pristine forests but also functionally viable forest remnants. PMID:22114695

Development of an accurate and high-throughput methodology for structural comprehension of chlorophylls derivatives. (I) Phytylated derivatives.

PubMed

Chen, Kewei; Ríos, José Julián; Pérez-Gálvez, Antonio; Roca, María

2015-08-07

Phytylated chlorophyll derivatives undergo specific oxidative reactions through the natural metabolism or during food processing or storage, and consequently pyro-, 13(2)-hydroxy-, 15(1)-hydroxy-lactone chlorophylls, and pheophytins (a and b) are originated. New analytical procedures have been developed here to reproduce controlled oxidation reactions that specifically, and in reasonable amounts, produce those natural target standards. At the same time and under the same conditions, 16 natural chlorophyll derivatives have been analyzed by APCI-HPLC-hrMS(2) and most of them by the first time. The combination of the high-resolution MS mode with powerful post-processing software has allowed the identification of new fragmentation patterns, characterizing specific product ions for some particular standards. In addition, new hypotheses and reaction mechanisms for the established MS(2)-based reactions have been proposed. As a general rule, the main product ions involve the phytyl and the propionic chains but the introduction of oxygenated functional groups at the isocyclic ring produces new and specific productions and at the same time inhibits some particular fragmentations. It is noteworthy that all b derivatives, except 15(1)-hydroxy-lactone compounds, undergo specific CO losses. We propose a new reaction mechanism based in the structural configuration of a and b chlorophyll derivatives that explain the exclusive CO fragmentation in all b series except for 15(1)-hydroxy-lactone b and all a series compounds. Copyright © 2015 Elsevier B.V. All rights reserved.

Secreted Amyloid β-Proteins in a Cell Culture Model Include N-Terminally Extended Peptides That Impair Synaptic Plasticity

PubMed Central

2014-01-01

Evidence for a central role of amyloid β-protein (Aβ) in the genesis of Alzheimer’s disease (AD) has led to advanced human trials of Aβ-lowering agents. The “amyloid hypothesis” of AD postulates deleterious effects of small, soluble forms of Aβ on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aβ could be therapeutically advantageous, it is important to understand the full range of soluble Aβ derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aβ species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aβ40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aβ-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aβ. These N-terminally extended Aβ-containing monomeric fragments are distinct from soluble Aβ oligomers formed from Aβ1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing β-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aβ-containing species can arise from APP processing events N-terminal to the classical β-secretase cleavage site. PMID:24840308

Direct processes in 54-MeV Li-7 breakup reactions on C-12 and Au-197 targets, and the extraction of astrophysical cross sections

NASA Astrophysics Data System (ADS)

Gazes, S. B.; Mason, J. E.; Roberts, R. B.; Teichmann, S. G.

1992-01-01

Strong direct processes were observed for elastic breakup in 54-MeV Li-7 + C-12, Au-197 reactions. In the case of C-12, the observed Li-7 to alpha + t direct-breakup yield was significantly larger than predicted by a Coulomb-breakup calculation, indicating the importance of the nuclear field. For Au-197, final-state interactions produced a strong distortion in the fragment energy spectra, as well as a modulation of the coincidence efficiency for different detector geometries. Such Coulomb effects are found to severely complicate the extraction of radiative-capture cross sections from direct-breakup data.

Route to three-dimensional fragments using diversity-oriented synthesis

PubMed Central

Hung, Alvin W.; Ramek, Alex; Wang, Yikai; Kaya, Taner; Wilson, J. Anthony; Clemons, Paul A.; Young, Damian W.

2011-01-01

Fragment-based drug discovery (FBDD) has proven to be an effective means of producing high-quality chemical ligands as starting points for drug-discovery pursuits. The increasing number of clinical candidate drugs developed using FBDD approaches is a testament of the efficacy of this approach. The success of fragment-based methods is highly dependent on the identity of the fragment library used for screening. The vast majority of FBDD has centered on the use of sp2-rich aromatic compounds. An expanded set of fragments that possess more 3D character would provide access to a larger chemical space of fragments than those currently used. Diversity-oriented synthesis (DOS) aims to efficiently generate a set of molecules diverse in skeletal and stereochemical properties. Molecules derived from DOS have also displayed significant success in the modulation of function of various “difficult” targets. Herein, we describe the application of DOS toward the construction of a unique set of fragments containing highly sp3-rich skeletons for fragment-based screening. Using cheminformatic analysis, we quantified the shapes and physical properties of the new 3D fragments and compared them with a database containing known fragment-like molecules. PMID:21482811

Route to three-dimensional fragments using diversity-oriented synthesis.

PubMed

Hung, Alvin W; Ramek, Alex; Wang, Yikai; Kaya, Taner; Wilson, J Anthony; Clemons, Paul A; Young, Damian W

2011-04-26

Fragment-based drug discovery (FBDD) has proven to be an effective means of producing high-quality chemical ligands as starting points for drug-discovery pursuits. The increasing number of clinical candidate drugs developed using FBDD approaches is a testament of the efficacy of this approach. The success of fragment-based methods is highly dependent on the identity of the fragment library used for screening. The vast majority of FBDD has centered on the use of sp(2)-rich aromatic compounds. An expanded set of fragments that possess more 3D character would provide access to a larger chemical space of fragments than those currently used. Diversity-oriented synthesis (DOS) aims to efficiently generate a set of molecules diverse in skeletal and stereochemical properties. Molecules derived from DOS have also displayed significant success in the modulation of function of various "difficult" targets. Herein, we describe the application of DOS toward the construction of a unique set of fragments containing highly sp(3)-rich skeletons for fragment-based screening. Using cheminformatic analysis, we quantified the shapes and physical properties of the new 3D fragments and compared them with a database containing known fragment-like molecules.

[Fragment-based drug discovery: concept and aim].

PubMed

Tanaka, Daisuke

2010-03-01

Fragment-Based Drug Discovery (FBDD) has been recognized as a newly emerging lead discovery methodology that involves biophysical fragment screening and chemistry-driven fragment-to-lead stages. Although fragments, defined as structurally simple and small compounds (typically <300 Da), have not been employed in conventional high-throughput screening (HTS), the recent significant progress in the biophysical screening methods enables fragment screening at a practical level. The intention of FBDD primarily turns our attention to weakly but specifically binding fragments (hit fragments) as the starting point of medicinal chemistry. Hit fragments are then promoted to more potent lead compounds through linking or merging with another hit fragment and/or attaching functional groups. Another positive aspect of FBDD is ligand efficiency. Ligand efficiency is a useful guide in screening hit selection and hit-to-lead phases to achieve lead-likeness. Owing to these features, a number of successful applications of FBDD to "undruggable targets" (where HTS and other lead identification methods failed to identify useful lead compounds) have been reported. As a result, FBDD is now expected to complement more conventional methodologies. This review, as an introduction of the following articles, will summarize the fundamental concepts of FBDD and will discuss its advantages over other conventional drug discovery approaches.

Renal targeted delivery of triptolide by conjugation to the fragment peptide of human serum albumin.

PubMed

Yuan, Zhi-xiang; Wu, Xiao-juan; Mo, Jingxin; Wang, Yan-li; Xu, Chao-qun; Lim, Lee Yong

2015-08-01

We have previously demonstrated that peptide fragments (PFs) of the human serum albumin could be developed as potential renal targeting carriers, in particular, the peptide fragment, PF-A299-585 (A299-585 representing the amino acid sequence of the human serum albumin). In this paper, we conjugated triptolide (TP), the anti-inflammatory Chinese traditional medicine, to PF-A299-585 via a succinic acid spacer to give TPS-PF-A299-585 (TP loading 2.2% w/w). Compared with the free TP, TPS-PF-A299-585 exhibited comparable anti-inflammatory activity in the lipopolysaccharide stimulated MDCK cells, but was significantly less cytotoxic than the free drug. Accumulation of TPS-PF-A299-585 in the MDCK cells in vitro and in rodent kidneys in vivo was demonstrated using FITC-labeled TPS-PF-A299-585. Renal targeting was confirmed in vivo in a membranous nephropathic (MN) rodent model, where optical imaging and analyses of biochemical markers were combined to show that TPS-PF-A299-585 was capable of alleviating the characteristic symptoms of MN. The collective data affirm PF-A299-585 to be a useful carrier for targeting TP to the kidney. Copyright © 2015 Elsevier B.V. All rights reserved.

Computational Fragment-Based Drug Design: Current Trends, Strategies, and Applications.

PubMed

Bian, Yuemin; Xie, Xiang-Qun Sean

2018-04-09

Fragment-based drug design (FBDD) has become an effective methodology for drug development for decades. Successful applications of this strategy brought both opportunities and challenges to the field of Pharmaceutical Science. Recent progress in the computational fragment-based drug design provide an additional approach for future research in a time- and labor-efficient manner. Combining multiple in silico methodologies, computational FBDD possesses flexibilities on fragment library selection, protein model generation, and fragments/compounds docking mode prediction. These characteristics provide computational FBDD superiority in designing novel and potential compounds for a certain target. The purpose of this review is to discuss the latest advances, ranging from commonly used strategies to novel concepts and technologies in computational fragment-based drug design. Particularly, in this review, specifications and advantages are compared between experimental and computational FBDD, and additionally, limitations and future prospective are discussed and emphasized.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

SPH simulations of high-speed collisions

NASA Astrophysics Data System (ADS)

Rozehnal, Jakub; Broz, Miroslav

2016-10-01

Our work is devoted to a comparison of: i) asteroid-asteroid collisions occurring at lower velocities (about 5 km/s in the Main Belt), and ii) mutual collisions of asteroids and cometary nuclei usually occurring at significantly higher relative velocities (> 10 km/s).We focus on differences in the propagation of the shock wave, ejection of the fragments and possible differences in the resultingsize-frequency distributions of synthetic asteroid families. We also discuss scaling with respect to the "nominal" target diameter D = 100 km, projectile velocity 3-7 km/s, for which a number of simulations were done so far (Durda et al. 2007, Benavidez et al. 2012).In the latter case of asteroid-comet collisions, we simulate the impacts of brittle or pre-damaged impactors onto solid monolithic targets at high velocities, ranging from 10 to 15 km/s. The purpose of this numerical experiment is to better understand impact processes shaping the early Solar System, namely the primordial asteroid belt during during the (late) heavy bombardment (as a continuation of Broz et al. 2013).For all hydrodynamical simulations we use a smoothed-particle hydrodynamics method (SPH), namely the lagrangian SPH3D code (Benz & Asphaug 1994, 1995). The gravitational interactions between fragments (re-accumulation) is simulated with the Pkdgrav tree-code (Richardson et al. 2000).

Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

PubMed

Alaimo, Agustina; Gorojod, Roxana M; Miglietta, Esteban A; Villarreal, Alejandro; Ramos, Alberto J; Kotler, Mónica L

2013-10-25

Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Fragmentation under the Scaling Symmetry and Turbulent Cascade with Intermittency

NASA Technical Reports Server (NTRS)

Gorokhovski, M.

2003-01-01

Fragmentation plays an important role in a variety of physical, chemical, and geological processes. Examples include atomization in sprays, crushing of rocks, explosion and impact of solids, polymer degradation, etc. Although each individual action of fragmentation is a complex process, the number of these elementary actions is large. It is natural to abstract a simple 'effective' scenario of fragmentation and to represent its essential features. One of the models is the fragmentation under the scaling symmetry: each breakup action reduces the typical length of fragments, r (right arrow) alpha r, by an independent random multiplier alpha (0 < alpha < 1), which is governed by the fragmentation intensity spectrum q(alpha), integral(sup 1)(sub 0) q(alpha)d alpha = 1. This scenario has been proposed by Kolmogorov (1941), when he considered the breakup of solid carbon particle. Describing the breakup as a random discrete process, Kolmogorov stated that at latest times, such a process leads to the log-normal distribution. In Gorokhovski & Saveliev, the fragmentation under the scaling symmetry has been reviewed as a continuous evolution process with new features established. The objective of this paper is twofold. First, the paper synthesizes and completes theoretical part of Gorokhovski & Saveliev. Second, the paper shows a new application of the fragmentation theory under the scale invariance. This application concerns the turbulent cascade with intermittency. We formulate here a model describing the evolution of the velocity increment distribution along the progressively decreasing length scale. The model shows that when the turbulent length scale gets smaller, the velocity increment distribution has central growing peak and develops stretched tails. The intermittency in turbulence is manifested in the same way: large fluctuations of velocity provoke highest strain in narrow (dissipative) regions of flow.

The importance of hydration thermodynamics in fragment-to-lead optimization.

PubMed

Ichihara, Osamu; Shimada, Yuzo; Yoshidome, Daisuke

2014-12-01

Using a computational approach to assess changes in solvation thermodynamics upon ligand binding, we investigated the effects of water molecules on the binding energetics of over 20 fragment hits and their corresponding optimized lead compounds. Binding activity and X-ray crystallographic data of published fragment-to-lead optimization studies from various therapeutically relevant targets were studied. The analysis reveals a distinct difference between the thermodynamic profile of water molecules displaced by fragment hits and those displaced by the corresponding optimized lead compounds. Specifically, fragment hits tend to displace water molecules with notably unfavorable excess entropies-configurationally constrained water molecules-relative to those displaced by the newly added moieties of the lead compound during the course of fragment-to-lead optimization. Herein we describe the details of this analysis with the goal of providing practical guidelines for exploiting thermodynamic signatures of binding site water molecules in the context of fragment-to-lead optimization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Forest Fragmentation as Cause of Bacterial Transmission among Nonhuman Primates, Humans, and Livestock, Uganda

PubMed Central

Gillespie, Thomas R.; Rwego, Innocent B.; Estoff, Elizabeth L.; Chapman, Colin A.

2008-01-01

We conducted a prospective study of bacterial transmission among humans, nonhuman primates (primates hereafter), and livestock in western Uganda. Humans living near forest fragments harbored Escherichia coli bacteria that were ≈75% more similar to bacteria from primates in those fragments than to bacteria from primates in nearby undisturbed forests. Genetic similarity between human/livestock and primate bacteria increased ≈3-fold as anthropogenic disturbance within forest fragments increased from moderate to high. Bacteria harbored by humans and livestock were approximately twice as similar to those of red-tailed guenons, which habitually enter human settlements to raid crops, than to bacteria of other primate species. Tending livestock, experiencing gastrointestinal symptoms, and residing near a disturbed forest fragment increased genetic similarity between a participant’s bacteria and those of nearby primates. Forest fragmentation, anthropogenic disturbance within fragments, primate ecology, and human behavior all influence bidirectional, interspecific bacterial transmission. Targeted interventions on any of these levels should reduce disease transmission and emergence. PMID:18760003

Crystallographic fragment-based drug discovery: use of a brominated fragment library targeting HIV protease.

PubMed

Tiefenbrunn, Theresa; Forli, Stefano; Happer, Meaghan; Gonzalez, Ana; Tsai, Yingssu; Soltis, Michael; Elder, John H; Olson, Arthur J; Stout, Charles D

2014-02-01

A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV-1 protease in order to probe surface sites in soaking experiments. Often, fragments are weak binders with partial occupancy, resulting in weak, difficult-to-fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3-bromo-2,6-dimethoxybenzoic acid (Br6) in the flap site and 1-bromo-2-naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br-fragments provides further insight into the nature of these surface pockets. © 2013 John Wiley & Sons A/S.

Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments

PubMed Central

Chung, Lisa M.; Colangelo, Christopher M.; Zhao, Hongyu

2014-01-01

Multiple Reaction Monitoring (MRM) conducted on a triple quadrupole mass spectrometer allows researchers to quantify the expression levels of a set of target proteins. Each protein is often characterized by several unique peptides that can be detected by monitoring predetermined fragment ions, called transitions, for each peptide. Concatenating large numbers of MRM transitions into a single assay enables simultaneous quantification of hundreds of peptides and proteins. In recognition of the important role that MRM can play in hypothesis-driven research and its increasing impact on clinical proteomics, targeted proteomics such as MRM was recently selected as the Nature Method of the Year. However, there are many challenges in MRM applications, especially data pre‑processing where many steps still rely on manual inspection of each observation in practice. In this paper, we discuss an analysis pipeline to automate MRM data pre‑processing. This pipeline includes data quality assessment across replicated samples, outlier detection, identification of inaccurate transitions, and data normalization. We demonstrate the utility of our pipeline through its applications to several real MRM data sets. PMID:24905083

Data Pre-Processing for Label-Free Multiple Reaction Monitoring (MRM) Experiments.

PubMed

Chung, Lisa M; Colangelo, Christopher M; Zhao, Hongyu

2014-06-05

Multiple Reaction Monitoring (MRM) conducted on a triple quadrupole mass spectrometer allows researchers to quantify the expression levels of a set of target proteins. Each protein is often characterized by several unique peptides that can be detected by monitoring predetermined fragment ions, called transitions, for each peptide. Concatenating large numbers of MRM transitions into a single assay enables simultaneous quantification of hundreds of peptides and proteins. In recognition of the important role that MRM can play in hypothesis-driven research and its increasing impact on clinical proteomics, targeted proteomics such as MRM was recently selected as the Nature Method of the Year. However, there are many challenges in MRM applications, especially data pre‑processing where many steps still rely on manual inspection of each observation in practice. In this paper, we discuss an analysis pipeline to automate MRM data pre‑processing. This pipeline includes data quality assessment across replicated samples, outlier detection, identification of inaccurate transitions, and data normalization. We demonstrate the utility of our pipeline through its applications to several real MRM data sets.

Fundamental study of hydrogen-attachment-induced peptide fragmentation occurring in the gas phase and during the matrix-assisted laser desorption/ionization process.

PubMed

Asakawa, Daiki; Takahashi, Hidenori; Iwamoto, Shinichi; Tanaka, Koichi

2018-05-09

Mass spectrometry with hydrogen-radical-mediated fragmentation techniques has been used for the sequencing of proteins/peptides. The two methods, matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) and hydrogen attachment/abstraction dissociation (HAD) are known as hydrogen-radical-mediated fragmentation techniques. MALDI-ISD occurs during laser induced desorption processes, whereas HAD utilizes the association of hydrogen with peptide ions in the gas phase. In this study, the general mechanisms of MALDI-ISD and HAD of peptides were investigated. We demonstrated the fragmentation of four model peptides and investigated the fragment formation pathways using density functional theory (DFT) calculations. The current experimental and computational joint study indicated that MALDI-ISD and HAD produce aminoketyl radical intermediates, which immediately undergo radical-induced cleavage at the N-Cα bond located on the C-terminal side of the radical site, leading to the c'/z˙ fragment pair. In the case of MALDI-ISD, the z˙ fragments undergo a subsequent reaction with the matrix to give z' and matrix adducts of the z fragments. In contrast, the c' and z˙ fragments react with hydrogen atoms during the HAD processes, and various fragment species, such as c˙, c', z˙ and z', were observed in the HAD-MS/MS mass spectra.

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

Abraitiene, Asta; US Department of Agriculture, Agricultural Research Service, Molecular Plant Pathology Laboratory, Room 214 Building 004 BARC-West, 10300 Baltimore Avenue, Beltsville, MD 20705; Zhao Yan

Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequentmore » GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the nucleus.« less

Object formation in visual working memory: Evidence from object-based attention.

PubMed

Zhou, Jifan; Zhang, Haihang; Ding, Xiaowei; Shui, Rende; Shen, Mowei

2016-09-01

We report on how visual working memory (VWM) forms intact perceptual representations of visual objects using sub-object elements. Specifically, when objects were divided into fragments and sequentially encoded into VWM, the fragments were involuntarily integrated into objects in VWM, as evidenced by the occurrence of both positive and negative object-based attention effects: In Experiment 1, when subjects' attention was cued to a location occupied by the VWM object, the target presented at the location of that object was perceived as occurring earlier than that presented at the location of a different object. In Experiment 2, responses to a target were significantly slower when a distractor was presented at the same location as the cued object (Experiment 2). These results suggest that object fragments can be integrated into objects within VWM in a manner similar to that of visual perception. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Hecht, Adam

Within the 3 year POP we propose to continue to test and further develop the fission spectrometers, to do development tests and full data acquisition run at the national laboratory neutron beam facilities, to measure correlated fission fragment yields at low neutron energies with 235 U fission targets, and make these data available to the nuclear community. The spectrometer development will be both on the university based r\\prototype and on the National Laboratory Spectrometer, and measurements will be performed with both. Over the longer time frame of the collaboration, we will take data over a range of low energies, andmore » use other fission targets available to the laboratory. We will gather energy specific fragment distributions and reaction cross sections. We will further develop the data acquisition capabilities to take correlated fission fragment'gamma ray/neurton data, all on an event-by-event basis. This really is an enabling technology.« less

2000 Year-old ancient equids: an ancient-DNA lesson from pompeii remains.

PubMed

Di Bernardo, Giovanni; Del Gaudio, Stefania; Galderisi, Umberto; Cipollaro, Marilena

2004-11-15

Ancient DNA extracted from 2000 year-old equine bones was examined in order to amplify mitochondrial and nuclear DNA fragments. A specific equine satellite-type sequence representing 3.7%-11% of the entire equine genome, proved to be a suitable target to address the question of the presence of aDNA in ancient bones. The PCR strategy designed to investigate this specific target also allowed us to calculate the molecular weight of amplifiable DNA fragments. Sequencing of a 370 bp DNA fragment of mitochondrial control region allowed the comparison of ancient DNA sequences with those of modern horses to assess their genetic relationship. The 16S rRNA mitochondrial gene was also examined to unravel the post-mortem base modification feature and to test the status of Pompeian equids taxon on the basis of a Mae III restriction site polymorphism. Copyright 2004 Wiley-Liss, Inc.

Azimuthal correlation and collective behavior in nucleus-nucleus collisions

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

Mali, P.; Mukhopadhyay, A., E-mail: amitabha-62@rediffmail.com; Sarkar, S.

2015-03-15

Various flow effects of nuclear and hadronic origin are investigated in nucleus-nucleus collisions. Nuclear emulsion data collected from {sup 84}Kr + Ag/Br interaction at an incident energy of 1.52 GeV per nucleon and from {sup 28}Si + Ag/Br interaction at an incident energy of 14.5 GeV per nucleon are used in the investigation. The transverse momentum distribution and the flow angle analysis show that collective behavior, like a bounce-off effect of the projectile spectators and a sidesplash effect of the target spectators, are present in our event samples. From an azimuthal angle analysis of the data we also see amore » direct flow of the projectile fragments and of the produced charged particles. On the other hand, for both data samples the target fragments exhibit a reverse flow, while the projectile fragments exhibit an elliptic flow. Relevant flow parameters are measured.« less

Identification of DNA primase inhibitors via a combined fragment-based and virtual screening

NASA Astrophysics Data System (ADS)

Ilic, Stefan; Akabayov, Sabine R.; Arthanari, Haribabu; Wagner, Gerhard; Richardson, Charles C.; Akabayov, Barak

2016-11-01

The structural differences between bacterial and human primases render the former an excellent target for drug design. Here we describe a technique for selecting small molecule inhibitors of the activity of T7 DNA primase, an ideal model for bacterial primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 primase were identified and then exploited in virtual filtration to select larger molecules from the ZINC database. The molecules were docked to the primase active site using the available primase crystal structure and ranked based on their predicted binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Our approach, which combines fragment based and virtual screening, is rapid and cost effective and can be applied to other targets.

Total reaction cross sections and neutron-removal cross sections of neutron-rich light nuclei measured by the COMBAS fragment-separator

NASA Astrophysics Data System (ADS)

Hue, B. M.; Isataev, T.; Erdemchimeg, B.; Artukh, A. G.; Aznabaev, D.; Davaa, S.; Klygin, S. A.; Kononenko, G. A.; Khuukhenkhuu, G.; Kuterbekov, K.; Lukyanov, S. M.; Mikhailova, T. I.; Maslov, V. A.; Mendibaev, K.; Sereda, Yu M.; Penionzhkevich, Yu E.; Vorontsov, A. N.

2017-12-01

Preliminary results of measurements of the total reaction cross sections σR and neutron removal cross section σ-xn for weakly bound 6He, 8Li, 9Be and 10Be nuclei at energy range (20-35) A MeV with 28Si target is presented. The secondary beams of light nuclei were produced by bombardment of the 22Ne (35 A MeV) primary beam on Be target and separated by COMBAS fragment-separator. In dispersive focal plane a horizontal slit defined the momentum acceptance as 1% and a wedge degrader of 200 μm Al was installed. The Bρ of the second section of the fragment-separator was adjusted for measurements in energy range (20-35) A MeV. Two-neutron removal cross sections for 6He and 10Be and one -neutron removal cross sections 8Li and 9Be were measured.

SPH/N-Body simulations of small (D = 10km) asteroidal breakups and improved parametric relations for Monte-Carlo collisional models

NASA Astrophysics Data System (ADS)

Ševeček, P.; Brož, M.; Nesvorný, D.; Enke, B.; Durda, D.; Walsh, K.; Richardson, D. C.

2017-11-01

We report on our study of asteroidal breakups, i.e. fragmentations of targets, subsequent gravitational reaccumulation and formation of small asteroid families. We focused on parent bodies with diameters Dpb = 10km . Simulations were performed with a smoothed-particle hydrodynamics (SPH) code combined with an efficient N-body integrator. We assumed various projectile sizes, impact velocities and impact angles (125 runs in total). Resulting size-frequency distributions are significantly different from scaled-down simulations with Dpb = 100km targets (Durda et al., 2007). We derive new parametric relations describing fragment distributions, suitable for Monte-Carlo collisional models. We also characterize velocity fields and angular distributions of fragments, which can be used as initial conditions for N-body simulations of small asteroid families. Finally, we discuss a number of uncertainties related to SPH simulations.

Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

PubMed

Badrinarayan, Preethi; Sastry, G Narahari

2012-04-01

In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.

Protein kinase C-ε activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules

PubMed Central

Bakajsova, Diana; Samarel, Allen M.

2011-01-01

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307–F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F0F1-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F0F1-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC. PMID:21289057

Preliminary Characterization Results from the DebriSat Project

NASA Technical Reports Server (NTRS)

Rivero, M.; Shiotani, B.; Kleespies, J.; Toledo-Burdett, R.; Moraguez, M.; Carrasquila, M.; Fitz-Coy, N.; Liou, J.-C.; Sorge, M.; Huynh, T.

2016-01-01

The DebriSat project is a continuing effort sponsored by NASA and DoD to update existing break-up models using data obtained from two separate hypervelocity impact tests used to simulate on-orbit collisions. To protect the fragments resulting from the impact tests, "soft-catch" arenas made of polyurethane foam panels were utilized. After each impact test, the test chamber was cleaned and debris resulting from the catastrophic demise of the test article were collected and shipped to the University of Florida for post-impact processing. The post-impact processing activities include collecting, characterizing, and cataloging of the fragments. Since the impact tests, a team of students has been working to characterize the fragments in terms of their mass, size, shape, color and material content. The focus of the 20 months since the impact tests has been on the collection of 2 millimeters- and larger fragments resulting from impact test on the 56 kilogram-representative LEO (Low Earth Orbit) satellite referred to as DebriSat. To date we have recovered in excess of 115,000 fragments, 30,000 more than the prediction of 85,000 fragments from the existing model. We continue to collect fragments but have transitioned to the characterization phase of the post-impact activities. Since the start of the characterization phase, the focus has been to utilize automation to (i) expedite fragment characterization process and (ii) minimize human-in-the- loop. We have developed and implemented such automated processes; e.g., we have automated the data entry process to reduce operator errors during transcription of the measurement data. However, at all steps of the process, there is human oversight to ensure the integrity of the data. Additionally, we have developed and implemented repeatability and reproducibility tests to ensure that the instrumentation used in the characterization process is accurate and properly calibrated. In this paper, the implemented processes are described and preliminary results presented. Additionally, lessons learned from the implemented automations and their impacts on the integrity of the results are discussed.

Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

PubMed

Molino, Yves; David, Marion; Varini, Karine; Jabès, Françoise; Gaudin, Nicolas; Fortoul, Aude; Bakloul, Karima; Masse, Maxime; Bernard, Anne; Drobecq, Lucile; Lécorché, Pascaline; Temsamani, Jamal; Jacquot, Guillaume; Khrestchatisky, Michel

2017-05-01

The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals ( ldlr -/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier. © FASEB.

OBSIFRAC: database-supported software for 3D modeling of rock mass fragmentation

NASA Astrophysics Data System (ADS)

Empereur-Mot, Luc; Villemin, Thierry

2003-03-01

Under stress, fractures in rock masses tend to form fully connected networks. The mass can thus be thought of as a 3D series of blocks produced by fragmentation processes. A numerical model has been developed that uses a relational database to describe such a mass. The model, which assumes the fractures to be plane, allows data from natural networks to test theories concerning fragmentation processes. In the model, blocks are bordered by faces that are composed of edges and vertices. A fracture can originate from a seed point, its orientation being controlled by the stress field specified by an orientation matrix. Alternatively, it can be generated from a discrete set of given orientations and positions. Both kinds of fracture can occur together in a model. From an original simple block, a given fracture produces two simple polyhedral blocks, and the original block becomes compound. Compound and simple blocks created throughout fragmentation are stored in the database. Several fragmentation processes have been studied. In one scenario, a constant proportion of blocks is fragmented at each step of the process. The resulting distribution appears to be fractal, although seed points are random in each fragmented block. In a second scenario, division affects only one random block at each stage of the process, and gives a Weibull volume distribution law. This software can be used for a large number of other applications.

Tafenoquine, an Antiplasmodial 8-Aminoquinoline, Targets Leishmania Respiratory Complex III and Induces Apoptosis ▿

PubMed Central

Carvalho, Luis; Luque-Ortega, Juan Román; Manzano, José Ignacio; Castanys, Santiago; Rivas, Luis; Gamarro, Francisco

2010-01-01

Tafenoquine (TFQ), an 8-aminoquinoline analogue of primaquine, which is currently under clinical trial (phase IIb/III) for the treatment and prevention of malaria, may represent an alternative treatment for leishmaniasis. In this work, we have studied the mechanism of action of TFQ against Leishmania parasites. TFQ impaired the overall bioenergetic metabolism of Leishmania promastigotes, causing a rapid drop in intracellular ATP levels without affecting plasma membrane permeability. TFQ induced mitochondrial dysfunction through the inhibition of cytochrome c reductase (respiratory complex III) with a decrease in the oxygen consumption rate and depolarization of mitochondrial membrane potential. This was accompanied by ROS production, elevation of intracellular Ca2+ levels and concomitant nuclear DNA fragmentation. We conclude that TFQ targets Leishmania mitochondria, leading to an apoptosis-like death process. PMID:20837758

Tafenoquine, an antiplasmodial 8-aminoquinoline, targets leishmania respiratory complex III and induces apoptosis.

PubMed

Carvalho, Luis; Luque-Ortega, Juan Román; Manzano, José Ignacio; Castanys, Santiago; Rivas, Luis; Gamarro, Francisco

2010-12-01

Tafenoquine (TFQ), an 8-aminoquinoline analogue of primaquine, which is currently under clinical trial (phase IIb/III) for the treatment and prevention of malaria, may represent an alternative treatment for leishmaniasis. In this work, we have studied the mechanism of action of TFQ against Leishmania parasites. TFQ impaired the overall bioenergetic metabolism of Leishmania promastigotes, causing a rapid drop in intracellular ATP levels without affecting plasma membrane permeability. TFQ induced mitochondrial dysfunction through the inhibition of cytochrome c reductase (respiratory complex III) with a decrease in the oxygen consumption rate and depolarization of mitochondrial membrane potential. This was accompanied by ROS production, elevation of intracellular Ca(2+) levels and concomitant nuclear DNA fragmentation. We conclude that TFQ targets Leishmania mitochondria, leading to an apoptosis-like death process.

Composing compound libraries for hit discovery--rationality-driven preselection or random choice by structural diversity?

PubMed

Weidel, Elisabeth; Negri, Matthias; Empting, Martin; Hinsberger, Stefan; Hartmann, Rolf W

2014-01-01

In order to identify new scaffolds for drug discovery, surface plasmon resonance is frequently used to screen structurally diverse libraries. Usually, hit rates are low and identification processes are time consuming. Hence, approaches which improve hit rates and, thus, reduce the library size are required. In this work, we studied three often used strategies for their applicability to identify inhibitors of PqsD. In two of them, target-specific aspects like inhibition of a homologous protein or predicted binding determined by virtual screening were used for compound preselection. Finally, a fragment library, covering a large chemical space, was screened and served as comparison. Indeed, higher hit rates were observed for methods employing preselected libraries indicating that target-oriented compound selection provides a time-effective alternative.

Fragment screening by SPR and advanced application to GPCRs.

PubMed

Shepherd, Claire A; Hopkins, Andrew L; Navratilova, Iva

2014-01-01

Surface plasmon resonance (SPR) is one of the primary biophysical methods for the screening of low molecular weight 'fragment' libraries, due to its low protein consumption and 'label-free' methodology. SPR biosensor interaction analysis is employed to both screen and confirm the binding of compounds in fragment screening experiments, as it provides accurate information on the affinity and kinetics of molecular interactions. The most advanced application of the use of SPR for fragment screening is against membrane protein drug targets, such G-protein coupled receptors (GPCRs). Biophysical GPCR assays using SPR have been validated with pharmacological measurements approximate to cell-based methods, yet provide the advantage of biophysical methods in their ability to measure the weak affinities of low molecular weight fragments. A number of SPR fragment screens against GPCRs have now been disclosed in the literature. SPR fragment screening is proving versatile to screen both thermostabilised GPCRs and solubilised wild type receptors. In this chapter, we discuss the state-of-the-art in GPCR fragment screening by SPR and the technical considerations in performing such experiments. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ultrasensitive electrochemical detection of nucleic acids by template enhanced hybridization followed with rolling circle amplification.

PubMed

Ji, Hanxu; Yan, Feng; Lei, Jianping; Ju, Huangxian

2012-08-21

An ultrasensitive protocol for electrochemical detection of DNA is designed with quantum dots (QDs) as a signal tag by combining the template enhanced hybridization process (TEHP) and rolling circle amplification (RCA). Upon the recognition of the molecular beacon (MB) to target DNA, the MB hybridizes with assistants and target DNA to form a ternary ''Y-junction''. The target DNA can be dissociated from the structure under the reaction of nicking endonuclease to initiate the next hybridization process. The template enhanced MB fragments further act as the primers of the RCA reaction to produce thousands of repeated oligonucleotide sequences, which can bind with oligonucleotide functionalized QDs. The attached signal tags can be easily read out by square-wave voltammetry after dissolving with acid. Because of the cascade signal amplification and the specific TEHP and RCA reaction, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the attomolar level (11 aM) with a linear range of 6 orders of magnitude (from 1 × 10(-17) to 1 × 10(-11) M) and can discriminate mismatched DNA from perfect matched target DNA with high selectivity. The high sensitivity and specificity make this method a great potential for early diagnosis in gene-related diseases.

Giant plasmon excitation in single and double ionization of C60 by fast highly charged Si and O ions

NASA Astrophysics Data System (ADS)

Kelkar, A. H.; Kadhane, U.; Misra, D.; Tribedi, L. C.

2007-09-01

Se have investigated single and double ionization of C60 molecule in collisions with 2.33 MeV/u Siq+ (q=6-14) and 3.125 MeV/u Oq+ (q=5-8) projectiles. The projectile charge state dependence of the single and double ionization yields of C60 are then compared to those for an ion-atom collision system using Ne gas as a target. A large difference between the gas and the cluster target behaviour was partially explained in terms of a model based on collective excitation namely the giant dipole plasmon resonance (GDPR). The qualitative agreement between the data and GDPR model prediction for single and double ionization signifies the importance of single and double plasmon excitations in the ionization process. A large deviation of the GDPR model for triple and quadruple ionization from the experimental data imply the importance of the other low impact parameter processes such as evaporation, fragmentation and a possible solid-like dynamical screening.

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

Syed, M. Bukhari; Blum, J.; Jansson, K. Wahlberg

Previous work on protoplanetary dust growth shows a halt at centimeter sizes owing to the occurrence of bouncing at velocities of ≳0.1 m s{sup −1} and fragmentation at velocities ≳1 m s{sup −1}. To overcome these barriers, spatial concentration of centimeter-sized dust pebbles and subsequent gravitational collapse have been proposed. However, numerical investigations have shown that dust aggregates may undergo fragmentation during the gravitational collapse phase. This fragmentation in turn changes the size distribution of the solids and thus must be taken into account in order to understand the properties of the planetesimals that form. To explore the fate of dustmore » pebbles undergoing fragmenting collisions, we conducted laboratory experiments on dust-aggregate collisions with a focus on establishing a collision model for this stage of planetesimal formation. In our experiments, we analyzed collisions of dust aggregates with masses between 0.7 and 91 g mass ratios between target and projectile from 1 to 126 at a fixed porosity of 65%, within the velocity range of 1.5–8.7 m s{sup −1}, at low atmospheric pressure of ∼10{sup −3} mbar, and in free-fall conditions. We derived the mass of the largest fragment, the fragment size/mass distribution, and the efficiency of mass transfer as a function of collision velocity and projectile/target aggregate size. Moreover, we give recipes for an easy-to-use fragmentation and mass-transfer model for further use in modeling work. In a companion paper, we use the experimental findings and the derived dust-aggregate collision model to investigate the fate of dust pebbles during gravitational collapse.« less

Development of a systematic approach to rapid classification and identification of notoginsenosides and metabolites in rat feces based on liquid chromatography coupled triple time-of-flight mass spectrometry.

PubMed

Xing, Rong; Zhou, Lijun; Xie, Lin; Hao, Kun; Rao, Tai; Wang, Qian; Ye, Wei; Fu, Hanxu; Wang, Xinwen; Wang, Guangji; Liang, Yan

2015-03-31

The present work contributes to the development of a powerful technical platform to rapidly identify and classify complicated components and metabolites for traditional Chinese medicines. In this process, notoginsenosides, the main active ingredients in Panaxnotoginseng, were chosen as model compounds. Firstly, the fragmental patterns, diagnostic product ions and neutral loss of each subfamily of notoginsenosides were summarized by collision-induced dissociation analysis of representative authentic standards. Next, in order to maximally cover low-concentration components which could otherwise be omitted from previous diagnostic fragment-ion method using only single product ion of notoginsenosides, a multiple product ions filtering strategy was proposed and utilized to identify and classify both non-target and target notoginsenosides of P.notoginseng extract (in vitro). With this strategy, 13 protopanaxadiol-type notoginsenosides and 30 protopanaxatriol-type notoginsenosides were efficiently extracted. Then, a neutral loss filtering technique was employed to trace prototype components and metabolites in rats (in vivo) since diagnostic product ions might shift therefore become unpredictable when metabolic reactions occurred on the mother skeleton of notoginsenosides. After comparing the constitute profiles in vitro with in vivo, 62 drug-related components were identified from rat feces, and these components were classified into 27 prototype compounds and 35 metabolites. Lastly, all the metabolites were successfully correlated to their parent compounds based on chemicalome-metabolome matching approach which was previously built by our group. This study provided a generally applicable approach to global metabolite identification for the complicated components in complex matrices. Copyright © 2015 Elsevier B.V. All rights reserved.

CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope

PubMed Central

1992-01-01

Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202- 221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site. PMID:1383384

Is stellar multiplicity universal? Tight stellar binaries in the Orion Nebula Cluster

NASA Astrophysics Data System (ADS)

Duchêne, Gaspard; Lacour, S.; Moraux, E.; Goodwin, S.; Bouvier, J.

2018-05-01

We present a survey for the tightest visual binaries among 0.3-2 M⊙ members the Orion Nebula Cluster (ONC). Among 42 targets, we discovered 13 new 0{^''.}025-0{^''.}15 companions. Accounting for the Branch bias, we find a companion star fraction (CSF) in the 10-60 au range of 21^{+8}_{-5}%, consistent with that observed in other star-forming regions (SFRs) and twice as high as among field stars; this excess is found with a high level of confidence. Since our sample is dominated by disk-bearing targets, this indicates that disk disruption by close binaries is inefficient, or has not yet taken place, in the ONC. The resulting separation distribution in the ONC drops sharply outside 60 au. These findings are consistent with a scenario in which the initial multiplicity properties, set by the star formation process itself, are identical in the ONC and in other SFRs and subsequently altered by the cluster's dynamical evolution. This implies that the fragmentation process does not depend on the global properties of a molecular cloud, but on the local properties of prestellar cores, and that the latter are self-regulated to be nearly identical in a wide range of environments. These results, however, raise anew the question of the origin of field stars as the tight binaries we have discovered will not be destroyed as the ONC dissolves into the galactic field. It thus appears that most field stars formed in regions that differ from well-studied SFRs in the Solar neighborhood, possibly due to changes in core fragmentation on Gyr timescales.

Fabrication and characterization of carbon-backed thin 208Pb targets.

PubMed

Thakur, Meenu; Dubey, R; Abhilash, S R; Behera, B R; Mohanty, B P; Kabiraj, D; Ojha, Sunil; Duggal, Heena

2016-01-01

Thin carbon-backed isotopically enriched 208 Pb targets were required for our experiment aimed to study the reaction dynamics for 48 Ti +  208 Pb system, populating the near super-heavy nucleus 256 Rf, through mass-energy correlation of the fission fragments. Purity and thickness of the targets are of utmost importance in such studies as these factors have strong influence on the measurement accuracy of mass and energy distribution of fission fragments. 208 Pb targets with thickness ranging from 60 μg/cm 2 to 250 μg/cm 2 have been fabricated in high vacuum environment using physical vapor deposition method. Important points in the method are as follows: • 208 Pb was deposited using resistive heating method, whereas carbon (backing foil) deposition was performed by using the electron beam bombardment technique.•Different characterization techniques such as Particle Induced X-ray Emission (PIXE), Energy Dispersive X-Ray Fluorescence (EDXRF) and Rutherford Backscattering Spectrometry (RBS) were used to assert the purity and thickness of the targets.•These targets have successfully been used to accomplish our experimental objectives.

Ionophore-A23187-induced cellular cytotoxicity: a cell fragment mediated process.

PubMed Central

Nash, G S; Niedt, G W; MacDermott, R P

1980-01-01

Calcium ionophore A23187 was found to induce human white blood cells to kill human red blood cells. Optimal conditions for ionophore-induced cellular cytotoxicity (IICC) included an 18 h time period, an incubation temperature of 25 degrees, a 25:1 or 50:1 killer:target cell ratio,and a final ionophore concentration of 2 . 5 microgram/ml. WBC or granulocytes which were either frozen and thawed three times or sonicated were capable of mediating IICC. As intact cells, granulocytes (67 . 2% cytotoxicity), monocytes (34 . 8%), B cells (22 . 0%) and Null cells (19 . 3%) were effector cells but T cells (7 . 4%) were not. After fragmenting these cells, all cell types including T cells were able to mediate IICC. When cell lines (K562, Chang, and NCTC) were used as effectors, none would mediate IICC when intact. After freezing and thawing, Chang and NCTC would not mediate IICC, whereas K562 cells did. These studies may be indicative of a calcium-dependent, membrane-localized mechanism in cellular cytotoxic processes, and may provide a useful indicator system for isolation of the enzyme systems involved in cellular cytotoxicity. PMID:6773881

Targeting FR-expressing cells in ovarian cancer with Fab-functionalized nanoparticles: a full study to provide the proof of principle from in vitro to in vivo

NASA Astrophysics Data System (ADS)

Quarta, Alessandra; Bernareggi, Davide; Benigni, Fabio; Luison, Elena; Nano, Giuseppe; Nitti, Simone; Cesta, Maria Candida; di Ciccio, Luciano; Canevari, Silvana; Pellegrino, Teresa; Figini, Mariangela

2015-01-01

Efficient targeting in tumor therapies is still an open issue: systemic biodistribution and poor specific accumulation of drugs weaken efficacy of treatments. Engineered nanoparticles are expected to bring benefits by allowing specific delivery of drug to the tumor or acting themselves as localized therapeutic agents. In this study we have targeted epithelial ovarian cancer with inorganic nanoparticles conjugated to a human antibody fragment against the folate receptor over-expressed on cancer cells. The conjugation approach is generally applicable. Indeed several types of nanoparticles (either magnetic or fluorescent) were engineered with the fragment, and their biological activity was preserved as demonstrated by biochemical methods in vitro. In vivo studies with mice bearing orthotopic and subcutaneous tumors were performed. Elemental and histological analyses showed that the conjugated magnetic nanoparticles accumulated specifically and were retained at tumor sites longer than the non-conjugated nanoparticles.Efficient targeting in tumor therapies is still an open issue: systemic biodistribution and poor specific accumulation of drugs weaken efficacy of treatments. Engineered nanoparticles are expected to bring benefits by allowing specific delivery of drug to the tumor or acting themselves as localized therapeutic agents. In this study we have targeted epithelial ovarian cancer with inorganic nanoparticles conjugated to a human antibody fragment against the folate receptor over-expressed on cancer cells. The conjugation approach is generally applicable. Indeed several types of nanoparticles (either magnetic or fluorescent) were engineered with the fragment, and their biological activity was preserved as demonstrated by biochemical methods in vitro. In vivo studies with mice bearing orthotopic and subcutaneous tumors were performed. Elemental and histological analyses showed that the conjugated magnetic nanoparticles accumulated specifically and were retained at tumor sites longer than the non-conjugated nanoparticles. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04426f

Effects of target plasma electron-electron collisions on correlated motion of fragmented protons.

PubMed

Barriga-Carrasco, Manuel D

2006-02-01

The objective of the present work is to examined the effects of plasma target electron-electron collisions on H2 + protons traversing it. Specifically, the target is deuterium in a plasma state with temperature Te=10 eV and density n=10(23) cm(-3), and proton velocities are vp=vth, vp=2vth, and vp=3vth, where vth is the electron thermal velocity of the target plasma. Proton interactions with plasma electrons are treated by means of the dielectric formalism. The interactions among close protons through plasma electronic medium are called vicinage forces. It is checked that these forces always screen the Coulomb explosions of the two fragmented protons from the same H2 + ion decreasing their relative distance. They also align the interproton vector along the motion direction, and increase the energy loss of the two protons at early dwell times while for longer times the energy loss tends to the value of two isolated protons. Nevertheless, vicinage forces and effects are modified by the target electron collisions. These collisions enhance the calculated self-stopping and vicinage forces over the collisionless results. Regarding proton correlated motion, when these collisions are included, the interproton vector along the motion direction overaligns at slower proton velocities (vp=vth) and misaligns for faster ones (vp=2vth, vp=3vth). They also contribute to a great extend to increase the energy loss of the fragmented H2 + ion. This later effect is more significant in reducing projectile velocity.

Identifying protein domains by global analysis of soluble fragment data.

PubMed

Bulloch, Esther M M; Kingston, Richard L

2014-11-15

The production and analysis of individual structural domains is a common strategy for studying large or complex proteins, which may be experimentally intractable in their full-length form. However, identifying domain boundaries is challenging if there is little structural information concerning the protein target. One experimental procedure for mapping domains is to screen a library of random protein fragments for solubility, since truncation of a domain will typically expose hydrophobic groups, leading to poor fragment solubility. We have coupled fragment solubility screening with global data analysis to develop an effective method for identifying structural domains within a protein. A gene fragment library is generated using mechanical shearing, or by uracil doping of the gene and a uracil-specific enzymatic digest. A split green fluorescent protein (GFP) assay is used to screen the corresponding protein fragments for solubility when expressed in Escherichia coli. The soluble fragment data are then analyzed using two complementary approaches. Fragmentation "hotspots" indicate possible interdomain regions. Clustering algorithms are used to group related fragments, and concomitantly predict domain location. The effectiveness of this Domain Seeking procedure is demonstrated by application to the well-characterized human protein p85α. Copyright © 2014 Elsevier Inc. All rights reserved.

Development of a potent and selective FLT3 kinase inhibitor by systematic expansion of a non-selective fragment-screening hit.

PubMed

Nakano, Hirofumi; Hasegawa, Tsukasa; Imamura, Riyo; Saito, Nae; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo

2016-05-01

A non-selective inhibitor (1) of FMS-like tyrosine kinase-3 (FLT3) was identified by fragment screening and systematically modified to afford a potent and selective inhibitor 26. We confirmed that 26 inhibited the growth of FLT-3-activated human acute myeloid leukemia cell line MV4-11. Our design strategy enabled rapid development of a novel type of FLT3 inhibitor from the hit fragment in the absence of target-structural information. Copyright © 2016 Elsevier Ltd. All rights reserved.

Customizing Monoclonal Antibodies for the Treatment of Methamphetamine Abuse: Current and Future Applications

PubMed Central

Peterson, Eric C.; Gentry, W. Brooks

2015-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse, and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggest a scientific vision for how intact mAb (singular and plural) or small antigen binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution x-ray crystal structure of a high affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen binding fragments (Fab) and single chain variable fragments (scFv) are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which the METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites and neuronal connections. PMID:24484976

Customizing monoclonal antibodies for the treatment of methamphetamine abuse: current and future applications.

PubMed

Peterson, Eric C; Gentry, W Brooks; Owens, S Michael

2014-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggests a scientific vision for how intact monoclonal antibody (mAb) (singular and plural) or small antigen-binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review, we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution X-ray crystal structure of a high-affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen-binding fragments and single-chain variable fragments are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites, and neuronal connections. © 2014 Elsevier Inc. All rights reserved.

Extraction and recovery of pectic fragments from citrus processing waste for co-production with ethanol

USDA-ARS?s Scientific Manuscript database

Steam treatment of citrus processing waste (CPW) at 160°C followed by a rapid decompression (steam explosion) at either pH 2.8 or 4.5 provides an efficient and rapid fragmentation of protopectin in CPW and renders a large fraction of fragmented pectins, arabinans, galactans and arabinogalactans solu...

Salt weathering on Mars

NASA Astrophysics Data System (ADS)

Jagoutz, E.

Large well rounded boulders and angular rock fragments characterizes the Martian landscape as seen on the recent excellent quality photos. Analyzing the different rock-shapes indicates a time sequence of emplacement, fragmentation and transport of different rocks on Mars, which might give interesting insight into transport and weathering processes. Larger commonly well rounded boulders were emplaced onto gravel plains. After emplacement, these rocks were fragmented and disassembled. Nests of angular rock fragments are marking the locations of preexisting larger rocks. Frequently it is possible to reconstruct larger rounded rocks from smaller angular fragments. In other cases transport after fragmentation obscured the relationship of the fragments. However, a strewn field of fragments is still reminiscent of the preexisting rock. Mechanical salt weathering could be a plausible explanation for the insitu fragmentation of larger rounded blocks into angular fragments. Impact or secondary air fall induced fragmentation produces very different patterns, as observed around impact crates on Earth. Salt weathering of rocks is a common process in terrestrial environments. Salt crystallization in capillaries causes fragmentation of rocks, irrespective of the process of salt transportation and concentration. On Earth significant salt weathering can be observed in different climatic environments: in the transition zone of alluvial aprons and salt playas in desserts and in dry valleys of Antarctica. In terrestrial semi-arid areas the salt is transported by salt solution, which is progressively concentrated by evaporation. In Antarctic dry valleys freeze-thaw cycles causes salt transportation and crystallization resulting in rock fragmentation. This salt induced process can lead to complete destruction of rocks and converts rocks to fine sand. The efficient breakdown of rocks is dominating the landscape in some dry valleys of the Earth but possibly also on Mars. (Malin, 1974). However, irrespectively of the climatic environment a liquid brine is a necessity for salt induced fragmentation of rocks. If salt weathering is responsible for the fragmented rocks on the Martian surface it implies a temporary present of liquid H_2O. However, due to the present dry atmosphere on Mars brines can only be present in restricted places without being in equilibrium with the atmosphere (Clark and van Hart 1980). M. C. Malin (1974) JGR Vol 79,26 p 3888-3894 B. C. Clark and D. C. vanHart (1980) ICARUS 45, 370-378

Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

PubMed

Amano, Yasushi; Tanabe, Eiki; Yamaguchi, Tomohiko

2015-05-15

Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800μM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51μM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits. Copyright © 2015 Elsevier Ltd. All rights reserved.

Discerning fragmentation dynamics of tropical forest and wetland during reforestation, urban sprawl, and policy shifts.

PubMed

Gao, Qiong; Yu, Mei

2014-01-01

Despite the overall trend of worldwide deforestation over recent decades, reforestation has also been found and is expected in developing countries undergoing fast urbanization and agriculture abandonment. The consequences of reforestation on landscape patterns are seldom addressed in the literature, despite their importance in evaluating biodiversity and ecosystem functions. By analyzing long-term land cover changes in Puerto Rico, a rapidly reforested (6 to 42% during 1940-2000) and urbanized tropical island, we detected significantly different patterns of fragmentation and underlying mechanisms among forests, urban areas, and wetlands. Forest fragmentation is often associated with deforestation. However, we also found significant fragmentation during reforestation. Urban sprawl and suburb development have a dominant impact on forest fragmentation. Reforestation mostly occurs along forest edges, while significant deforestation occurs in forest interiors. The deforestation process has a much stronger impact on forest fragmentation than the reforestation process due to their different spatial configurations. In contrast, despite the strong interference of coastal urbanization, wetland aggregation has occurred due to the effective implementation of laws/regulations for wetland protection. The peak forest fragmentation shifted toward rural areas, indicating progressively more fragmentation in forest interiors. This shift is synchronous with the accelerated urban sprawl as indicated by the accelerated shift of the peak fragmentation index of urban cover toward rural areas, i.e., 1.37% yr-1 in 1977-1991 versus 2.17% yr-1 in 1991-2000. Based on the expected global urbanization and the regional forest transition from deforested to reforested, the fragmented forests and aggregated wetlands in this study highlight possible forest fragmentation processes during reforestation in an assessment of biodiversity and functions and suggest effective laws/regulations in land planning to reduce future fragmentation.

Discerning Fragmentation Dynamics of Tropical Forest and Wetland during Reforestation, Urban Sprawl, and Policy Shifts

PubMed Central

Gao, Qiong; Yu, Mei

2014-01-01

Despite the overall trend of worldwide deforestation over recent decades, reforestation has also been found and is expected in developing countries undergoing fast urbanization and agriculture abandonment. The consequences of reforestation on landscape patterns are seldom addressed in the literature, despite their importance in evaluating biodiversity and ecosystem functions. By analyzing long-term land cover changes in Puerto Rico, a rapidly reforested (6 to 42% during 1940–2000) and urbanized tropical island, we detected significantly different patterns of fragmentation and underlying mechanisms among forests, urban areas, and wetlands. Forest fragmentation is often associated with deforestation. However, we also found significant fragmentation during reforestation. Urban sprawl and suburb development have a dominant impact on forest fragmentation. Reforestation mostly occurs along forest edges, while significant deforestation occurs in forest interiors. The deforestation process has a much stronger impact on forest fragmentation than the reforestation process due to their different spatial configurations. In contrast, despite the strong interference of coastal urbanization, wetland aggregation has occurred due to the effective implementation of laws/regulations for wetland protection. The peak forest fragmentation shifted toward rural areas, indicating progressively more fragmentation in forest interiors. This shift is synchronous with the accelerated urban sprawl as indicated by the accelerated shift of the peak fragmentation index of urban cover toward rural areas, i.e., 1.37% yr−1 in 1977–1991 versus 2.17% yr−1 in 1991–2000. Based on the expected global urbanization and the regional forest transition from deforested to reforested, the fragmented forests and aggregated wetlands in this study highlight possible forest fragmentation processes during reforestation in an assessment of biodiversity and functions and suggest effective laws/regulations in land planning to reduce future fragmentation. PMID:25409016

In search of new lead compounds for trypanosomiasis drug design: A protein structure-based linked-fragment approach

NASA Astrophysics Data System (ADS)

Verlinde, Christophe L. M. J.; Rudenko, Gabrielle; Hol, Wim G. J.

1992-04-01

A modular method for pursuing structure-based inhibitor design in the framework of a design cycle is presented. The approach entails four stages: (1) a design pathway is defined in the three-dimensional structure of a target protein; (2) this pathway is divided into subregions; (3) complementary building blocks, also called fragments, are designed in each subregion; complementarity is defined in terms of shape, hydrophobicity, hydrogen bond properties and electrostatics; and (4) fragments from different subregions are linked into potential lead compounds. Stages (3) and (4) are qualitatively guided by force-field calculations. In addition, the designed fragments serve as entries for retrieving existing compounds from chemical databases. This linked-fragment approach has been applied in the design of potentially selective inhibitors of triosephosphate isomerase from Trypanosoma brucei, the causative agent of sleeping sickness.

Analyzing Internal Fragmentation of Electrosprayed Ubiquitin Ions During Beam-Type Collisional Dissociation

NASA Astrophysics Data System (ADS)

Durbin, Kenneth R.; Skinner, Owen S.; Fellers, Ryan T.; Kelleher, Neil L.

2015-05-01

Gaseous fragmentation of intact proteins is multifaceted and can be unpredictable by current theories in the field. Contributing to the complexity is the multitude of precursor ion states and fragmentation channels. Terminal fragment ions can be re-fragmented, yielding product ions containing neither terminus, termed internal fragment ions. In an effort to better understand and capitalize upon this fragmentation process, we collisionally dissociated the high (13+), middle (10+), and low (7+) charge states of electrosprayed ubiquitin ions. Both terminal and internal fragmentation processes were quantified through step-wise increases of voltage potential in the collision cell. An isotope fitting algorithm matched observed product ions to theoretical terminal and internal fragment ions. At optimal energies for internal fragmentation of the 10+, nearly 200 internal fragments were observed; on average each of the 76 residues in ubiquitin was covered by 24.1 internal fragments. A pertinent finding was that formation of internal ions occurs at similar energy thresholds as terminal b- and y-ion types in beam-type activation. This large amount of internal fragmentation is frequently overlooked during top-down mass spectrometry. As such, we present several new approaches to visualize internal fragments through modified graphical fragment maps. With the presented advances of internal fragment ion accounting and visualization, the total percentage of matched fragment ions increased from approximately 40% to over 75% in a typical beam-type MS/MS spectrum. These sequence coverage improvements offer greater characterization potential for whole proteins with no needed experimental changes and could be of large benefit for future high-throughput intact protein analysis.

Hydrocode predictions of collisional outcomes: Effects of target size

NASA Technical Reports Server (NTRS)

Ryan, Eileen V.; Asphaug, Erik; Melosh, H. J.

1991-01-01

Traditionally, laboratory impact experiments, designed to simulate asteroid collisions, attempted to establish a predictive capability for collisional outcomes given a particular set of initial conditions. Unfortunately, laboratory experiments are restricted to using targets considerably smaller than the modelled objects. It is therefore necessary to develop some methodology for extrapolating the extensive experimental results to the size regime of interest. Results are reported obtained through the use of two dimensional hydrocode based on 2-D SALE and modified to include strength effects and the fragmentation equations. The hydrocode was tested by comparing its predictions for post-impact fragment size distributions to those observed in laboratory impact experiments.

The dominance of the ν(0d5/2) 2 configuration in the N = 8 shell in 12Be from the breakup reaction on a proton target at intermediate energy

NASA Astrophysics Data System (ADS)

Chung, Le Xuan; Bertulani, Carlos A.; Egelhof, Peter; Ilieva, Stoyanka; Khoa, Dao T.; Kiselev, Oleg A.

2017-11-01

The momentum distribution of 11Be fragments produced by the breakup of 12Be interacting with a proton target at 700.5 MeV/u energy has been measured at GSI Darmstadt. To obtain the structure information on the anomaly of the N = 8 neutron shell, the momentum distribution of 11Be fragments from the one-neutron knockout 12Be (p , pn) reaction, measured in inverse kinematics, has been analysed in the distorted wave impulse approximation (DWIA) based on a quasi-free scattering scenario. The DWIA analysis shows a surprisingly strong contribution of the neutron 0d5/2 orbital in 12Be to the transverse momentum distribution of the 11Be fragments. The single-neutron 0d5/2 spectroscopic factor deduced from the present knock-out data is 1.39(10), which is significantly larger than that deduced recently from data of 12Be breakup on a carbon target. This result provides a strong experimental evidence for the dominance of the neutron ν(0d5/2) 2 configuration in the ground state of 12Be.

Forward-backward multiplicity correlations of target fragments in nucleus-emulsion collisions at a few hundred MeV/u

NASA Astrophysics Data System (ADS)

Zhang, Dong-Hai; Chen, Yan-Ling; Wang, Guo-Rong; Li, Wang-Dong; Wang, Qing; Yao, Ji-Jie; Zhou, Jian-Guo; Li, Rong; Li, Jun-Sheng; Li, Hui-Ling

2015-01-01

The forward-backward multiplicity and correlations of a target evaporated fragment (black track particle) and target recoiled proton (grey track particle) emitted from 150 A MeV 4He, 290 A MeV 12C, 400 A MeV 12C, 400 A MeV 20Ne and 500 A MeV 56Fe induced different types of nuclear emulsion target interactions are investigated. It is found that the forward and backward averaged multiplicity of a grey, black and heavily ionized track particle increases with the increase of the target size. The averaged multiplicity of a forward black track particle, backward black track particle, and backward grey track particle do not depend on the projectile size and energy, but the averaged multiplicity of a forward grey track particle increases with an increase of projectile size and energy. The backward grey track particle multiplicity distribution follows an exponential decay law and the decay constant decreases with an increase of target size. The backward-forward multiplicity correlations follow linear law which is independent of the projectile size and energy, and the saturation effect is observed in some heavy target data sets.

Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

PubMed

Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

2016-01-01

Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

A nuclear fragmentation energy deposition model

NASA Technical Reports Server (NTRS)

Ngo, D. M.; Wilson, J. W.; Fogarty, T. N.; Buck, W. W.; Townsend, L. W. (Principal Investigator)

1991-01-01

A formalism for target fragment transport is presented with application to energy loss spectra in thin silicon devices. A nuclear data base is recommended that agrees well with the measurements of McNulty et al. using surface barrier detectors. High-energy events observed by McNulty et al., which are not predicted by intranuclear cascade models, are well represented by the present work.

Meta-analysis of the effects of forest fragmentation on interspecific interactions.

PubMed

Magrach, Ainhoa; Laurance, William F; Larrinaga, Asier R; Santamaria, Luis

2014-10-01

Forest fragmentation dramatically alters species persistence and distribution and affects many ecological interactions among species. Recent studies suggest that mutualisms, such as pollination and seed dispersal, are more sensitive to the negative effects of forest fragmentation than antagonisms, such as predation or herbivory. We applied meta-analytical techniques to evaluate this hypothesis and quantified the relative contributions of different components of the fragmentation process (decreases in fragment size, edge effects, increased isolation, and habitat degradation) to the overall effect. The effects of fragmentation on mutualisms were primarily driven by habitat degradation, edge effects, and fragment isolation, and, as predicted, they were consistently more negative on mutualisms than on antagonisms. For the most studied interaction type, seed dispersal, only certain components of fragmentation had significant (edge effects) or marginally significant (fragment size) effects. Seed size modulated the effect of fragmentation: species with large seeds showed stronger negative impacts of fragmentation via reduced dispersal rates. Our results reveal that different components of the habitat fragmentation process have varying impacts on key mutualisms. We also conclude that antagonistic interactions have been understudied in fragmented landscapes, most of the research has concentrated on particular types of mutualistic interactions such as seed dispersal, and that available studies of interspecific interactions have a strong geographical bias (arising mostly from studies carried out in Brazil, Chile, and the United States). © 2014 Society for Conservation Biology.

Experimental modelling of fragmentation applied to volcanic explosions

NASA Astrophysics Data System (ADS)

Haug, Øystein Thordén; Galland, Olivier; Gisler, Galen R.

2013-12-01

Explosions during volcanic eruptions cause fragmentation of magma and host rock, resulting in fragments with sizes ranging from boulders to fine ash. The products can be described by fragment size distributions (FSD), which commonly follow power laws with exponent D. The processes that lead to power-law distributions and the physical parameters that control D remain unknown. We developed a quantitative experimental procedure to study the physics of the fragmentation process through time. The apparatus consists of a Hele-Shaw cell containing a layer of cohesive silica flour that is fragmented by a rapid injection of pressurized air. The evolving fragmentation of the flour is monitored with a high-speed camera, and the images are analysed to obtain the evolution of the number of fragments (N), their average size (A), and the FSD. Using the results from our image-analysis procedure, we find transient empirical laws for N, A and the exponent D of the power-law FSD as functions of the initial air pressure. We show that our experimental procedure is a promising tool for unravelling the complex physics of fragmentation during phreatomagmatic and phreatic eruptions.

[Comparative study of substance P and its fragments: analgesic properties, effect on behavior and monoaminergic processes].

PubMed

Klusha, V E; Abissova, N A; Mutsenietse, R K; Svirskis, Sh V; Binert, M

1981-12-01

The effect of substance P (SP) and of its fragments 5-11, 8-11, 9-11, 10-11 administered into the brain ventricles in doses of 5, 25 and 50 nM on the behavior and content of biogenic monoamines of the rat brain was studied. The analgetic properties of the substances under consideration and those of fragment SP 10-11 in doses of 5, 25, 50 and 100 nM were also subjected to examination. It was found that SP and fragment 5-11 stimulate and enhance the locomotor activity in rats, while fragments 8-11 and 9-11 provoke hypoactivity. The substances under study increase the serotonin and dopamine turnover, whereas SP and fragment 8-11 lower the serotonin content as well. After administration of SP and fragment 5-11 analgesia was seen to transform to hyperalgesia depending on the dose. Fragments 8-11 and 9-11 produce analgetic effect. It is suggested that both SP fragments and the whole SP molecule can influence the neurochemical process that regulate behavior and pain perception.

A comparison of charcoal measurements for reconstruction of Mediterranean paleo-fire frequency in the mountains of Corsica

NASA Astrophysics Data System (ADS)

Leys, Bérangère; Carcaillet, Christopher; Dezileau, Laurent; Ali, Adam A.; Bradshaw, Richard H. W.

2013-05-01

Fire-history reconstructions inferred from sedimentary charcoal records are based on measuring sieved charcoal fragment area, estimating fragment volume, or counting fragments. Similar fire histories are reconstructed from these three approaches for boreal lake sediment cores, using locally defined thresholds. Here, we test the same approach for a montane Mediterranean lake in which taphonomical processes might differ from boreal lakes through fragmentation of charcoal particles. The Mediterranean charcoal series are characterized by highly variable charcoal accumulation rates. Results there indicate that the three proxies do not provide comparable fire histories. The differences are attributable to charcoal fragmentation. This could be linked to fire type (crown or surface fires) or taphonomical processes, including charcoal transportation in the catchment area or in the sediment. The lack of correlation between the concentration of charcoal and of mineral matter suggests that fragmentation is not linked to erosion. Reconstructions based on charcoal area are more robust and stable than those based on fragment counts. Area-based reconstructions should therefore be used instead of the particle-counting method when fragmentation may influence the fragment abundance.

Using In Silico Fragmentation to Improve Routine Residue Screening in Complex Matrices.

PubMed

Kaufmann, Anton; Butcher, Patrick; Maden, Kathryn; Walker, Stephan; Widmer, Mirjam

2017-12-01

Targeted residue screening requires the use of reference substances in order to identify potential residues. This becomes a difficult issue when using multi-residue methods capable of analyzing several hundreds of analytes. Therefore, the capability of in silico fragmentation based on a structure database ("suspect screening") instead of physical reference substances for routine targeted residue screening was investigated. The detection of fragment ions that can be predicted or explained by in silico software was utilized to reduce the number of false positives. These "proof of principle" experiments were done with a tool that is integrated into a commercial MS vendor instrument operating software (UNIFI) as well as with a platform-independent MS tool (Mass Frontier). A total of 97 analytes belonging to different chemical families were separated by reversed phase liquid chromatography and detected in a data-independent acquisition (DIA) mode using ion mobility hyphenated with quadrupole time of flight mass spectrometry. The instrument was operated in the MS E mode with alternating low and high energy traces. The fragments observed from product ion spectra were investigated using a "chopping" bond disconnection algorithm and a rule-based algorithm. The bond disconnection algorithm clearly explained more analyte product ions and a greater percentage of the spectral abundance than the rule-based software (92 out of the 97 compounds produced ≥1 explainable fragment ions). On the other hand, tests with a complex blank matrix (bovine liver extract) indicated that the chopping algorithm reports significantly more false positive fragments than the rule based software. Graphical Abstract.

Using In Silico Fragmentation to Improve Routine Residue Screening in Complex Matrices

NASA Astrophysics Data System (ADS)

Kaufmann, Anton; Butcher, Patrick; Maden, Kathryn; Walker, Stephan; Widmer, Mirjam

2017-12-01

Targeted residue screening requires the use of reference substances in order to identify potential residues. This becomes a difficult issue when using multi-residue methods capable of analyzing several hundreds of analytes. Therefore, the capability of in silico fragmentation based on a structure database ("suspect screening") instead of physical reference substances for routine targeted residue screening was investigated. The detection of fragment ions that can be predicted or explained by in silico software was utilized to reduce the number of false positives. These "proof of principle" experiments were done with a tool that is integrated into a commercial MS vendor instrument operating software (UNIFI) as well as with a platform-independent MS tool (Mass Frontier). A total of 97 analytes belonging to different chemical families were separated by reversed phase liquid chromatography and detected in a data-independent acquisition (DIA) mode using ion mobility hyphenated with quadrupole time of flight mass spectrometry. The instrument was operated in the MSE mode with alternating low and high energy traces. The fragments observed from product ion spectra were investigated using a "chopping" bond disconnection algorithm and a rule-based algorithm. The bond disconnection algorithm clearly explained more analyte product ions and a greater percentage of the spectral abundance than the rule-based software (92 out of the 97 compounds produced ≥1 explainable fragment ions). On the other hand, tests with a complex blank matrix (bovine liver extract) indicated that the chopping algorithm reports significantly more false positive fragments than the rule based software. [Figure not available: see fulltext.

Correlation between y-type ions observed in ion trap and triple quadrupole mass spectrometers.

PubMed

Sherwood, Carly A; Eastham, Ashley; Lee, Lik Wee; Risler, Jenni; Vitek, Olga; Martin, Daniel B

2009-09-01

Multiple reaction monitoring mass spectrometry (MRM-MS) is a technique for high-sensitivity targeted analysis. In proteomics, MRM-MS can be used to monitor and quantify a peptide based on the production of expected fragment peaks from the selected peptide precursor ion. The choice of which fragment ions to monitor in order to achieve maximum sensitivity in MRM-MS can potentially be guided by existing MS/MS spectra. However, because the majority of discovery experiments are performed on ion trap platforms, there is concern in the field regarding the generalizability of these spectra to MRM-MS on a triple quadrupole instrument. In light of this concern, many operators perform an optimization step to determine the most intense fragments for a target peptide on a triple quadrupole mass spectrometer. We have addressed this issue by targeting, on a triple quadrupole, the top six y-ion peaks from ion trap-derived consensus library spectra for 258 doubly charged peptides from three different sample sets and quantifying the observed elution curves. This analysis revealed a strong correlation between the y-ion peak rank order and relative intensity across platforms. This suggests that y-type ions obtained from ion trap-based library spectra are well-suited for generating MRM-MS assays for triple quadrupoles and that optimization is not required for each target peptide.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

PubMed

Banner, David W; Gsell, Bernard; Benz, Jörg; Bertschinger, Julian; Burger, Dominique; Brack, Simon; Cuppuleri, Simon; Debulpaep, Maja; Gast, Alain; Grabulovski, Dragan; Hennig, Michael; Hilpert, Hans; Huber, Walter; Kuglstatter, Andreas; Kusznir, Eric; Laeremans, Toon; Matile, Hugues; Miscenic, Christian; Rufer, Arne C; Schlatter, Daniel; Steyaert, Jan; Stihle, Martine; Thoma, Ralf; Weber, Martin; Ruf, Armin

2013-06-01

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

rRNA fragmentation induced by a yeast killer toxin.

PubMed

Kast, Alene; Klassen, Roland; Meinhardt, Friedhelm

2014-02-01

Virus like dsDNA elements (VLE) in yeast were previously shown to encode the killer toxins PaT and zymocin, which target distinct tRNA species via specific anticodon nuclease (ACNase) activities. Here, we characterize a third member of the VLE-encoded toxins, PiT from Pichia inositovora, and identify PiOrf4 as the cytotoxic subunit by conditional expression in Saccharomyces cerevisiae. In contrast to the tRNA targeting toxins, however, neither a change of the wobble uridine modification status by introduction of elp3 or trm9 mutations nor tRNA overexpression rescued from PiOrf4 toxicity. Consistent with a distinct RNA target, expression of PiOrf4 causes specific fragmentation of the 25S and 18S rRNA. A stable cleavage product comprising the first ∼ 130 nucleotides of the 18S rRNA was purified and characterized by linker ligation and subsequent reverse transcription; 3'-termini were mapped to nucleotide 131 and 132 of the 18S rRNA sequence, a region showing some similarity to the anticodon loop of tRNA(Glu)(UUC), the zymocin target. PiOrf4 residues Glu9 and His214, corresponding to catalytic sites Glu9 and His209 in the ACNase subunit of zymocin are essential for in vivo toxicity and rRNA fragmentation, raising the possibility of functionally conserved RNase modules in both proteins. © 2013 John Wiley & Sons Ltd.

GERMcode: A Stochastic Model for Space Radiation Risk Assessment

NASA Technical Reports Server (NTRS)

Kim, Myung-Hee Y.; Ponomarev, Artem L.; Cucinotta, Francis A.

2012-01-01

A new computer model, the GCR Event-based Risk Model code (GERMcode), was developed to describe biophysical events from high-energy protons and high charge and energy (HZE) particles that have been studied at the NASA Space Radiation Laboratory (NSRL) for the purpose of simulating space radiation biological effects. In the GERMcode, the biophysical description of the passage of HZE particles in tissue and shielding materials is made with a stochastic approach that includes both particle track structure and nuclear interactions. The GERMcode accounts for the major nuclear interaction processes of importance for describing heavy ion beams, including nuclear fragmentation, elastic scattering, and knockout-cascade processes by using the quantum multiple scattering fragmentation (QMSFRG) model. The QMSFRG model has been shown to be in excellent agreement with available experimental data for nuclear fragmentation cross sections. For NSRL applications, the GERMcode evaluates a set of biophysical properties, such as the Poisson distribution of particles or delta-ray hits for a given cellular area and particle dose, the radial dose on tissue, and the frequency distribution of energy deposition in a DNA volume. By utilizing the ProE/Fishbowl ray-tracing analysis, the GERMcode will be used as a bi-directional radiation transport model for future spacecraft shielding analysis in support of Mars mission risk assessments. Recent radiobiological experiments suggest the need for new approaches to risk assessment that include time-dependent biological events due to the signaling times for activation and relaxation of biological processes in cells and tissue. Thus, the tracking of the temporal and spatial distribution of events in tissue is a major goal of the GERMcode in support of the simulation of biological processes important in GCR risk assessments. In order to validate our approach, basic radiobiological responses such as cell survival curves, mutation, chromosomal aberrations, and representative mouse tumor induction curves are implemented into the GERMcode. Extension of these descriptions to other endpoints related to non-targeted effects and biochemical pathway responses will be discussed.

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format.

PubMed

Ramlee, Muhammad Khairul; Wang, Jing; Cheung, Alice M S; Li, Shang

2017-04-08

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

Retrieval Mode Distinguishes the Testing Effect from the Generation Effect

ERIC Educational Resources Information Center

Karpicke, Jeffrey D.; Zaromb, Franklin M.

2010-01-01

A series of four experiments examined the effects of generation vs. retrieval practice on subsequent retention. Subjects were first exposed to a list of target words. Then the subjects were shown the targets again intact for Read trials or they were shown fragments of the targets. Subjects in Generate conditions were told to complete the fragments…

Laboratory tests of catastrophic disruption of rotating bodies

NASA Astrophysics Data System (ADS)

Morris, A. J. W.; Burchell, M. J.

2017-11-01

The results of catastrophic disruption experiments on static and rotating targets are reported. The experiments used cement spheres of diameter 10 cm as the targets. Impacts were by mm sized stainless steel spheres at speeds of between 1 and 7.75 km s-1. Energy densities (Q) in the targets ranged from 7 to 2613 J kg-1. The experiments covered both the cratering and catastrophic disruption regimes. For static, i.e. non-rotating targets the critical energy density for disruption (Q*, the value of Q when the largest surviving target fragment has a mass equal to one half of the pre-impact target mass) was Q* = 1447 ± 90 J kg-1. For rotating targets (median rotation frequency of 3.44 Hz) we found Q* = 987 ± 349 J kg-1, a reduction of 32% in the mean value. This lower value of Q* for rotating targets was also accompanied by a larger scatter on the data, hence the greater uncertainty. We suggest that in some cases the rotating targets behaved as static targets, i.e. broke up with the same catastrophic disruption threshold, but in other cases the rotation helped the break up causing a lower catastrophic disruption threshold, hence both the lower value of Q* and the larger scatter on the data. The fragment mass distributions after impact were similar in both the static and rotating target experiments with similar slopes.

Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

PubMed Central

Hwang, In Sun; Ahn, Il-Pyung

2016-01-01

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi. PMID:27298592

Smooth Particle Hydrodynamics GPU-Acceleration Tool for Asteroid Fragmentation Simulation

NASA Astrophysics Data System (ADS)

Buruchenko, Sergey K.; Schäfer, Christoph M.; Maindl, Thomas I.

2017-10-01

The impact threat of near-Earth objects (NEOs) is a concern to the global community, as evidenced by the Chelyabinsk event (caused by a 17-m meteorite) in Russia on February 15, 2013 and a near miss by asteroid 2012 DA14 ( 30 m diameter), on the same day. The expected energy, from either a low-altitude air burst or direct impact, would have severe consequences, especially in populated regions. To mitigate this threat one of the methods is employment of large kinetic-energy impactors (KEIs). The simulation of asteroid target fragmentation is a challenging task which demands efficient and accurate numerical methods with large computational power. Modern graphics processing units (GPUs) lead to a major increase 10 times and more in the performance of the computation of astrophysical and high velocity impacts. The paper presents a new implementation of the numerical method smooth particle hydrodynamics (SPH) using NVIDIA-GPU and the first astrophysical and high velocity application of the new code. The code allows for a tremendous increase in speed of astrophysical simulations with SPH and self-gravity at low costs for new hardware. We have implemented the SPH equations to model gas, liquids and elastic, and plastic solid bodies and added a fragmentation model for brittle materials. Self-gravity may be optionally included in the simulations.

Long-term effects of fragmentation and fragment properties on bird species richness in Hawaiian forests

Treesearch

David J. Flaspohler; Christian P. Giardina; Gregory P. Asner; Patrick Hart; Jonathan Price; Cassie Ka’apu Lyons; Xeronimo Castaneda

2010-01-01

Forest fragmentation is a common disturbance affecting biological diversity, yet the impacts of fragmentation on many forest processes remain poorly understood. Forest restoration is likely to be more successful when it proceeds with an understanding of how native and exotic vertebrates utilize forest patches of different size. We used a system of forest fragments...

Tube pumices as strain markers of the ductile-brittle transition during magma fragmentation

NASA Astrophysics Data System (ADS)

Martí, J.; Soriano, C.; Dingwell, D. B.

1999-12-01

Magma fragmentation-the process by which relatively slow-moving magma transforms into a violent gas flow carrying fragments of magma-is the defining feature of explosive volcanism. Yet of all the processes involved in explosively erupting systems, fragmentation is possibly the least understood. Several theoretical and laboratory studies on magma degassing and fragmentation have produced a general picture of the sequence of events leading to the fragmentation of silicic magma. But there remains a debate over whether magma fragmentation is a consequence of the textural evolution of magma to a foamed state where disintegration of walls separating bubbles becomes inevitable due to a foam-collapse criterion, or whether magma is fragmented purely by stresses that exceed its tensile strength. Here we show that tube pumice-where extreme bubble elongation is observed-is a well-preserved magmatic `strain marker' of the stress state immediately before and during fragmentation. Structural elements in the pumice record the evolution of the magma's mechanical response from viscous behaviour (foaming and foam elongation) through the plastic or viscoelastic stage, and finally to brittle behaviour. These observations directly support the hypothesis that fragmentation occurs when magma undergoes a ductile-brittle transition and stresses exceed the magma's tensile strength.

A closed curve is much more than an incomplete one: effect of closure in figure-ground segmentation.

PubMed

Kovács, I; Julesz, B

1993-08-15

Detection of fragmented closed contours against a cluttered background occurs much beyond the local coherence distance (maximal separation between segments) of nonclosed contours. This implies that the extent of interaction between locally connected detectors is boosted according to the global stimulus structure. We further show that detection of a target probe is facilitated when the probe is positioned inside a closed circle. To explain the striking contour segregation ability found here, and performance enhancement inside closed boundaries, we propose the existence of a synergetic process in early vision.

Effective progression of nuclear magnetic resonance-detected fragment hits.

PubMed

Eaton, Hugh L; Wyss, Daniel F

2011-01-01

Fragment-based drug discovery (FBDD) has become increasingly popular over the last decade as an alternate lead generation tool to HTS approaches. Several compounds have now progressed into the clinic which originated from a fragment-based approach, demonstrating the utility of this emerging field. While fragment hit identification has become much more routine and may involve different screening approaches, the efficient progression of fragment hits into quality lead series may still present a major bottleneck for the broadly successful application of FBDD. In our laboratory, we have extensive experience in fragment-based NMR screening (SbN) and the subsequent iterative progression of fragment hits using structure-assisted chemistry. To maximize impact, we have applied this approach strategically to early- and high-priority targets, and those struggling for leads. Its application has yielded a clinical candidate for BACE1 and lead series in about one third of the SbN/FBDD projects. In this chapter, we will give an overview of our strategy and focus our discussion on NMR-based FBDD approaches. Copyright © 2011 Elsevier Inc. All rights reserved.

Fragment-based virtual screening approach and molecular dynamics simulation studies for identification of BACE1 inhibitor leads.

PubMed

Manoharan, Prabu; Ghoshal, Nanda

2018-05-01

Traditional structure-based virtual screening method to identify drug-like small molecules for BACE1 is so far unsuccessful. Location of BACE1, poor Blood Brain Barrier permeability and P-glycoprotein (Pgp) susceptibility of the inhibitors make it even more difficult. Fragment-based drug design method is suitable for efficient optimization of initial hit molecules for target like BACE1. We have developed a fragment-based virtual screening approach to identify/optimize the fragment molecules as a starting point. This method combines the shape, electrostatic, and pharmacophoric features of known fragment molecules, bound to protein conjugate crystal structure, and aims to identify both chemically and energetically feasible small fragment ligands that bind to BACE1 active site. The two top-ranked fragment hits were subjected for a 53 ns MD simulation. Principle component analysis and free energy landscape analysis reveal that the new ligands show the characteristic features of established BACE1 inhibitors. The potent method employed in this study may serve for the development of potential lead molecules for BACE1-directed Alzheimer's disease therapeutics.

Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

PubMed

Szaszkó, Mária; Hajdú, István; Flachner, Beáta; Dobi, Krisztina; Magyar, Csaba; Simon, István; Lőrincz, Zsolt; Kapui, Zoltán; Pázmány, Tamás; Cseh, Sándor; Dormán, György

2017-02-01

A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

PubMed

Crivianu-Gaita, Victor; Thompson, Michael

2015-08-15

Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of Hypervelocity Metal Fragments for Explosive Initiation

NASA Astrophysics Data System (ADS)

Yeager, John; Bowden, Patrick; Guildenbecher, Daniel; Olles, Joseph

2017-06-01

The off-normal detonation behavior of two plastic-bonded explosive (PBX) formulations was studied using explosively-driven aluminum fragments moving at hypersonic velocity. Witness plate materials, including copper and polycarbonate, were used to characterize the distribution of particles, finding that the aluminum did not fragment homogeneously but rather with larger particles in a ring surrounding finer particles. Digital holography experiments were conducted to measure three-dimensional shape and size of the fastest-moving fragments, which ranged between 100 and 700 microns and traveled between 2 and 3.5 km/s. Crucially, these experiments showed variability in the fragmentation in terms of the number of fragments at the leading edge of the fragment field, indicating that both single and multiple shock impacts could be imparted to the target material. Lower density PBX 9407 (RDX-based) was initiable at up to 4.5 inches, while higher density PBX 9501 (HMX-based) was only initiable at up to 0.25 inches. This type of data is critical for safety experiments and hydrocode simulations to quantify shock-to-detonation transition mechanisms and the associated risk-margins for these materials.

The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation*

PubMed Central

Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F.

2015-01-01

During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

Tank fragmentation test

NASA Technical Reports Server (NTRS)

Daye, C. J.; Cooksey, D.; Walters, R. J.; Auble, A. E.

1973-01-01

A photographic study of a simulated tank fragmentation test was made. Sixteen disks and four spheres were ejected from a test article mounted in a vertical orientation 110 ft above a target installed on the test chamber floor. The test was performed at a chamber pressure of 25 microns. Velocities at impingement on the target ranged from 88 to 120 ft/sec; corresponding ejection velocities at the exit plane of the ejector assembly ranged from 29 to 87 ft/sec. Tumble axes of the disks were expected to be all in the north-south direction; the majority of those measured were, while some were skewed from this direction, the maximum observed being 90 deg. A typical measured tumble rate was 2.4 turns/sec. The dispersion pattern measured on the target was reasonably regular, and measured approximately 16 ft east-to-west by 11 ft north-to-south.

Promiscuous 2-aminothiazoles (PrATs): a frequent hitting scaffold.

PubMed

Devine, Shane M; Mulcair, Mark D; Debono, Cael O; Leung, Eleanor W W; Nissink, J Willem M; Lim, San Sui; Chandrashekaran, Indu R; Vazirani, Mansha; Mohanty, Biswaranjan; Simpson, Jamie S; Baell, Jonathan B; Scammells, Peter J; Norton, Raymond S; Scanlon, Martin J

2015-02-12

We have identified a class of molecules, known as 2-aminothiazoles (2-ATs), as frequent-hitting fragments in biophysical binding assays. This was exemplified by 4-phenylthiazol-2-amine being identified as a hit in 14/14 screens against a diverse range of protein targets, suggesting that this scaffold is a poor starting point for fragment-based drug discovery. This prompted us to analyze this scaffold in the context of an academic fragment library used for fragment-based drug discovery (FBDD) and two larger compound libraries used for high-throughput screening (HTS). This analysis revealed that such "promiscuous 2-aminothiazoles" (PrATs) behaved as frequent hitters under both FBDD and HTS settings, although the problem was more pronounced in the fragment-based studies. As 2-ATs are present in known drugs, they cannot necessarily be deemed undesirable, but the combination of their promiscuity and difficulties associated with optimizing them into a lead compound makes them, in our opinion, poor scaffolds for fragment libraries.

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

Target and Projectile: Material Effects on Crater Excavation and Growth

NASA Technical Reports Server (NTRS)

Anderson, J. L. B.; Burleson, T.; Cintala, Mark J.

2010-01-01

Scaling relationships allow the initial conditions of an impact to be related to the excavation flow and final crater size and have proven useful in understanding the various processes that lead to the formation of a planetary-scale crater. In addition, they can be examined and tested through laboratory experiments in which the initial conditions of the impact are known and ejecta kinematics and final crater morphometry are measured directly. Current scaling relationships are based on a point-source assumption and treat the target material as a continuous medium; however, in planetary-scale impacts, this may not always be the case. Fragments buried in a megaregolith, for instance, could easily approach or exceed the dimensions of the impactor; rubble-pile asteroids could present similar, if not greater, structural complexity. Experiments allow exploration into the effects of target material properties and projectile deformation style on crater excavation and dimensions. This contribution examines two of these properties: (1) the deformation style of the projectile, ductile (aluminum) or brittle (soda-lime glass) and (2) the grain size of the target material, 0.5-1 mm vs. 1-3 mm sand.

A Sensitive DNA Capacitive Biosensor Using Interdigitated Electrodes

PubMed Central

Wang, Lei; Veselinovic, Milena; Yang, Lang; Geiss, Brian J.; Dandy, David S.; Chen, Tom

2017-01-01

This paper presents a label-free affinity-based capacitive biosensor using interdigitated electrodes. Using an optimized process of DNA probe preparation to minimize the effect of contaminants in commercial thiolated DNA probe, the electrode surface was functionalized with the 24-nucleotide DNA probes based on the West Nile virus sequence (Kunjin strain). The biosensor has the ability to detect complementary DNA fragments with a detection limit down to 20 DNA target molecules (1.5 aM range), making it suitable for a practical point-of-care (POC) platform for low target count clinical applications without the need for amplification. The reproducibility of the biosensor detection was improved with efficient covalent immobilization of purified single-stranded DNA probe oligomers on cleaned gold microelectrodes. In addition to the low detection limit, the biosensor showed a dynamic range of detection from 1 μL−1 to 105 μL−1 target molecules (20 to 2 million targets), making it suitable for sample analysis in a typical clinical application environment. The binding results presented in this paper were validated using fluorescent oligomers. PMID:27619528

Fragment-Linking Approach Using (19)F NMR Spectroscopy To Obtain Highly Potent and Selective Inhibitors of β-Secretase.

PubMed

Jordan, John B; Whittington, Douglas A; Bartberger, Michael D; Sickmier, E Allen; Chen, Kui; Cheng, Yuan; Judd, Ted

2016-04-28

Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, β-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.

Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system

PubMed Central

2014-01-01

Background The plant pathogenic and saprophytic fungus Fusarium avenaceum causes considerable in-field and post-field losses worldwide due to its infections of a wide range of different crops. Despite its significant impact on the profitability of agriculture production and a desire to characterize the infection process at the molecular biological level, no genetic transformation protocol has yet been established for F. avenaceum. In the current study, it is shown that F. avenaceum can be efficiently transformed by Agrobacterium tumefaciens mediated transformation. In addition, an efficient and versatile single step vector construction strategy relying on Uracil Specific Excision Reagent (USER) Fusion cloning, is developed. Results The new vector construction system, termed USER-Brick, is based on a limited number of PCR amplified vector fragments (core USER-Bricks) which are combined with PCR generated fragments from the gene of interest. The system was found to have an assembly efficiency of 97% with up to six DNA fragments, based on the construction of 55 vectors targeting different polyketide synthase (PKS) and PKS associated transcription factor encoding genes in F. avenaceum. Subsequently, the ΔFaPKS3 vector was used for optimizing A. tumefaciens mediated transformation (ATMT) of F. avenaceum with respect to six variables. Acetosyringone concentration, co-culturing time, co-culturing temperature and fungal inoculum were found to significantly impact the transformation frequency. Following optimization, an average of 140 transformants per 106 macroconidia was obtained in experiments aimed at introducing targeted genome modifications. Targeted deletion of FaPKS6 (FA08709.2) in F. avenaceum showed that this gene is essential for biosynthesis of the polyketide/nonribosomal compound fusaristatin A. Conclusion The new USER-Brick system is highly versatile by allowing for the reuse of a common set of building blocks to accommodate seven different types of genome modifications. New USER-Bricks with additional functionality can easily be added to the system by future users. The optimized protocol for ATMT of F. avenaceum represents the first reported targeted genome modification by double homologous recombination of this plant pathogen and will allow for future characterization of this fungus. Functional linkage of FaPKS6 to the production of the mycotoxin fusaristatin A serves as a first testimony to this. PMID:25048842