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Sample records for macromolecular contrast medium

  1. A technique for determining the deuterium/hydrogen contrast map in neutron macromolecular crystallography.

    PubMed

    Chatake, Toshiyuki; Fujiwara, Satoru

    2016-01-01

    A difference in the neutron scattering length between hydrogen and deuterium leads to a high density contrast in neutron Fourier maps. In this study, a technique for determining the deuterium/hydrogen (D/H) contrast map in neutron macromolecular crystallography is developed and evaluated using ribonuclease A. The contrast map between the D2O-solvent and H2O-solvent crystals is calculated in real space, rather than in reciprocal space as performed in previous neutron D/H contrast crystallography. The present technique can thus utilize all of the amplitudes of the neutron structure factors for both D2O-solvent and H2O-solvent crystals. The neutron D/H contrast maps clearly demonstrate the powerful detectability of H/D exchange in proteins. In fact, alternative protonation states and alternative conformations of hydroxyl groups are observed at medium resolution (1.8 Å). Moreover, water molecules can be categorized into three types according to their tendency towards rotational disorder. These results directly indicate improvement in the neutron crystal structure analysis. This technique is suitable for incorporation into the standard structure-determination process used in neutron protein crystallography; consequently, more precise and efficient determination of the D-atom positions is possible using a combination of this D/H contrast technique and standard neutron structure-determination protocols.

  2. Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors

    PubMed Central

    Daldrup-Link, Heike E.; Mohanty, Suchismita; Ansari, Celina; Ito, Ken; Hong, Su Hyun; Hoffmann, Matthias; Pisani, Laura; Boudreau, Nancy; Gambhir, Sanjiv Sam; Coussens, Lisa M.

    2016-01-01

    Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-β receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies. PMID:27182558

  3. Incompatibility of Contrast Medium and Trisodium Citrate

    SciTech Connect

    Delcour, Christian Bruninx, Guy

    2013-02-15

    To test the compatibility of trisodium citrate, a catheter lock solution, with iodinated contrast medium. Iohexol, iobitridol, iodixanol, ioxaglate, ioxithalamate, iomeprol, and iopromide were tested. In all tests, 2 ml of contrast medium were mixed with 2 ml of trisodium citrate solution. Iodixanol and ioxaglate provoked a highly viscous gluelike precipitation when mixed with trisodium citrate. A brief transient precipitate was observed with iohexol, iomeprol, and ioxithalamate. Permanent precipitation occurred with iobitridol and iopromide. One must be aware of the potential for precipitation when contrast medium is mixed with trisodium citrate solution. Before trisodium citrate solution is injected, the catheter should be thoroughly flushed with saline if a contrast medium has previously been injected through it.

  4. Hytrast: A New Contrast Medium for Bronchography

    PubMed Central

    Misener, F. J.; Quinlan, J. J.; Hiltz, J. E.

    1965-01-01

    In 1962 Hytrast, an aqueous suspension containing 50% w/v of combined iodine as a mixture of N-(2,3-dihydroxypropyl)-3,5-diiodopyridone-4 and 3,5-diiodopyridone-4, was introduced as a contrast medium for bronchography. Extensive clinical trials had suggested that this agent was superior to products usually employed for this purpose. At the Nova Scotia Sanatorium, Hytrast was used as a bronchographic contrast medium in 31 consecutive cases. For comparison purposes, the records of the first 50 patients in whom another contrast medium, Dionosil Oily, was used were reviewed. In all cases the contrast medium was introduced through a catheter passed into the bronchus with the aid of a laryngeal mirror, after local anesthesia was induced by pontocaine 2%. Experience in this limited number of cases was at variance with most published results. Hytrast was more irritating than Dionosil Oily, had a greater tendency to produce alveolarization, caused more frequent undesirable sequelae, and was retained in the lung for prolonged periods. PMID:14264971

  5. Preclinical evaluation of biodegradable macromolecular contrast agents for magnetic resonance imaging

    NASA Astrophysics Data System (ADS)

    Feng, Yi

    Macromolecular contrast agents have been shown to be superior to small molecular weight contrast agents for MRI in blood pool imaging, tumor diagnosis and grading. However, none has been approved by the FDA because they circulate in the bloodstream much longer than small molecular weight contrast agents and result in high tissue accumulation of toxic Gd(III) ions. Biodegradable macromolecular contrast agents (BMCA) were invented to alleviate the toxic accumulation. They have a cleavable disulfide bond based backbone that can be degraded in vivo and excreted out of the body via renal filtration. Furthermore, the side chain of the backbone can be modified to achieve various degradation rates. Three BMCA, (Gd-DTPA)-cystamine copolymers (GDCC), Gd-DTPA cystine copolymers (GDCP), and Gd-DTPA cystine diethyl ester copolymers (GDCEP), were evaluated as blood pool contrast agents in a rat model. They have excellent blood pool enhancement, preferred pharmacokinetics, and only minimal long-term tissue retention of toxic Gd(III) ions. GDCC and GDCP, the lead agents with desired degradation rates, with molecular weights of 20 KDa and 70 KDa, were chosen for dynamic contrast enhanced MRI (DCE-MRI) to differentiate human prostate tumor models of different malignancy and growth rates. GDCC and GDCP could differentiate these tumor models, providing more accurate estimations of plasma volume, flow leakage rate, and permeability surface area product than a small molecular weight contrast agent Gd-DTPA-BMA when compared to the prototype macromolecular contrast agent albumin-Gd-DTPA. GDCC was favored for its neutral charge side chain and reasonable uptake rate by the tumors. GDCC with a molecular weight of 40 KDa (GDCC-40, above the renal filtration cutoff size) was used to assess the efficacy of two photothermal therapies (interstitial and indocyanine green enhanced). GDCC-40 provided excellent tumor enhancement shortly after its injection. Acute tumor response (4 hr) after therapies

  6. A new macromolecular paramagnetic MR contrast agent binds to activated human platelets.

    PubMed

    Chaubet, Frédéric; Bertholon, Isabelle; Serfaty, Jean-Michel; Bazeli, Ramin; Alsaid, Hasan; Jandrot-Perrus, Martine; Zahir, Charaf; Even, Pascale; Bachelet, Laure; Touat, Ziad; Lancelot, Eric; Corot, Claire; Canet-Soulas, Emmanuelle; Letourneur, Didier

    2007-07-01

    A new functionalized macromolecular magnetic resonance (MR) contrast agent has been developed from a carboxymethyldextran-Gd(DOTA) devoid of biospecificity. The functionalized contrast agent was synthesized in order to mimic PSGL-1, the main ligand of P-selectin, a glycoprotein mainly expressed on the surface of activated platelets. The starting compound, CM1, was first carboxymethylated by monochloroacetic acid leading to a series of 10 derivatives varying in their carboxymethyl content. CM8 derivative, with a degree of substitution in carboxymethyl of 0.84, was chosen for subsequent fluorolabeling and sulfation to give CM8FS. CM8FS has an average number molecular weight of 27 000 +/- 500 g/mol, a hydrodynamic radius of 5.7 +/- 0.2 nm and a high relaxivity (r(1) = 11.2/mM (Gd)/s at 60 MHz). Flow cytometry experiments on whole human blood or on isolated platelets evidenced in vitro a preferential binding of CM8FS on TRAP-activated human platelets. Interestingly, CM8FS did not bind to other blood cells or to resting platelets. Pellets of TRAP-activated human platelets have also been imaged in tubes with a 1.5 T MR imager. A MR signal was observed for activated platelets incubated with CM8FS. Altogether, these in vitro results evidenced the recognition of activated human platelets by a fluorescent paramagnetic contrast agent grafted with carboxyl and sulfate groups. This biomimetic approach associated with the versatile macromolecular platform appears promising for the development of new contrast agents for molecular imaging of activated platelets in cardiovascular diseases such as atherosclerosis and aneurysms.

  7. Contrast medium usage reduction in abdominal computed tomography by using high-iodinated concentration contrast medium

    NASA Astrophysics Data System (ADS)

    Suwannasri, A.; Kaewlai, R.; Asavaphatiboon, S.

    2016-03-01

    This study was to determine if administration of a low volume high-concentration iodinated contrast medium can preserve image quality in comparison with regular-concentration intravenous contrast medium in patient undergoing contrast-enhancement abdominal computed tomography (CT). Eighty-four patients were randomly divided into 3 groups of similar iodine delivery rate; A: 1.2 cc/kg of iomeprol-400, B: 1.0 cc/kg of iomeprol-400 and C: 1.5 cc/kg of ioversol-350. Contrast enhancement of the liver parenchyma, pancreas and aorta was quantitatively measured in Hounsfield units and qualitative assessed by a radiologist. T-test was used to evaluate contrast enhancement, and Chi-square test was used to evaluate qualitative image assessment, at significance level of 0.05 with 95% confidence intervals. There were no statistically significant differences in contrast enhancement of liver parenchyma and pancreas between group A and group C in both quantitative and qualitative analyses. Group C showed superior vascular enhancement to group A and B on quantitative analysis.

  8. A neutral polydisulfide containing Gd(III) DOTA monoamide as a redox-sensitive biodegradable macromolecular MRI contrast agent.

    PubMed

    Ye, Zhen; Zhou, Zhuxian; Ayat, Nadia; Wu, Xueming; Jin, Erlei; Shi, Xiaoyue; Lu, Zheng-Rong

    2016-01-01

    This work aims to develop safe and effective gadolinium (III)-based biodegradable macromolecular MRI contrast agents for blood pool and cancer imaging. A neutral polydisulfide containing macrocyclic Gd-DOTA monoamide (GOLS) was synthesized and characterized. In addition to studying the in vitro degradation of GOLS, its kinetic stability was also investigated in an in vivo model. The efficacy of GOLS for contrast-enhanced MRI was examined with female BALB/c mice bearing 4T1 breast cancer xenografts. The pharmacokinetics, biodistribution, and metabolism of GOLS were also determined in mice. GOLS has an apparent molecular weight of 23.0 kDa with T1 relaxivities of 7.20 mM(-1) s(-1) per Gd at 1.5 T, and 6.62 mM(-1) s(-1) at 7.0 T. GOLS had high kinetic inertness against transmetallation with Zn(2+) ions, and its polymer backbone was readily cleaved by L-cysteine. The agent showed improved efficacy for blood pool and tumor MR imaging. The structural effect on biodistribution and in vivo chelation stability was assessed by comparing GOLS with Gd(HP-DO3A), a negatively charged polydisulfide containing Gd-DOTA monoamide GODC, and a polydisulfide containing Gd-DTPA-bisamide (GDCC). GOLS showed high in vivo chelation stability and minimal tissue deposition of gadolinium. The biodegradable macromolecular contrast agent GOLS is a promising polymeric contrast agent for clinical MR cardiovascular imaging and cancer imaging.

  9. Contrast-Medium-Enhanced Digital Mammography: Contrast vs. Iodine Concentration Phantom Calibration

    SciTech Connect

    Rosado-Mendez, I.; Brandan, M. E.; Villasenor, Y.; Benitez-Bribiesca, L.

    2008-08-11

    This work deals with the application of the contrast-medium-enhanced digital subtraction mammography technique in order to calibrate the contrast level in subtracted phantom images as function of iodine concentration to perform dynamic studies of the contrast-medium uptake in the breast. Previously optimized dual-energy temporal subtraction modalities were used (a) to determine radiological parameters for a dynamic clinical study composed of 1 mask+3 post-contrast images limiting the total mean glandular dose to 2.5 mGy, and (b) to perform a contrast vs iodine concentration calibration using a custom-made phantom. Calculated exposure values were applied using a commercial full-field digital mammography unit. Contrast in subtracted phantom images (one mask and one post-CM) is linear as function of iodine concentration, although the sensitivity (contrast per iodine concentration) decreases beyond 8 mg/mL. This calibration seems to apply only to thin and normal thickness breasts.

  10. Investigation of a potential macromolecular MRI contrast agent prepared from PPI (G = 2, polypropyleneimine, generation 2) dendrimer bifunctional chelates

    NASA Astrophysics Data System (ADS)

    Wang, Jianxin Steven

    The long-term objective is to develop magnetic resonance (MR) contrast agents that actively and passively target tumors for diagnosis and therapy. Many diagnostic imaging techniques for cancer lack specificity. A dendrimer based magnetic resonance imaging contrast agent has been developed with large proton relaxation enhancements and high molecular relaxivities. A new type of linear dendrimer based MRI contrast agent that is built from the polypropyleneimine and polyamidoamine dendrimers in which free amines have been conjugated to the chelate DTPA, which further formed the complex with Gadolinium (Gd) was studied. The specific research goals were to test the hypothesis that a linear chelate with macromolecular agents can be used in vitro and in vivo. This work successfully examined the adequacy and viability of the application for this agent in vitro and in vivo. A small animal whole body counter was designed and constructed to allow us to monitor biodistribution and kinetic mechanisms using a radioisotope labeled complex. The procedures of metal labeling, separation and purification have been established from this work. A biodistribution study has been performed using radioisotope induced organ/tissue counting and gamma camera imaging. The ratio of percentage of injected dose per gram organ/tissue for kidney and liver is 3.71 from whole body counter and 3.77 from the gamma camera. The results suggested that retention of Gd (III) is too high and a more kinetically stable chelate should be developed. The pharmacokinetic was evaluated in the whole animal model with the whole body clearance, and a kinetics model was developed. The pharmacokinetic results showed a bi-exponential decay in the animal model with two component excretion constants 1.43e(-5) and 0.0038511, which give half-lives of 3 hours and 33.6 days, respectively. Magnetic resonance imaging of this complex resulted in a 52% contrast enhancement in the rat kidney following the agents' administration in

  11. Synthesis and evaluation of a polydisulfide with Gd-DOTA monoamide side chains as a biodegradable macromolecular contrast agent for MR blood pool imaging.

    PubMed

    Ye, Zhen; Wu, Xueming; Tan, Mingqian; Jesberger, Jack; Grisworld, Mark; Lu, Zheng-Rong

    2013-01-01

    Macromolecular Gd(III)-based contrast agents are effective for contrast-enhanced blood pool and cancer MRI in preclinical studies. However, their clinical applications are impeded by potential safety concerns associated with slow excretion and prolonged retention of these agents in the body. To minimize the safety concerns of macromolecular Gd contrast agents, we have developed biodegradable macromolecular Gd contrast agents based on polydisulfide Gd(III) complexes. In this study, we designed and synthesized a new generation of the polydisulfide Gd(III) complexes containing a macrocyclic Gd(III) chelate, Gd-DOTA monoamide, to improve the in vivo kinetic inertness of the Gd(III) chelates. (N6-Lysyl)lysine-(Gd-DOTA) monoamide and 3-(2-carboxyethyldisulfanyl)propanoic acid copolymers (GODC) were synthesized by copolymerization of (N6-lysyl)lysine DOTA monoamide and dithiobis(succinimidylpropionate), followed by complexation with Gd(OAc)3. The GODC had an apparent molecular weight of 26.4 kDa and T1 relaxivity of 8.25 mM(-1) s(-1) per Gd at 1.5 T. The polymer chains of GODC were readily cleaved by L-cysteine and the chelates had high kinetic stability against transmetallation in the presence of an endogenous metal ion Zn(2+). In vivo MRI study showed that GODC produced strong and prolonged contrast enhancement in the vasculature and tumor periphery of mice with breast tumor xenografts. GODC is a promising biodegradable macromolecular MRI contrast agent with high kinetic stability for MR blood pool imaging.

  12. Reduction of iodinated contrast medium in CT: feasibility study

    NASA Astrophysics Data System (ADS)

    Nasirudin, Radin A.; Mei, Kai; Kopp, Felix K.; Penchev, Petar; Rummeny, Ernst J.; Fiebich, Martin; Noël, Peter B.

    2015-03-01

    In CT, the magnitude of enhancement is proportional to the amount of contrast medium (CM) injected. However, high doses of iodinated CM pose health risks, ranging from mild side effects to serious complications such as contrast-induced nephropathy (CIN). This work presents a method that enables the reduction of CM dosage, without affecting the diagnostic image quality. The technique proposed takes advantage of the additional spectral information provided by photon-counting CT systems. In the first step, we apply a material decomposition technique on the projection data to discriminate iodine from other materials. Then, we estimated the noise of the decomposed image by calculating the Cramér-Rao lower bound of the parameter estimator. Next, we iteratively reconstruct the iodine-only image by using the decomposed image and the estimation of noise as an input into a maximum-likelihood iterative reconstruction algorithm. Finally, we combine the iodine-only image with the original image to enhance the contrast of low iodine concentrations. The resulting reconstructions show a notably improved contrast in the final images. Quantitatively, the combined image has a significantly improved CNR, while the measured concentrations are closer to the actual concentrations of the iodine. The preliminary results from our technique show the possibility of reducing the clinical dosage of iodine, without affecting the diagnostic image quality.

  13. Human pharmacokinetics of iohexol. A new nonionic contrast medium

    SciTech Connect

    Olsson, B.; Aulie, A.; Sveen, K.; Andrew, E.

    1983-03-01

    The pharmacokinetics of iohexol, a new nonionic, water-soluble contrast medium, have been determined after intravenous injection in 20 healthy volunteers, at four different dose levels (125-500 mg I/kg). The apparent volume of distribution was 0.27 1/kg, indicating distribution in the extracellular water. The biologic half-life was 121 minutes, comparable with that of other intravascular contrast media. Iohexol was excreted completely unmetabolized in the urine, with a 100% recovery 24 hours after injection. A comparison of iohexol and chromium-51 (/sup 51/Cr)-EDTA clearances indicates that iohexol is mainly excreted by glomerular filtration. The /sup 51/Cr-EDTA clearance was the same when injected separately and concomitantly with iohexol, indicating that glomerular filtration rate is not affected by iohexol. No dose dependency was observed in the investigated parameters t1/2 alpha, t1/2 beta, Vd, ClT or ClR. Iohexol pharmacokinetics are in correspondence with previously reported data on intravascular contrast media.

  14. Speckle contrast diffuse correlation tomography of complex turbid medium flow

    SciTech Connect

    Huang, Chong; Irwin, Daniel; Lin, Yu; Shang, Yu; He, Lian; Kong, Weikai; Yu, Guoqiang; Luo, Jia

    2015-07-15

    Purpose: Developed herein is a three-dimensional (3D) flow contrast imaging system leveraging advancements in the extension of laser speckle contrast imaging theories to deep tissues along with our recently developed finite-element diffuse correlation tomography (DCT) reconstruction scheme. This technique, termed speckle contrast diffuse correlation tomography (scDCT), enables incorporation of complex optical property heterogeneities and sample boundaries. When combined with a reflectance-based design, this system facilitates a rapid segue into flow contrast imaging of larger, in vivo applications such as humans. Methods: A highly sensitive CCD camera was integrated into a reflectance-based optical system. Four long-coherence laser source positions were coupled to an optical switch for sequencing of tomographic data acquisition providing multiple projections through the sample. This system was investigated through incorporation of liquid and solid tissue-like phantoms exhibiting optical properties and flow characteristics typical of human tissues. Computer simulations were also performed for comparisons. A uniquely encountered smear correction algorithm was employed to correct point-source illumination contributions during image capture with the frame-transfer CCD and reflectance setup. Results: Measurements with scDCT on a homogeneous liquid phantom showed that speckle contrast-based deep flow indices were within 12% of those from standard DCT. Inclusion of a solid phantom submerged below the liquid phantom surface allowed for heterogeneity detection and validation. The heterogeneity was identified successfully by reconstructed 3D flow contrast tomography with scDCT. The heterogeneity center and dimensions and averaged relative flow (within 3%) and localization were in agreement with actuality and computer simulations, respectively. Conclusions: A custom cost-effective CCD-based reflectance 3D flow imaging system demonstrated rapid acquisition of dense boundary

  15. Effects of contrast medium on radiation-induced chromosome aberrations

    SciTech Connect

    Matsubara, S.; Suzuki, S.; Suzuki, H.; Kuwabara, Y.; Okano, T.

    1982-07-01

    The effects of contrast material (meglumine iothalamate) on radiation-induced chromosome aberrations were investigated in studies on the lymphocytes of patients who had undergone diagnostic radiography and in in vitro experiments with diagnostic x rays and /sup 60/Co gamma rays. Chromosome and chromatid aberrations were found to increase significantly with increasing concentrations of contrast material that were added at irradiation. However, the aberrations were not associated with elevation of the ratio of dicentric and ring chromosomes to the number of cells with unstable chromosome aberrations at the first mitosis. Lymphocytes irradiated in the absence of contrast material did not show an increase in chromosome-type aberrations when the agent was given in increasing concentrations during subsequent incubation, but there were greater numbers of chromatid gaps and breaks. When lymphocytes were exposed to 400 R (103.2 mC/kg) of /sup 60/Co gamma rays, the presence of contrast agent did not increase the yield of dicentric and ring chromosomes, but induced a marked delay in cell proliferation, especially in lymphocytes with more heavily damaged chromosomes. In additional examination, the contrast agent itself induced sister chromatid exchanges in lymphocytes.

  16. MACROMOLECULAR THERAPEUTICS

    PubMed Central

    Yang, Jiyuan; Kopeček, Jindřich

    2014-01-01

    This review covers water-soluble polymer-drug conjugates and macromolecules that possess biological activity without attached low molecular weight drugs. The main design principles of traditional and backbone degradable polymer-drug conjugates as well as the development of a new paradigm in nanomedicines – (low molecular weight) drug-free macromolecular therapeutics are discussed. To address the biological features of cancer, macromolecular therapeutics directed to stem/progenitor cells and the tumor microenvironment are deliberated. Finally, the future perspectives of the field are briefly debated. PMID:24747162

  17. Macromolecular therapeutics.

    PubMed

    Yang, Jiyuan; Kopeček, Jindřich

    2014-09-28

    This review covers water-soluble polymer-drug conjugates and macromolecules that possess biological activity without attached low molecular weight drugs. The main design principles of traditional and backbone degradable polymer-drug conjugates as well as the development of a new paradigm in nanomedicines - (low molecular weight) drug-free macromolecular therapeutics are discussed. To address the biological features of cancer, macromolecular therapeutics directed to stem/progenitor cells and the tumor microenvironment are deliberated. Finally, the future perspectives of the field are briefly debated.

  18. In the eye of the storm: iodinated contrast medium induced thyroid storm presenting as cardiopulmonary arrest.

    PubMed

    Alkhuja, Samer; Pyram, Ronald; Odeyemi, Olutunde

    2013-01-01

    The administration of iodinated contrast medium may lead to excess free thyroid hormone release and cause thyroid storm. A woman presented to the emergency department with dyspnea, hemoptysis, and intermittent bilateral lower extremities edema. Physical examination revealed mildly enlarged thyroid. Patient underwent a computed tomography scan of the chest with intravenous iodinated contrast medium to rule out pulmonary embolism, the patient developed a thyroid storm second to iodinated contrast medium injection. Proper treatment was provided and the patient had a good outcome. We present this case of an unusual presentation of a thyroid storm with cardiac arrest. This case illustrates that evaluating thyroid function tests in patients with an enlarged thyroid prior to the administration of iodinated contrast medium could prevent the development of thyroid storm.

  19. Multi-modal magnetic resonance imaging and histology of vascular function in xenografts using macromolecular contrast agent hyperbranched polyglycerol (HPG-GdF).

    PubMed

    Baker, Jennifer H E; McPhee, Kelly C; Moosvi, Firas; Saatchi, Katayoun; Häfeli, Urs O; Minchinton, Andrew I; Reinsberg, Stefan A

    2016-01-01

    Macromolecular gadolinium (Gd)-based contrast agents are in development as blood pool markers for MRI. HPG-GdF is a 583 kDa hyperbranched polyglycerol doubly tagged with Gd and Alexa 647 nm dye, making it both MR and histologically visible. In this study we examined the location of HPG-GdF in whole-tumor xenograft sections matched to in vivo DCE-MR images of both HPG-GdF and Gadovist. Despite its large size, we have shown that HPG-GdF extravasates from some tumor vessels and accumulates over time, but does not distribute beyond a few cell diameters from vessels. Fractional plasma volume (fPV) and apparent permeability-surface area product (aPS) parameters were derived from the MR concentration-time curves of HPG-GdF. Non-viable necrotic tumor tissue was excluded from the analysis by applying a novel bolus arrival time (BAT) algorithm to all voxels. aPS derived from HPG-GdF was the only MR parameter to identify a difference in vascular function between HCT116 and HT29 colorectal tumors. This study is the first to relate low and high molecular weight contrast agents with matched whole-tumor histological sections. These detailed comparisons identified tumor regions that appear distinct from each other using the HPG-GdF biomarkers related to perfusion and vessel leakiness, while Gadovist-imaged parameter measures in the same regions were unable to detect variation in vascular function. We have established HPG-GdF as a biocompatible multi-modal high molecular weight contrast agent with application for examining vascular function in both MR and histological modalities.

  20. Power port contrast medium flushing and trapping: impact of temperature, an in vitro experimental study

    PubMed Central

    Guiffant, Gérard; Durussel, Jean Jacques; Flaud, Patrice; Royon, Laurent; Marcy, Pierre Yves; Merckx, Jacques

    2013-01-01

    Purpose The use of totally implantable venous access devices (TIVADs) certified as “high pressure resistant” or “power port” has begun to spread worldwide as a safe procedure for power contrast injection. Owing to the thermo-rheological properties of the contrast media, the primary aim of this work is to present an in vitro experimental impact study concerning the impact of the temperature level on flushing efficiency after contrast medium injection. Moreover, we report experimental data that confirms the role of needle bevel orientation. The secondary aim is to answer the following questions: Is there significant device contrast medium trapping after contrast medium injection? Is saline flushing efficient? And, finally, is it safe to inject contrast medium through an indwelled port catheter? Results The experimental results show that in addition to hydrodynamics, temperature is a key parameter for the efficiency of device flushing after contrast medium injection. It appears that this is the case when the cavity is incompletely rinsed after three calibrated flushing volumes of 10 mL saline solution, even by using the Huber needle bevel opposite to the port exit. This leads to a potentially important trapped volume of contrast medium in the port, and consequently to the possibility of subsequent salt precipitates and long term trisubstituted benzene nuclei delivery that might impair the solute properties, which may be further injected via the power port later on. Conclusion We thus suggest, in TIVADS patients, the use of a temporary supplementary intravenous line rather than the port to perform contrast medium injections in daily radiology routine practice. PMID:24043959

  1. Macromolecular recognition.

    PubMed

    Deremble, Cyril; Lavery, Richard

    2005-04-01

    Computational methods are being developed both to detect the binding surfaces of individual macromolecules and to predict the structure of binary macromolecular complexes. Speeding up and refining this process has required work on search algorithms, molecular representations and interaction potentials. Although backbone flexibility and solvent effects continue to pose problems, encouraging results have been obtained for both protein-protein and protein-DNA complexes.

  2. Quantitative Assessment of Macromolecular Concentration during Direct Infusion into an Agarose Hydrogel Phantom using Contrast-Enhanced MRI

    PubMed Central

    Chen, Xiaoming; Astary, Garrett W.; Sepulveda, Hector; Mareci, Thomas H.; Sarntinoranont, Malisa

    2011-01-01

    Convection-enhanced delivery (CED), i.e., direct tissue infusion, has emerged as a promising local drug delivery method for treating diseases of the nervous system. Determination of the spatial distribution of therapeutic agents after infusion is important in evaluating the efficacy of treatment, optimizing infusion protocols, and improving the understanding of drug pharmacokinetics. In this study, we provide a methodology to determine the concentration distribution of Gd-labeled tracers during infusion using contrast-enhanced MR imaging. To the best of our knowledge, MR studies that quantify concentration profiles for CED have not been previously reported. The methodology utilizes intrinsic material properties (T1 and R1) and reduces the effect of instrumental factors (e.g., inhomogeneity of MR detection field). As a methodology investigation, this study used an agarose hydrogel phantom as a tissue substitute for infusion. An 11.1 T magnet system was used to image infusion of Gd-DTPA labeled albumin (Gd-albumin) into the hydrogel. By using data from preliminary scans, Gd-albumin distribution was determined from the signal intensity of the MR images. As a validation test, MR-derived concentration profiles were found comparable to both results measured directly using quantitative optical imaging and results from a computational transport model in porous media. In future studies, the developed methodology will be used to quantitatively monitor the distribution of Gd-tracer following infusion directly into tissues. PMID:18583082

  3. Optical tracking of contrast medium bolus to optimize bolus shape and timing in dynamic computed tomography

    NASA Astrophysics Data System (ADS)

    Eisa, Fabian; Brauweiler, Robert; Peetz, Alexander; Hupfer, Martin; Nowak, Tristan; Kalender, Willi A.

    2012-05-01

    One of the biggest challenges in dynamic contrast-enhanced CT is the optimal synchronization of scan start and duration with contrast medium administration in order to optimize image contrast and to reduce the amount of contrast medium. We present a new optically based approach, which was developed to investigate and optimize bolus timing and shape. The time-concentration curve of an intravenously injected test bolus of a dye is measured in peripheral vessels with an optical sensor prior to the diagnostic CT scan. The curves can be used to assess bolus shapes as a function of injection protocols and to determine contrast medium arrival times. Preliminary results for phantom and animal experiments showed the expected linear behavior between dye concentration and absorption. The kinetics of the dye was compared to iodinated contrast medium and was found to be in good agreement. The contrast enhancement curves were reliably detected in three mice with individual bolus shapes and delay times of 2.1, 3.5 and 6.1 s, respectively. The optical sensor appears to be a promising approach to optimize injection protocols and contrast enhancement timing and is applicable to all modalities without implying any additional radiation dose. Clinical tests are still necessary.

  4. Contrast medium administration in the elderly patient: is advancing age an independent risk factor for contrast nephropathy after angiographic procedures?

    PubMed

    Detrenis, Simona; Meschi, Michele; Bertolini, Laura; Savazzi, Giorgio

    2007-02-01

    Contrast medium-induced nephropathy (CMIN) is the third leading cause of hospital-acquired acute renal dysfunction. Even if the number of patients over 75 years of age undergoing diagnostic and/or interventional procedures and requiring administration of contrast medium (CM) is growing constantly, at present there is no definitive consensus regarding the role of advancing age and related morphologic or functional renal changes as an independent risk factor for CMIN. The authors review the evidence from recent medical literature on the definition, pathophysiology, and clinical presentation of CMIN as well as therapeutic approaches to its prophylaxis. Attention is focused on advancing age as a preexisting physiologic condition that is, per se, able to predispose the patient to CM-induced renal impairment, assuming that every elderly patient is potentially at risk for CMIN.

  5. Liver-specific magnetic resonance contrast medium in the evaluation of chronic liver disease

    PubMed Central

    dos Reis, Marcio Augusto Correia Rodrigues; Baroni, Ronaldo Hueb

    2015-01-01

    ABSTRACT The hepatobiliary-specific contrast medium (gadoxetic acid – Primovist®) is primarily used to improve detection and characterization of focal hepatic lesions, such as in chronic liver disease patients with suspected hepatocellular carcinoma. Since the contrast medium is selectively taken up by functioning hepatocytes in the late hepatobiliary phase, it helps to detect typical hepatocellular carcinoma, which show low signal intensity on this phase. This imaging feature also assists in differentiating regenerative/dysplastic nodules from early hepatocellular carcinomas (with over 90% accuracy), as well as hypervascular hepatocellular carcinomas from arterial pseudo-enhancement foci. Future perspectives include its use in quantification of hepatic function and fibrosis. PMID:26154554

  6. A method to evaluate the dose increase in CT with iodinated contrast medium

    SciTech Connect

    Amato, Ernesto; Lizio, Domenico; Settineri, Nicola; Di Pasquale, Andrea; Salamone, Ignazio; Pandolfo, Ignazio

    2010-08-15

    Purpose: The objective of this study is to develop a method to calculate the relative dose increase when a computerized tomography scan (CT) is carried out after administration of iodinated contrast medium, with respect to the same CT scan in absence of contrast medium. Methods: A Monte Carlo simulation in GEANT4 of anthropomorphic neck and abdomen phantoms exposed to a simplified model of CT scanner was set up in order to calculate the increase of dose to thyroid, liver, spleen, kidneys, and pancreas as a function of the quantity of iodine accumulated; a series of experimental measurements of Hounsfield unit (HU) increment for known concentrations of iodinated contrast medium was carried out on a Siemens Sensation 16 CT scanner in order to obtain a relationship between the increment in HU and the relative dose increase in the organs studied. The authors applied such a method to calculate the average dose increase in three patients who underwent standard CT protocols consisting of one native scan in absence of contrast, followed by a contrast-enhanced scan in venous phase. Results: The authors validated their GEANT4 Monte Carlo simulation by comparing the resulting dose increases for iodine solutions in water with the ones presented in literature and with their experimental data obtained through a Roentgen therapy unit. The relative dose increases as a function of the iodine mass fraction accumulated and as a function of the Hounsfield unit increment between the contrast-enhanced scan and the native scan are presented. The data shown for the three patients exhibit an average relative dose increase between 22% for liver and 74% for kidneys; also, spleen (34%), pancreas (28%), and thyroid (48%) show a remarkable average increase. Conclusions: The method developed allows a simple evaluation of the dose increase when iodinated contrast medium is used in CT scans, basing on the increment in Hounsfield units observed on the patients' organs. Since many clinical protocols

  7. Thyroid function in very low birthweight infants after intravenous administration of the iodinated contrast medium iopromide

    PubMed Central

    Dembinski, J; Arpe, V; Kroll, M; Hieronimi, G; Bartmann, P

    2000-01-01

    BACKGROUND—Thyroid function disorders have often been observed in preterm infants after intravenous administration of iodinated contrast medium. The effect on thyroid function depends on the dosage, but the choice of the contrast medium may be equally important, as there are appreciable pharmacological differences between them.
METHOD—Thyroid function was analysed in 20 very low birthweight infants of gestational age less than 30 weeks after injection of iopromide, a monomeric non-ionic iodinated contrast medium. Levels of free thyroxine and thyroid stimulating hormone were compared with those in 26 control infants.
RESULTS—Free thyroxine levels in all study infants ranged from 9.0 to 25.7 pmol/l (days 14-21) and 9.0 to 23.2 pmol/l (days 35-49), and thyroid stimulating hormone levels ranged from 0.13 to 0.26mU/l (days 14-21) and 0.26 to 11.11 mU/l (days 35-49). These levels were not altered after injection of iopromide.
CONCLUSION—The risk of transient hypothyroidism or hyperthyrotropinaemia may be reduced with the use of iopromide compared with other contrast media.

 PMID:10794789

  8. Relationship between ventral lumbar disc protrusion and contrast medium leakage during sympathetic nerve block.

    PubMed

    Tazawa, Toshiharu; Kamiya, Yoshinori; Takamori, Mina; Ogawa, Ken-Ichi; Goto, Takahisa

    2015-02-01

    Ventral disc protrusions have been neglected because they are asymptomatic. Lumbar sympathetic nerve block (LSNB) is one of the clinical choices for refractory low back pain treatment. Leakage of the contrast medium may occur and lead to complications, especially when using a neurolytic agent. In this study, we retrospectively reviewed the magnetic resonance images (MRIs) of 52 consecutive patients with refractory low back pain due to lumbar spinal canal stenosis who underwent LSNB, and graded ventral disc protrusion at the L1/2 to L5/S1 vertebral discs on a three-point scale (grade 0 = no protrusion, grade 1 = protrusion without migration, grade 2 = protrusion with migration). We also determined if there was leakage of contrast medium in LSNB. Ventral disc protrusion was observed in all patients, and 75 % (39/52) had grade 2 protrusion in the L1/2-L3/4 vertebral discs. Moreover, the incidence of contrast medium leakage was significantly higher at the vertebrae that had grade 2 protrusion than at those with less protrusion. We revealed a higher incidence of ventral disc protrusion of the lumbar vertebrae than previously reported, and that the incidence of leakage in LSNB increased when ventral disc protrusion was present. To avoid complications, attention should be paid to ventral disc protrusions before performing LSNB.

  9. Relative diffusion of paramagnetic metal complexes of MRI contrast agents in an isotropic hydrogel medium.

    PubMed

    Weerakoon, Bimali Sanjeevani; Osuga, Toshiaki

    2017-03-01

    The observation of molecular diffusion by means of magnetic resonance imaging (MRI) is significant in the evaluation of the metabolic activity of living tissues. Series of MRI examinations were conducted on a diffusion model to study the behaviour of the diffusion process of different-molecular-weight (MW) paramagnetic MRI contrast agents in an isotropic agar hydrogel medium. The model consisted of a solidified 1 % agar gel with an initial concentration of 0.5 mmol/L contrast solution layered on top of the gel. The diffusion process was monitored at pre-determined time intervals of immediately, 1, 6, 9, 23, and 48 h after introduction of the contrast agents onto the agar gel with a T1-weighted spin-echo (SE) pulse sequence. Three types of paramagnetic contrast agents, Gd-DTPA with a MW of 547.57 g/mol, Prohance with a MW of 558.69 g/mol and MnCl2 with a MW of 125.84 g/mol, resulted in an approximate average diffusional displacement ratio of 1:1:2 per hour, respectively, within 48 h of the experiment. Therefore, the results of this study supported the hypothesis that the rate of the diffusion process of MRI contrast agents in the agar hydrogel medium is inversely related to their MWs. However, more repetitions are necessary under various types of experimental conditions and also with various types of contrast media of different MWs for further confirmation and validation of these results.

  10. Efficacy of coronary fractional flow reserve using contrast medium compared to adenosine

    PubMed Central

    Tanboğa, Ibrahim Halil; Aksakal, Enbiya; Aksu, Uğur; Gulcu, Oktay; Birdal, Oğuzhan; Arısoy, Arif; Kalaycı, Arzu; Ulusoy, Fatih Rifat; Sevimli, Serdar

    2016-01-01

    Introduction Coronary fractional flow reserve (FFR) is recommended as the gold standard method in evaluating intermediate coronary stenoses. However, there are significant debates concerning the agents and the timing of the measurement. Aim To compare the contrast medium induced Pd/Pa ratio (CMR) with the FFR. Material and methods We enrolled 28 consecutive patients with 34 intermediate lesions who underwent coronary FFR measurement by intracoronary (i.c.) adenosine. After baseline Pd/Pa was calculated, a single contrast medium (Iomeron) injection of 6 ml (3 ml/s) was performed manually. Within 10 s after the contrast medium injection, the CMR was calculated. Bolus injection of i.c. adenosine was performed to induce maximal hyperemia (from 60 µg to 600 µg), and when it was ≤ 0.80, the intermediate lesion was considered as significant. Results After bolus i.c. adenosine, 12 lesions of 34 (35.3%) were identified as significant. The CMR value was 0.86 ±0.06 (range: 0.71–0.97). There were no significant differences between FFR and CMR values (p = 0.108). A substantial positive correlation between adenosine and contrast values was detected (0.886 and p < 0.001). Good agreement in Bland-Altman analysis was revealed (mean bias was 0.027, 95% confidence interval 0.038–0.092). Receiver operating characteristics curve analysis showed 90.9% sensitivity and 91.7% specificity for a cut-off value of 0.85 for the CMR compared to FFR (≤ 0.80). Conclusions Our study showed that measuring the CMR is a feasible method compared to FFR. The CMR may be used in situations where adenosine cannot be administered. PMID:27625683

  11. Iodinated contrast medium as an aid to gallstone dissolution with methyl tert-butyl ether: in vitro study.

    PubMed

    Zhou, J; Lee, S H; Rawat, B; Fache, J S; Maciejewska, U; Burhenne, H J

    1990-05-01

    Methyl tert-butyl ether (MTBE) floats on bile, whereas gallstones sink. Therefore, stones and MTBE are separated by a layer of bile. This study investigates the effect of contrast medium on flotation of gallstones in bile and its role in stone and fragment dissolution with MTBE. Fresh human gallstones, both calcified and noncalcified, from different patients were tested in vitro for flotation in bile, with and without addition of contrast medium. All gallstones or fragments sank in bile before the introduction of contrast medium. Noncalcified stones floated when the contrast medium-bile volume ratio was 1:6 or more, while double this amount of contrast medium was required to float calcified stones. Fragments did dissolve somewhat in MTBE in the presence of bile alone, but when contrast medium was added, almost complete dissolution occurred. This is thought to be due to increased contact between the fragments and MTBE, both floating on the contrast medium-bile mixture. Contrast material may be a useful adjuvant in gallstone dissolution therapy with MTBE in vivo.

  12. Extensive exfoliative dermatitis induced by non-ionic contrast medium Iodixanol (Visipaque) used during percutaneous coronary intervention.

    PubMed

    Choi, Cheol Ung; Rha, Seung-Woon; Suh, Soon Yong; Kim, Jin Won; Kim, Eung Ju; Park, Chang Gyu; Seo, Hong Seog; Oh, Dong Joo

    2008-02-29

    We report a case of extensive exfoliative dermatitis in a patient appearing 3 days after intracoronary administration of non-ionic contrast medium Iodixanol (Visipaque) during the primary percutaneous coronary intervention. The patient presented with acute myocardial infarction and has never exposed to any X-ray contrast medium. The patient was successfully treated with corticosteroid, antihistamines and antibiotics for the prevention of secondary bacterial infection. The patient was recovered 8 days after the anti-allergic medical management. This case can be a rare example of late-onset allergic reaction to a non-ionic contrast medium Iodixanol presented with extensive exfoliative dermatitis.

  13. Bilateral Renal Fornix Rupture Following Intraarterial Contrast Medium Application for Infrarenal Aortic Stent Placement

    SciTech Connect

    Niggemann, Pascal Brehmer, Bernhard; Schuermann, Karl

    2006-02-15

    A 74-year-old male claudicant who had a significant abdominal aortic stenosis was hydrated before aortic stent placement because of an elevated creatinine level. During the intervention the patient experienced acute abdominal pain with vomiting. No vascular cause was detected. Due to persistant pain, plain radiography and an abdominal CT scan were performed a few hours after the procedure. Images revealed a bilateral renal fornix rupture with a large retroperitoneal fluid collection. The patient was treated conservatively with ureteral double-J placement and percutaneous nephrostomy. The further course was uneventful and the patient was discharged 2 weeks later free of symptoms. Renal fornix rupture is a very rare complication after contrast medium application that can be treated without surgery.

  14. Contrasting metabolic effects of medium- versus long-chain fatty acids in skeletal muscle[S

    PubMed Central

    Montgomery, Magdalene K.; Osborne, Brenna; Brown, Simon H. J.; Small, Lewin; Mitchell, Todd W.; Cooney, Gregory J.; Turner, Nigel

    2013-01-01

    Dietary intake of long-chain fatty acids (LCFAs) plays a causative role in insulin resistance and risk of diabetes. Whereas LCFAs promote lipid accumulation and insulin resistance, diets rich in medium-chain fatty acids (MCFAs) have been associated with increased oxidative metabolism and reduced adiposity, with few deleterious effects on insulin action. The molecular mechanisms underlying these differences between dietary fat subtypes are poorly understood. To investigate this further, we treated C2C12 myotubes with various LCFAs (16:0, 18:1n9, and 18:2n6) and MCFAs (10:0 and 12:0), as well as fed mice diets rich in LCFAs or MCFAs, and investigated fatty acid-induced changes in mitochondrial metabolism and oxidative stress. MCFA-treated cells displayed less lipid accumulation, increased mitochondrial oxidative capacity, and less oxidative stress than LCFA-treated cells. These changes were associated with improved insulin action in MCFA-treated myotubes. MCFA-fed mice exhibited increased energy expenditure, reduced adiposity, and better glucose tolerance compared with LCFA-fed mice. Dietary MCFAs increased respiration in isolated mitochondria, with a simultaneous reduction in reactive oxygen species generation, and subsequently low oxidative damage. Collectively our findings indicate that in contrast to LCFAs, MCFAs increase the intrinsic respiratory capacity of mitochondria without increasing oxidative stress. These effects potentially contribute to the beneficial metabolic actions of dietary MCFAs. PMID:24078708

  15. Transformation of the X-ray contrast medium iopromide in soil and biological wastewater treatment.

    PubMed

    Schulz, Manoj; Löffler, Dirk; Wagner, Manfred; Ternes, Thomas A

    2008-10-01

    In water/soil systems, the iodinated contrast medium iopromide was quantitatively biotransformed into several transformation products (TPs). Twelve TPs were identified via HPLC-UV and LC tandem MS. The chemical structures of the TPs were elucidated via fragmentation in MS2 and MS3 of LC tandem MS with a linear ion trap and 1H and 13C NMR analyses. All TPs exhibited transformations at the side chains containing either carboxylic moieties and/or primary and secondary amide moieties, while the triiodoisophthalic acid structure remained unaltered. A transformation pathway was proposed based on the sequence of TP formation in aerobic batch experiments. Additionally, the occurrence of iopromide TPs was investigated in native water samples. All TPs identified were found in municipal WWTP effluents because of their formation during biological wastewater treatment with maximum concentrations of up to 3.7 +/- 0.9 microg/L (TP 819). Predominantly, those TPs were present at higher concentrations in WWTP effluents which were formed at the beginning of the transformation pathway. Furthermore, four TPs formed at the end of the transformation pathway (TP 759, 701A/B, and 643) were also found in bank filtrate up to 0.050 microg/L and in groundwater of an wastewater irrigation area up to 4.6 microg/L.

  16. Separation and characterization of the two diastereomers for [Gd(DTPA-bz-NH2)(H2O)]2-, a common synthon in macromolecular MRI contrast agents: their water exchange and isomerization kinetics.

    PubMed

    Burai, László; Tóth, Eva; Sour, Angélique; Merbach, André E

    2005-05-16

    Chiral, bifunctional poly(amino carboxylate) ligands are commonly used for the synthesis of macromolecular, Gd(III)-based MRI contrast agents, prepared in the objective of increasing relaxivity or delivering the paramagnetic Gd(III) to a specific site (targeting). Complex formation with such ligands results in two diastereomeric forms for the complex which can be separated by HPLC. We demonstrated that the diastereomer ratio for Ln(III) DTPA derivatives (approximately 60:40) remains constant throughout the lanthanide series, in contrast to Ln(III) EPTPA derivatives, where it varies as a function of the cation size with a maximum for the middle lanthanides (DTPA(5-) = diethylenetriaminepentaacetate; EPTPA(5-) = ethylenepropylenetriaminepentaacetate). The interconversion of the two diastereomers, studied by HPLC, is a proton-catalyzed process (k(obs) = k(1)[H(+)]). It is relatively fast for [Gd(EPTPA-bz-NH(2))(H(2)O)](2-) but slow enough for [Gd(DTPA-bz-NH(2))(H(2)O)](2-) to allow investigation of pure individual isomers (isomerization rate constants are k(1) = (3.03 +/- 0.07) x 10(4) and 11.6 +/- 0.5 s(-1) M(-1) for [Gd(EPTPA-bz-NH(2))(H(2)O)](2)(-) and [Gd(DTPA-bz-NH(2))(H(2)O)](2-), respectively). Individual water exchange rates have been determined for both diastereomers of [Gd(DTPA-bz-NH(2))(H(2)O)](2-) by a variable-temperature (17)O NMR study. Similarly to Ln(III) EPTPA derivatives, k(ex) values differ by a factor of 2 (k(ex)(298) = (5.7 +/- 0.2) x 10(6) and (3.1 +/- 0.1) x 10(6) s(-1)). This variance in the exchange rate has no consequence on the proton relaxivity of the two diastereomers, since it is solely limited by fast rotation. However, such difference in k(ex) will affect proton relaxivity when these diastereomers are linked to a slowly rotating macromolecule. Once the rotation is optimized, slow water exchange will limit relaxivity; thus, a factor of 2 in the exchange rate can lead to a remarkably different relaxivity for the diastereomer complexes

  17. Aggravation of Pre-Existing Atrioventricular Block, Wenckebach Type, Provoked by Application of X-Ray Contrast Medium

    SciTech Connect

    Brodmann, Marianne Seinost, Gerald; Stark, Gerhard; Pilger, Ernst

    2006-12-15

    Background. Significant bradycardia followed by cardiac arrest related to single bolus administration of X-ray contrast medium into a peripheral artery has not, to our knowledge, been described in the literature. Methods and Results. While performing a percutaneous transluminal angioplasty of the left superficial femoral artery in a 68-year old patient with a pre-existing atrioventricular (AV) block, Wenckebach type, he developed an AV block III after a single bolus injection of intra-arterial X-ray contrast medium. Conclusion. We believe that application of contrast medium causes a transitory ischemia in the obstructed vessel and therefore elevation of endogenous adenosine. In the case of a previously damaged AV node this elevation of endogenous adenosine may be responsible for the development of a short period of third-degree AV block.

  18. Establishment of the intracranial hemodynamic model based on contrast medium and clinical applications

    PubMed Central

    Cheng, Yaoer; He, Wen

    2016-01-01

    Abstract Ischemic cerebrovascular diseases are one of the most common vascular diseases in aged people and CT perfusion (CTP) is a very popular tool to detect the ischemic changes in brain vascular. The present study aims to establish a novel intracranial hemodynamic model to simulate anterior cerebral artery blood flow, and compare the actual and simulated hemodynamic parameters of healthy people and patients with carotid stenosis or occlusion. A mathematical model of the intracranial hemodynamic was generated using MATLAB software, and data from patients with or without infarct disease (57 and 44 cases, respectively) were retrospectively collected to test the new model. The actual time-density curve (TDC) of anterior cerebral artery was obtained from the original intracranial CTP data, and simulated TDC was calculated from our intracranial hemodynamic model. All model parameters were adjusted according to patients’ sex, height, and weight. Time to peak enhancement (TTP), maximum enhancement (ME), and mean transit time (MTT) were selected to evaluate the status of hemodynamics. In healthy people, there were no significant differences of TTP and ME between actual and simulated curves. For patients with infarct symptoms, ME was significantly decreased in actual data compared with simulated curve, while there was no obvious difference of TTP between actual and simulated data. Moreover, MTT was delayed in infarct patients compared with healthy people. Our group generated a computer-based, physiologic model to simulate intracranial hemodynamics. The model successfully simulated anterior cerebral artery hemodynamics in normal healthy people and showed noncompliant ME and MTT in infarct patients, reflecting their abnormal cerebral hemodynamic status. The digital model is reliable and may help optimize the protocol of contrast medium enhancement in intracranial CT, and provide a solid tool to study intracranial hemodynamics. PMID:27930555

  19. Contrast evaluation of the polarimetric images of different targets in turbid medium: possible sources of systematic errors

    NASA Astrophysics Data System (ADS)

    Novikova, T.; Bénière, A.; Goudail, F.; De Martino, A.

    2010-04-01

    Subsurface polarimetric (differential polarization, degree of polarization or Mueller matrix) imaging of various targets in turbid media shows image contrast enhancement compared with total intensity measurements. The image contrast depends on the target immersion depth and on both target and background medium optical properties, such as scattering coefficient, absorption coefficient and anisotropy. The differential polarization image contrast is usually not the same for circularly and linearly polarized light. With linearly and circularly polarized light we acquired the orthogonal state contrast (OSC) images of reflecting, scattering and absorbing targets. The targets were positioned at various depths within the container filled with polystyrene particle suspension in water. We also performed numerical Monte Carlo modelling of backscattering Mueller matrix images of the experimental set-up. Quite often the dimensions of container, its shape and optical properties of container walls are not reported for similar experiments and numerical simulations. However, we found, that depending on the photon transport mean free path in the scattering medium, the above mentioned parameters, as well as multiple target design could all be sources of significant systematic errors in the evaluation of polarimetric image contrast. Thus, proper design of experiment geometry is of prime importance in order to remove the sources of possible artefacts in the image contrast evaluation and to make a correct choice between linear and circular polarization of the light for better target detection.

  20. Intense femtosecond laser driven collimated fast electron transport in a dielectric medium-role of intensity contrast.

    PubMed

    Dey, Indranuj; Adak, Amitava; Singh, Prashant Kumar; Shaikh, Moniruzzaman; Chatterjee, Gourab; Sarkar, Deep; Lad, Amit D; Kumar, G Ravindra

    2016-12-12

    Ultra-high intensity (> 1018 W/cm2), femtosecond (~30 fs) laser induced fast electron transport in a transparent dielectric has been studied for two laser systems having three orders of magnitude different peak to pedestal intensity contrast, using ultrafast time-resolved shadowgraphy. Use of a 400 nm femtosecond pulse as a probe enables the exclusive visualization of the dynamics of highest density electrons (> 7 × 1021 cm-3) observed so far. High picosecond contrast (~109) results in greater coupling of peak laser energy to the plasma electrons, enabling long (~1 mm), collimated (divergence angle ~2°) transport of fast electrons inside the dielectric medium at relativistic speeds (~0.66c). In comparison, the laser system with a contrast of ~106 has a large pre-plasma, limiting the coupling of laser energy to the solid and yielding limited fast electron injection into the dielectric. In the lower contrast case, bulk of the electrons expand as a cloud inside the medium with an order of magnitude lower speed than that of the fast electrons obtained with the high contrast laser. The expansion speed of the plasma towards vacuum is similar for the two contrasts.

  1. Penetration of subarachnoid contrast medium into rabbit spinal cord. Comparison between metrizamide and iohexol

    SciTech Connect

    Holtas, S.; Morris, T.W.; Ekholm, S.E.; Isaac, L.; Fonte, D.

    1986-02-01

    The penetration into rabbit spinal cord of two nonionic contrast media, iohexol and metrizamide, and a reference tracer, technetium DTPA, were compared. The spinal subarachnoid space was perfused for 4 hours with a CSF solution to which technetium DTPA and either iohexol or metrizamide had been added. The contrast media and technetium DTPA concentrations reached a plateau level in CSF outflow within 80 minutes. The contrast media concentrations in CSF were higher than the technetium DTPA (P less than .001). In the cord tissue, technetium DTPA reached higher concentrations than the contrast media (P less than .001), and iohexol reached higher concentrations relative to technetium DTPA than metrizamide (P less than .001). The mean contrast media distribution volumes in the thoracic cord were 13% (iohexol) and 12% (metrizamide). The smaller distribution volume observed for metrizamide could be related to the larger effective size of associated metrizamide molecules or an interference with diffusion perhaps related to binding to glucose carriers.

  2. Improvement of the optical imaging of objects in a strongly scattering medium by means of contrast-enhancing dyes

    SciTech Connect

    Vorob'ev, Nikolai S; Smirnov, A V; Podgaetskii, Vitalii M; Tereshchenko, Sergei A; Tomilova, Larisa G

    1999-12-31

    The problem of enhancing the contrast of optical images in a strongly scattering medium by means of luminescent and absorbing dyes, topical in laser tomography, is examined. Preparations based on diphthalocyanine compounds were selected on the grounds of their tropism and resistance to the action of heat and light. Images with enhanced contrast in model scattering media (an aqueous solution of milk and margarine) were obtained in the IR region of the spectrum using the radiation of a picosecond neodymium laser. (laser applications and other topics in quantum electronics)

  3. Effects of contrast medium on radiation-induced chromosome aberrations. [X-ray; /sup 60/Co

    SciTech Connect

    Matsubara, S.; Suzuki, S.; Suzuki, H.; Kuwabara, Y.; Okano, T.

    1982-07-01

    The effects of contrast material (meglumine iothalamate) on radiation-induced chromosome aberrations were investigated in studies on the lymphocytes of patients who had undergone diagnostic radiography and in vitro experiments with diagnostic x rays and /sup 60/Co ..gamma.. rays. Chromosome and chromatic aberrations were found to increase significantly with increasing concentrations of contrast material that were added at irradiation. However, the aberrations were not associated with elevation of the ratio of dicentric and ring chromosomes to the number of cells with unstable chromosome aberrations at the first mitosis. Lymphocytes irradiated in the absence of contrast material did not show an increase in chromosome-type aberrations when the agent was given in increasing concentrations during subsequent incubation, but there were greater numbers of chromatid gaps and breaks. When lymphocytes were exposed to 400 R (103.2 mC/kg) of /sup 60/Co ..gamma.. rays, the presence of contrast agent did not increase the yield of dicentric and ring chromosomes, but induced a marked delay in cell proliferation, especially in lymphocytes with more heavily damaged chromosomes. In additional examination, the contrast agent itself induced sister chromatid exchanges in lymphocytes.

  4. Macromolecular Crystallization in Microgravity

    NASA Technical Reports Server (NTRS)

    Snell, Edward H.; Helliwell, John R.

    2004-01-01

    The key concepts that attracted crystal growers, macromolecular or solid state, to microgravity research is that density difference fluid flows and sedimentation of the growing crystals are greatly reduced. Thus, defects and flaws in the crystals can be reduced, even eliminated, and crystal volume can be increased. Macromolecular crystallography differs from the field of crystalline semiconductors. For the latter, crystals are harnessed for their electrical behaviors. A crystal of a biological macromolecule is used instead for diffraction experiments (X-ray or neutron) to determine the three-dimensional structure of the macromolecule. The better the internal order of the crystal of a biological macromolecule then the more molecular structure detail that can be extracted. This structural information that enables an understanding of how the molecule functions. This knowledge is changing the biological and chemical sciences with major potential in understanding disease pathologies. Macromolecular structural crystallography in general is a remarkable field where physics, biology, chemistry, and mathematics meet to enable insight to the basic fundamentals of life. In this review, we examine the use of microgravity as an environment to grow macromolecular crystals. We describe the crystallization procedures used on the ground, how the resulting crystals are studied and the knowledge obtained from those crystals. We address the features desired in an ordered crystal and the techniques used to evaluate those features in detail. We then introduce the microgravity environment, the techniques to access that environment, and the theory and evidence behind the use of microgravity for crystallization experiments. We describe how ground-based laboratory techniques have been adapted to microgravity flights and look at some of the methods used to analyze the resulting data. Several case studies illustrate the physical crystal quality improvements and the macromolecular structural

  5. Does iodinated contrast medium amplify DNA damage during exposure to radiation

    PubMed Central

    2015-01-01

    There is a recognized increased risk of cancer following exposure of humans to ionizing radiation; this is felt to be most likely due to damage to DNA strands during exposure. Damage to DNA strands can be demonstrated microscopically following exposure to X-rays, and new evidence is emerging that this effect may be compounded by administration of iodinated contrast agents. PMID:26234959

  6. Thromboelastographic Changes Following Nonionic Contrast Medium Injection During Transfemoral Angiography in Patients with Peripheral Arterial Occlusive Disease

    SciTech Connect

    Shankar, V.K. Handa, A.; Philips-Hughes, J.; Boardman, P.; Uberoi, R.; Hands, L.J.

    2006-12-15

    Background/Purpose. Patients with peripheral arterial occlusive disease (PAOD) are known to be systemically hypercoagulable and there is concern that exposing them to contrast media during angiography may exacerbate that thrombotic tendency. Many in vitro studies in which blood is exposed to contrast media suggest that nonionic contrast medium (NICM) has a weaker anticoagulant effect than ionic contrast medium (ICM) and some studies suggest that NICM can lead to activation of coagulation thus increasing the risk of thrombotic events where it is employed. We have looked at the changes in coagulation adjacent to the site of contrast injection/potential angioplasty to determine the magnitude of change locally. Methods. We measured changes in the coagulability of aortic blood samples immediately before and within 2 min after injection of the last bolus of iohexol (NICM) prior to any intervention procedure in 30 patients with PAOD. Samples were analyzed using thromboelastography (TEG) to identify changes in the coagulability of the aortic blood samples. Results. TEG tracings of samples taken from the aorta after injection of NICM showed a significant increase in R time (time to fibrin formation) (p = 0.036) and in k time (dynamics of clot formation) (p = 0.028) and a reduction in Angle (decreased acceleration of fibrin build-up) (p = 0.013), Maximal amplitude (MA) (reduced ultimate clot strength) (p = 0.018) and Coagulation Index (CI) (p = 0.032). Conclusion. These changes in TEG parameters show that the local effect of NICM is a reduction in coagulation activity rather than the activation suggested by some previous studies.

  7. Direct cardiac effects of an ionic and a non-ionic contrast medium in dogs.

    PubMed

    Palik, I; Szente, A; Köszeghy, G A; Koltai, M Z; Kiss, V; Pogátsa, G

    1986-11-01

    Direct cardiac effects of ionic diatrizoate (Uromiro) and non-ionic iopamidol (Iopamiro) were investigated in "in situ" heart-lung preparation of 19 vagotomized dogs. Diatrizoate was found to induce considerably greater alteration in plasma osmolality and subsequent dehydration of the myocardium compared with iopamidol. Myocardial dehydration resulted in a decrease of left ventricular compliance and in that of cardiac performance. Diatrizoate was shown to influence the myocardium not only by its hyperosmolarity but also by a direct action. Heart rate was reduced by both contrast media.

  8. Automated macromolecular crystallization screening

    DOEpatents

    Segelke, Brent W.; Rupp, Bernhard; Krupka, Heike I.

    2005-03-01

    An automated macromolecular crystallization screening system wherein a multiplicity of reagent mixes are produced. A multiplicity of analysis plates is produced utilizing the reagent mixes combined with a sample. The analysis plates are incubated to promote growth of crystals. Images of the crystals are made. The images are analyzed with regard to suitability of the crystals for analysis by x-ray crystallography. A design of reagent mixes is produced based upon the expected suitability of the crystals for analysis by x-ray crystallography. A second multiplicity of mixes of the reagent components is produced utilizing the design and a second multiplicity of reagent mixes is used for a second round of automated macromolecular crystallization screening. In one embodiment the multiplicity of reagent mixes are produced by a random selection of reagent components.

  9. Blueprinting macromolecular electronics.

    PubMed

    Palma, Carlos-Andres; Samorì, Paolo

    2011-06-01

    Recently, by mastering either top-down or bottom-up approaches, tailor-made macromolecular nano-objects with semiconducting properties have been fabricated. These engineered nanostructures for organic electronics are based on conjugated systems predominantly made up of sp²-hybridized carbon, such as graphene nanoribbons. Here, we describe developments in a selection of these nanofabrication techniques, which include graphene carving, stimulus-induced synthesis of conjugated polymers and surface-assisted synthesis. We also assess their potential to reproduce chemically and spatially precise molecular arrangements, that is, molecular blueprints. In a broad context, the engineering of a molecular blueprint represents the fabrication of an integrated all-organic macromolecular electronic circuit. In this Perspective, we suggest chemical routes, as well as convergent on-surface synthesis and microfabrication approaches, for the ultimate goal of bringing the field closer to technology.

  10. Practical macromolecular cryocrystallography

    SciTech Connect

    Pflugrath, J. W.

    2015-05-27

    Current methods, reagents and experimental hardware for successfully and reproducibly flash-cooling macromolecular crystals to cryogenic temperatures for X-ray diffraction data collection are reviewed. Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented.

  11. Practical macromolecular cryocrystallography

    PubMed Central

    Pflugrath, J. W.

    2015-01-01

    Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented. PMID:26057787

  12. Microgravity and Macromolecular Crystallography

    NASA Technical Reports Server (NTRS)

    Kundrot, Craig E.; Judge, Russell A.; Pusey, Marc L.; Snell, Edward H.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Macromolecular crystal growth has been seen as an ideal experiment to make use of the reduced acceleration environment provided by an orbiting spacecraft. The experiments are small, simply operated and have a high potential scientific and economic impact. In this review we examine the theoretical reasons why microgravity should be a beneficial environment for crystal growth and survey the history of experiments on the Space Shuttle Orbiter, on unmanned spacecraft, and on the Mir space station. Finally we outline the direction for optimizing the future use of orbiting platforms.

  13. Practical macromolecular cryocrystallography.

    PubMed

    Pflugrath, J W

    2015-06-01

    Cryocrystallography is an indispensable technique that is routinely used for single-crystal X-ray diffraction data collection at temperatures near 100 K, where radiation damage is mitigated. Modern procedures and tools to cryoprotect and rapidly cool macromolecular crystals with a significant solvent fraction to below the glass-transition phase of water are reviewed. Reagents and methods to help prevent the stresses that damage crystals when flash-cooling are described. A method of using isopentane to assess whether cryogenic temperatures have been preserved when dismounting screened crystals is also presented.

  14. Clinical analysis of contributors to the delayed gallbladder opacification following the use of water-soluble contrast medium

    PubMed Central

    Ku, Ming-Chang; Kok, Victor C; Lee, Ming-Yung; Hsu, Soa-Min; Lee, Pei-Yu; Chang, Che-Wei; Tyan, Yeu-Sheng; Juan, Chi-Wen

    2016-01-01

    Objectives Gallbladder opacification (GBO) on computed tomography (CT) imaging may obscure certain pathological or emergent conditions in the gallbladder, such as neoplasms, stones, and hemorrhagic cholecystitis. This study aimed to investigate the clinical contributing factors that could predict the presence of delayed GBO determined by CT. Methods This study retrospectively evaluated 243 consecutive patients who received enhanced CT or intravenous pyelography imaging and then underwent abdominal CT imaging within 5 days. According to the interval between imaging, the patients were divided into group A (1 day), group B (2 or 3 days), and group C (4 or 5 days). Three radiologists evaluated CT images to determine GBO. Fisher’s exact test and multivariate backward stepwise elimination logistic regression were performed. Results Positive GBO was significantly associated with the interval between imaging studies, contrast type, contrast volume, renal function, and hypertransaminasemia (P<0.05). Multivariate backward stepwise elimination logistic regression analysis of the three groups identified contrast type and hypertransaminasemia as independent predictors of GBO in group B patients (odds ratio [OR], 13.52, 95% confidence interval [CI], 1.72–106.38 and OR, 3.43, 95% CI, 1.31–8.98, respectively; P<0.05). Hypertransaminasemia was the only independent predictor of GBO in group C patients with an OR of 7.2 (95% CI, 1.62–31.73). Hypertransaminasemia was noted in three patients (100%) who initially underwent imaging 5 days prior to GBO. Conclusion Delayed GBO on CT imaging may be associated with laboratory hypertransaminasemia, particularly in patients receiving contrast medium over a period of ≥4 days. A detailed clinical history, physical examination, and further workup are of paramount importance for investigating the underlying cause behind the hypertransaminasemia. PMID:27660453

  15. Quantitative analysis applied to contrast medium extravasation by using the computed-tomography number within the region of interest

    NASA Astrophysics Data System (ADS)

    Lee, Jae-Seung; Im, In-Chul; Kim, Moon-Jib; Goo, Eun-Hoe; Kim, Sun-Ju; Kim, Kwang; Kwak, Byung-Joon

    2014-02-01

    The present study was carried out to present a method to analyze extravasation quantitatively by measuring the computed tomography (CT) number after determining the region of interest (ROI) in the CT images obtained from patients suspected of extravasation induced by contrast medium auto-injection. To achieve this, we divided the study subjects into a group of patients who incurred extravasation and a group of patients who underwent routine scans without incurring extravasation. The CT numbers at IV sites were obtained as reference values, and CT numbers at extravasation sites and hepatic portal veins, respectively, were obtained as relative values. Thereupon, the predicted time for extravasation ( T EP ) and the predicted ratio for extravasation ( R EP ) of an extravasation site were obtained and analyzed quantitatively. In the case of extravasation induced by a dual auto-injector, the values of the CT numbers were confirmed to be lower and the extravasation site to be enlarged when compared to the extravasation induced by a single autoinjector. This is because the physiological saline introduced after the injection of the contrast agent diluted the concentration of the extravasated contrast agent. Additionally, the T EP caused by the auto-injector was about 40 seconds, and we could perform a precise quantitative assessment of the site suspected of extravasation. In conclusion, the dual auto-injection method, despite its advantage of reducing the volume of contrast agent and improving the quality of images for patients with good vascular integrity, was judged to be likely to increase the risk of extravasation and aggravate outcomes for patients with poor vascular integrity by enlarging extravasation sites.

  16. Myocardial rupture associated with bolus injection of contrast medium during computed tomographic study in a patient with acute myocardial infarction: a rare but lethal complication.

    PubMed

    Lai, Vincent; Hau, K C; Lau, H Y; Chan, W C

    2009-08-01

    Well-documented potential cardiovascular complications associated with the use of contrast media include bradycardia, hypotension, arrhythmia, and conduction disturbances. Rupture of the myocardium after acute myocardial infarction is a known cause of death, but has yet to be recognised as a potential complication of the use of a bolus injection of contrast medium. On the contrary, contrast-enhanced computed tomographic studies have been performed widely for the diagnosis and evaluation of myocardial infarction. We report a case of complicated myocardial rupture after a single bolus injection of contrast medium during a computed tomographic study in an elderly woman with acute myocardial infarction, which led to cardiac tamponade and rapid death. Although rare, this should alert us to the need for cautious use of contrast medium in patients with acute myocardial infarction.

  17. Annealing macromolecular crystals.

    PubMed

    Hanson, B Leif; Bunick, Gerard J

    2007-01-01

    The process of crystal annealing has been used to improve the quality of diffraction from crystals that would otherwise be discarded for displaying unsatisfactory diffraction after flash cooling. Although techniques and protocols vary, macromolecular crystals are annealed by warming the flash-cooled crystal, then flash cooling it again. To apply macromolecular crystal annealing, a flash-cooled crystal displaying unacceptably high mosaicity or diffraction from ice is removed from the goniometer and immediately placed in cryoprotectant buffer. The crystal is incubated in the buffer at either room temperature or the temperature at which the crystal was grown. After about 3 min, the crystal is remounted in the loop and flash cooled. In situ annealing techniques, where the cold stream is diverted and the crystal allowed to warm on the loop prior to flash cooling, are variations of annealing that appears to work best when large solvent channels are not present in the crystal lattice or the solvent content of the crystal is relatively low.

  18. Investigations into the environmental fate and effects of iopromide (ultravist), a widely used iodinated X-ray contrast medium.

    PubMed

    Steger-Hartmann, Thomas; Länge, Reinhard; Schweinfurth, Hermann; Tschampel, Matthias; Rehmann, Irmgard

    2002-01-01

    lodinated X-ray contrast media are pharmaceuticals which are biologically inert and metabolically stable during their passage through the body and are excreted almost completely within a day into waste water. They are not readily biodegradable. However, in a test system simulating sewage treatment, we were able to show that the model compound iopromide (N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-methoxyacetylamino-N-methyliso-phthalamide) was amenable to primary degradation. The resulting degradation product (5-amino-N'N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-N-methyliso-phthalamide) showed a faster photolysis than the parent compound. Additionally this product was further degraded in a test system simulating surface water conditions. Short-term toxicity of the primary degradation product was low, i.e. no effects on any of various aquatic species could be found even at concentrations of 1 gl(-1). Additionally no chronic toxicity of the degradation product was observed in an early-life stage test with zebrafish at the highest tested concentration of 100mgl(-1). Based on the results from model systems a degradation pathway for iopromide is postulated. Though further work showing the transferability of the laboratory results to environmental conditions is necessary the presently available information on the environmental fate and effects of iopromide and its degradation products do not provide evidence of a risk for aquatic life caused by the introduction of this contrast medium into waste water.

  19. Dose perturbations due to contrast medium and air in MammoSite registered treatment: An experimental and Monte Carlo study

    SciTech Connect

    Cheng, C.-W.; Mitra, R.; Allen Li, X.; Das, Indra J.

    2005-07-15

    In the management of early breast cancer, a partial breast irradiation technique called MammoSite registered (Proxima Therapeutic Inc., Alpharetta, GA) has been advocated in recent years. In MammoSite, a balloon implanted at the surgical cavity during tumor excision is filled with a radio-opaque solution, and radiation is delivered via a high dose rate brachytherapy source situated at the center of the balloon. Frequently air may be introduced during placement of the balloon and/or injection of the contrast solution into the balloon. The purpose of this work is to quantify as well as to understand dose perturbations due to the presence of a high-Z contrast medium and/or an air bubble with measurements and Monte Carlo calculations. In addition, the measured dose distribution is compared with that obtained from a commercial treatment planning system (Nucletron PLATO system). For a balloon diameter of 42 mm, the dose variation as a function of distance from the balloon surface is measured for various concentrations of a radio-opaque solution (in the range 5%-25% by volume) with a small volume parallel plate ion chamber and a micro-diode detector placed perpendicular to the balloon axis. Monte Carlo simulations are performed to provide a basic understanding of the interaction mechanism and the magnitude of dose perturbation at the interface near balloon surface. Our results show that the radio-opaque concentration produces dose perturbation up to 6%. The dose perturbation occurs mostly within the distances <1 mm from the balloon surface. The Plato system that does not include heterogeneity correction may be sufficient for dose planning at distances {>=}10 mm from the balloon surface for the iodine concentrations used in the MammoSite procedures. The dose enhancement effect near the balloon surface (<1 mm) due to the higher iodine concentration is not correctly predicted by the Plato system. The dose near the balloon surface may be increased by 0.5% per cm{sup 3} of air

  20. Macromolecular crystal growing system

    NASA Technical Reports Server (NTRS)

    Snyder, Robert S. (Inventor); Herren, Blair J. (Inventor); Carter, Daniel C. (Inventor); Yost, Vaughn H. (Inventor); Bugg, Charles E. (Inventor); Delucas, Lawrence J. (Inventor); Suddath, Fred L. (Inventor)

    1991-01-01

    A macromolecular crystal growing system especially designed for growing crystals in the low gravity of space as well as the gravity of earth includes at least one tray assembly, a carrier assembly which receives the tray, and a refrigeration-incubation module in which the carrier assembly is received. The tray assembly includes a plurality of sealed chambers with a plastic syringe and a plug means for the double tip of the syringe provided therein. Ganging mechanisms operate the syringes and plugs simultaneously in a precise and smooth operation. Preferably, the tray assemblies are mounted on ball bearing slides for smooth operation in inserting and removing the tray assemblies into the carrier assembly. The plugging mechanism also includes a loading control mechanism. A mechanism for leaving a syringe unplugged is also provided.

  1. SU-C-12A-03: The Impact of Contrast Medium On Radiation Dose in CT: A Systematic Evaluation Across 58 Patient Models

    SciTech Connect

    Sahbaee, P; Samei, E; Segars, W

    2014-06-01

    Purpose: To assess the effect of contrast medium on radiation dose as a function of time via Monte Carlo simulation from the liver CT scan across a library of 5D XCAT models Methods: A validated Monte Carlo simulation package (PENELOPE) was employed to model a CT system (LightSpeed 64 VCT, GE Healthcare). The radiation dose was estimated from a common abdomen CT examination. The dose estimation was performed on a library of adult extended cardiac-torso (5D XCAT) phantoms (35 male, 23 female, mean age 51.5 years, mean weight 80.2 kg). The 5D XCAT models were created based on patient-specific iodine concentration-time results from our computational contrast medium propagation model for different intravenous injection protocols. To enable a dynamic estimation of radiation dose, each organ in the model was assigned to its own time-concentration curve via the PENELOPE package, material.exe. Using the Monte Carlo, for each scan time point after the injection, 80 million photons were initiated and tracked through the phantoms. Finally, the dose to the liver was tallied from the deposited energy. Results: Monte Carlo simulation results of radiation dose delivered to the liver from the XCAT models indicated up to 30% increase in dose for different time after the administration of contrast medium. Conclusion: The contrast enhancement is employed in over 60% of imaging modalities, which not only remarkably affects the CT image quality, but also increases the radiation dose by as much as 70%. The postinjection multiple acquisition in several enhanced CT protocols, makes the radiation dose increment through the use of contrast medium, an inevitable factor in optimization of these protocols. The relationship between radiation dose and injected contrast medium as a function of time studied in this work allows optimization of contrast administration for vulnerable individuals.

  2. Evaluation of the Dark-Medium Objective Lens in Counting Asbestos Fibers by Phase-Contrast Microscopy

    PubMed Central

    Lee, Eun Gyung; Nelson, John H.; Kashon, Michael L.; Harper, Martin

    2015-01-01

    A Japanese round-robin study revealed that analysts who used a dark-medium (DM) objective lens reported higher fiber counts from American Industrial Hygiene Association (AIHA) Proficiency Analytical Testing (PAT) chrysotile samples than those using a standard objective lens, but the cause of this difference was not investigated at that time. The purpose of this study is to determine any major source of this difference by performing two sets of round-robin studies. For the first round-robin study, 15 AIHA PAT samples (five each of chrysotile and amosite generated by water-suspended method, and five chrysotile generated by aerosolization method) were prepared with relocatable cover slips and examined by nine laboratories. A second round-robin study was then performed with six chrysotile field sample slides by six out of nine laboratories who participated in the first round-robin study. In addition, two phase-shift test slides to check analysts’ visibility and an eight-form diatom test plate to compare resolution between the two objectives were examined. For the AIHA PAT chrysotile reference slides, use of the DM objective resulted in consistently higher fiber counts (1.45 times for all data) than the standard objective (P-value < 0.05), regardless of the filter generation (water-suspension or aerosol) method. For the AIHA PAT amosite reference and chrysotile field sample slides, the fiber counts between the two objectives were not significantly different. No statistically significant differences were observed in the visibility of blocks of the test slides between the two objectives. Also, the DM and standard objectives showed no pattern of differences in viewing the fine lines and/or dots of each species images on the eight-form diatom test plate. Among various potential factors that might affect the analysts’ performance of fiber counts, this study supports the greater contrast caused by the different phase plate absorptions as the main cause of high counts for

  3. Evaluation of the dark-medium objective lens in counting asbestos fibers by phase-contrast microscopy.

    PubMed

    Lee, Eun Gyung; Nelson, John H; Kashon, Michael L; Harper, Martin

    2015-06-01

    A Japanese round-robin study revealed that analysts who used a dark-medium (DM) objective lens reported higher fiber counts from American Industrial Hygiene Association (AIHA) Proficiency Analytical Testing (PAT) chrysotile samples than those using a standard objective lens, but the cause of this difference was not investigated at that time. The purpose of this study is to determine any major source of this difference by performing two sets of round-robin studies. For the first round-robin study, 15 AIHA PAT samples (five each of chrysotile and amosite generated by water-suspended method, and five chrysotile generated by aerosolization method) were prepared with relocatable cover slips and examined by nine laboratories. A second round-robin study was then performed with six chrysotile field sample slides by six out of nine laboratories who participated in the first round-robin study. In addition, two phase-shift test slides to check analysts' visibility and an eight-form diatom test plate to compare resolution between the two objectives were examined. For the AIHA PAT chrysotile reference slides, use of the DM objective resulted in consistently higher fiber counts (1.45 times for all data) than the standard objective (P-value < 0.05), regardless of the filter generation (water-suspension or aerosol) method. For the AIHA PAT amosite reference and chrysotile field sample slides, the fiber counts between the two objectives were not significantly different. No statistically significant differences were observed in the visibility of blocks of the test slides between the two objectives. Also, the DM and standard objectives showed no pattern of differences in viewing the fine lines and/or dots of each species images on the eight-form diatom test plate. Among various potential factors that might affect the analysts' performance of fiber counts, this study supports the greater contrast caused by the different phase plate absorptions as the main cause of high counts for the

  4. LASER APPLICATIONS AND OTHER TOPICS IN QUANTUM ELECTRONICS: Improvement of the optical imaging of objects in a strongly scattering medium by means of contrast-enhancing dyes

    NASA Astrophysics Data System (ADS)

    Vorob'ev, Nikolai S.; Podgaetskii, Vitalii M.; Smirnov, A. V.; Tereshchenko, Sergei A.; Tomilova, Larisa G.

    1999-12-01

    The problem of enhancing the contrast of optical images in a strongly scattering medium by means of luminescent and absorbing dyes, topical in laser tomography, is examined. Preparations based on diphthalocyanine compounds were selected on the grounds of their tropism and resistance to the action of heat and light. Images with enhanced contrast in model scattering media (an aqueous solution of milk and margarine) were obtained in the IR region of the spectrum using the radiation of a picosecond neodymium laser.

  5. Solvent isotope effect on macromolecular dynamics in E. coli.

    PubMed

    Jasnin, Marion; Tehei, Moeava; Moulin, Martine; Haertlein, Michael; Zaccai, Giuseppe

    2008-06-01

    Elastic incoherent neutron scattering was used to explore solvent isotope effects on average macromolecular dynamics in vivo. Measurements were performed on living E. coli bacteria containing H2O and D2O, respectively, close to physiological conditions of temperature. Global macromolecular flexibility, expressed as mean square fluctuation (MSF) values, and structural resilience in a free energy potential, expressed as a mean effective force constant, [Symbol: see text]k'[Symbol: see text], were extracted in the two solvent conditions. They referred to the average contribution of all macromolecules inside the cell, mostly dominated by the internal motions of the protein fraction. Flexibility and resilience were both found to be smaller in D2O than in H2O. A difference was expected because the driving forces behind macromolecular stabilization and dynamics are different in H2O and D2O. In D2O, the hydrophobic effect is known to be stronger than in H2O: it favours the burial of non-polar surfaces as well as their van der Waals' packing in the macromolecule cores. This may lead to the observed smaller MSF values. In contrast, in H2O, macromolecules would present more water-exposed surfaces, which would give rise to larger MSF values, in particular at the macromolecular surface. The smaller [Symbol: see text]k'[Symbol: see text] value suggested a larger entropy content in the D2O case due to increased sampling of macromolecular conformational substates.

  6. Contrast Medium Exposure During Computed Tomography and Risk of Development of End-Stage Renal Disease in Patients With Chronic Kidney Disease

    PubMed Central

    Hsieh, Ming-Shun; Chiu, Chien-Shan; How, Chorng-Kuang; Chiang, Jen-Huai; Sheu, Meei-Ling; Chen, Wen-Chi; Lin, Hsuan-Jen; Hsieh, Vivian Chia-Rong; Hu, Sung-Yuan

    2016-01-01

    Abstract The aim of the study was to investigate the long-term association between contrast medium exposure during computed tomography (CT) and the subsequent development of end-stage renal disease (ESRD) in patients with chronic kidney disease (CKD). We conducted a population-based cohort study using Taiwan's National Health Insurance Research Database. A total of 7100 patients with nonadvanced CKD who underwent contrast medium-enhanced CT were identified and served as the study cohort. To avoid selection bias, we used the propensity score to match 7100 nonadvanced CKD patients, who underwent noncontrast medium-enhanced CT to serve as the comparison cohort. The age, sex, index year, and frequency of undergoing CTs were also matched between the study and comparison cohorts. Participants were followed until a new diagnosis of ESRD or December 31, 2011. Hazard ratios (HRs) with 95% confidence interval (95% CI) were calculated using the Cox proportional hazards regression. Contrast medium exposure was not identified as a risk factor for developing ESRD in nonadvanced CKD patients after confounders adjustment (adjusted HR = 0.91; 95% CI, 0.66–1.26; P = 0.580). We further divided the patients who underwent CTs with contrast medium use into ≤1 exposure per year on average, >1 and <2 exposure per year on average, and ≥2 exposure per year on average. After adjusting for confounders, we identified a much higher risk for developing ESRD in the 2 groups of >1 and <2 exposure per year on average and ≥2 exposure per year on average (adjusted HR = 8.13; 95% CI, 5.57–11.87 and adjusted HR = 12.08; 95% CI, 7.39–19.75, respectively) compared with the patients who underwent CTs without contrast medium use. This long-term follow-up study demonstrated that contrast medium exposure was not associated with an increased risk of ESRD development in nonadvanced CKD patients. PMID:27100424

  7. MO-E-17A-02: Incorporation of Contrast Medium Dynamics in Anthropomorphic Phantoms: The Advent of 5D XCAT Models

    SciTech Connect

    Sahbaee, P; Samei, E; Segars, W

    2014-06-15

    Purpose: To develop a unique method to incorporate the dynamics of contrast-medium propagation into the anthropomorphic phantom, to generate a five-dimensional (5D) patient model for multimodality imaging studies. Methods: A compartmental model of blood circulation network within the body was embodied into an extended cardiac-torso (4D-XCAT) patient model. To do so, a computational physiologic model of the human cardiovascular system was developed which includes a series of compartments representing heart, vessels, and organs. Patient-specific cardiac output and blood volume were used as inputs influenced by the weight, height, age, and gender of the patient's model. For a given injection protocol and given XCAT model, the contrast-medium transmission within the body was described by a series of mass balance differential equations, the solutions to which provided the contrast enhancement-time curves for each organ; thereby defining the tissue materials including the contrastmedium within the XCAT model. A library of time-dependent organ materials was then defined. Each organ in each voxelized 4D-XCAT phantom was assigned to a corresponding time-varying material to create the 5D-XCAT phantom in which the fifth dimension is blood/contrast-medium within the temporal domain. Results: The model effectively predicts the time-varying concentration behavior of various contrast-medium administration in each organ for different patient models as function of patient size (weight/height) and different injection protocol factors (injection rate and pattern, iodine concentration or volume). The contrast enhanced XCAT patient models was developed based on the concentration of iodine as a function of time after injection. Conclusion: Majority of medical imaging systems take advantage of contrast-medium administration in terms of better image quality, the effect of which was ignored in previous optimization studies. The study enables a comprehensive optimization of contrast

  8. [CT evaluation of extravascular perfusion of contrast medium and its potential to a new method of diagnosis: an experimental study using macro, micro-molecular contrast media].

    PubMed

    Sako, M; Sugimoto, K; Matsumoto, S; Hirota, S; Fujita, Y; Hasegawa, Y; Kuwata, Y; Tomita, M; Murakami, T; Kono, M

    1994-03-25

    To evaluate the dynamics of extravascular perfusion, dynamic CT with two different molecular sized contrast media was performed on VX2 tumor of rabbit. The first dynamic CT was performed with a bolus injection of iopamidol (IP:120 mgI/ml, 5 ml). After ascertaining that the tumor attenuation had returned to the pre-contrast level, the second dynamic CT was performed on the same slice with bolus injection of iodoethylated starch (IES:120 mgI/ml). The time-density (T-D) curves of the same tumor area on the images obtained by two contrast media were compared. The T-D curve with IP showed definitely higher level than that with IES. This occurrence can be explained that IP, 13 A in size, has higher permeability distributing not only in the intravascular space, but also into the extravascular space. On the other hand, IES, 200 A in size, will stay mostly in the intravascular space. From this, we consider that the attenuation difference between the two curves will be an indicator for the dynamics of extravascular perfusion, suggesting to become a new method for CT diagnosis.

  9. Transmucosal macromolecular drug delivery.

    PubMed

    Prego, C; García, M; Torres, D; Alonso, M J

    2005-01-03

    Mucosal surfaces are the most common and convenient routes for delivering drugs to the body. However, macromolecular drugs such as peptides and proteins are unable to overcome the mucosal barriers and/or are degraded before reaching the blood stream. Among the approaches explored so far in order to optimize the transport of these macromolecules across mucosal barriers, the use of nanoparticulate carriers represents a challenging but promising strategy. The present paper aims to compare the characteristics and potential of nanostructures based on the mucoadhesive polysaccharide chitosan (CS). These are CS nanoparticles, CS-coated oil nanodroplets (nanocapsules) and CS-coated lipid nanoparticles. The characteristics and behavior of CS nanoparticles and CS-coated lipid nanoparticles already reported [A. Vila, A. Sanchez, M. Tobio, P. Calvo, M.J. Alonso, Design of biodegradable particles for protein delivery, J. Control. Rel. 78 (2002) 15-24; R. Fernandez-Urrusuno, P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res. 16 (1999) 1576-1581; M. Garcia-Fuentes, D. Torres, M.J. Alonso, New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin (submitted for publication).] are compared with those of CS nanocapsules originally reported here. The three types of systems have a size in the nanometer range and a positive zeta potential that was attributed to the presence of CS on their surface. They showed an important capacity for the association of peptides such as insulin, salmon calcitonin and proteins, such as tetanus toxoid. Their mechanism of interaction with epithelia was investigated using the Caco-2 model cell line. The results showed that CS-coated systems caused a concentration-dependent reduction in the transepithelial resistance of the cell monolayer. Moreover, within the range of concentrations investigated, these systems were internalized in the

  10. [Significance of a nonionic renographic contrast medium (Iopamidol 300) in the roentgen diagnosis of the kidneys and urinary tract in children].

    PubMed

    Schneider, K; Fendel, H

    1984-10-01

    The ionic contrast media used so far have been associated with considerable risks in infants and children. The high osmolality of these media did not always permit a dosage sufficient for kidney imaging in the nephrographic and in the pyelographic phase. The new non-ionic contrast media have largely reduced these risks so that their general application in infants and young children should be recommended. Intravenous urographies using the non-ionic contrast medium Iopamidol 300 (Solutrast 300) were performed in 195 children aged one day to 12 years. Good to superior urograms were obtained in 85% of the investigations. No adverse reactions caused by the osmolality of the contrast media had been observed in spite of the relative high dosage. As a result of their low osmolality and non-ionic property a safe application of high doses was possible.

  11. The influence of buoyancy contrasts on miscible source sink flows in a porous medium with thermal inertia

    NASA Astrophysics Data System (ADS)

    Nigam, Mats S.; Woods, Andrew W.

    We investigate the displacement of one fluid through an inclined porous sheet by the injection of a second fluid of different density. Using numerical simulation we explore the role of the density contrast between the injected and the reservoir fluid on the displacement process, in the cases where the density contrast originates from either compositional contrasts and/or temperature contrasts between the fluids. In the case where the density contrast originates from compositional differences between the fluids, the density front moves with the fluid fluid front, and gravity may accelerate or decelerate the time for the injected liquid to reach the sink. In the case where the density contrast originates from a temperature contrast between the injected fluid and the reservoir fluid, then the density front follows the thermal front. Therefore, owing to thermal inertia, it lags behind the fluid fluid front. This has a quantitative impact on the time required for the injected liquid to reach the sink. If there are both thermal and compositional contrasts between the injected and reservoir fluid, then the thermal and compositional fronts become decoupled in space. The two fronts may lead to complementary or opposing density changes; the different cases lead to vastly different patterns of displacement and time at which the injected liquid reaches the sink, even if the net change in density between reservoir and the injected fluid is the same. We discuss the implications of these phenomena for water injection in sub-surface hydrocarbon and geothermal reservoirs. In an Appendix, we note how a viscosity across both the thermal front and the fluid fluid front can also lead to a rich spectrum of flow patterns, especially if one front is stable and the other unstable to viscous instability.

  12. Medium-dependent control of the bacterial growth rate.

    PubMed

    Ehrenberg, Måns; Bremer, Hans; Dennis, Patrick P

    2013-04-01

    By combining results from previous studies of nutritional up-shifts we here re-investigate how bacteria adapt to different nutritional environments by adjusting their macromolecular composition for optimal growth. We demonstrate that, in contrast to a commonly held view the macromolecular composition of bacteria does not depend on the growth rate as an independent variable, but on three factors: (i) the genetic background (i.e. the strain used), (ii) the physiological history of the bacteria used for inoculation of a given growth medium, and (iii) the kind of nutrients in the growth medium. These factors determine the ribosome concentration and the average rate of protein synthesis per ribosome, and thus the growth rate. Immediately after a nutritional up-shift, the average number of ribosomes in the bacterial population increases exponentially with time at a rate which eventually is attained as the final post-shift growth rate of all cell components. After a nutritional up-shift from one minimal medium to another minimal medium of higher nutritional quality, ribosome and RNA polymerase syntheses are co-regulated and immediately increase by the same factor equal to the increase in the final growth rate. However, after an up-shift from a minimal medium to a medium containing all 20 amino acids, RNA polymerase and ribosome syntheses are no longer coregulated; a smaller rate of synthesis of RNA polymerase is compensated by a gradual increase in the fraction of free RNA polymerase, possibly due to a gradual saturation of mRNA promoters. We have also analyzed data from a recent publication, in which it was concluded that the macromolecular composition in terms of RNA/protein and RNA/DNA ratios is solely determined by the effector molecule ppGpp. Our analysis indicates that this is true only in special cases and that, in general, medium adaptation also depends on factors other than ppGpp.

  13. Radiation damage in macromolecular cryocrystallography.

    PubMed

    Ravelli, Raimond B G; Garman, Elspeth F

    2006-10-01

    X-ray radiation damage to cryocooled ( approximately 100 K) macromolecular crystals has emerged as a general problem, especially since the advent of third generation synchrotron undulator sources. Interest in understanding the physical and chemical phenomena behind the observed effects is growing rapidly. The specific structural damage seen in electron density maps has to be accounted for when studying intermediates, and can sometimes be related to biological function. Radiation damage induces non-isomorphism, thus hampering traditional phasing methods. However, specific damage can also be used to obtain phases. With an increased knowledge of expected crystal lifetime, beamline characteristics and types of damage, macromolecular crystallographers might soon be able to account for radiation damage in data collection, processing and phasing.

  14. Computed tomography pulmonary angiography using a 20% reduction in contrast medium dose delivered in a multiphasic injection

    PubMed Central

    Chen, Mitchell; Mattar, Gaith; Abdulkarim, Jamal A

    2017-01-01

    AIM To evaluate the feasibility of reducing the dose of iodinated contrast agent in computed tomography pulmonary angiography (CTPA). METHODS One hundred and twenty-seven patients clinically suspected of having pulmonary embolism underwent spiral CTPA, out of whom fifty-seven received 75 mL and the remaining seventy a lower dose of 60 mL of contrast agent. Both doses were administered in a multiphasic injection. A minimum opacification threshold of 250 Hounsfield units (HU) in the main pulmonary artery is used for assessing the technical adequacy of the scans. RESULTS Mean opacification was found to be positively correlated to patient age (Pearson’s correlation 0.4255, P < 0.0001) and independent of gender (male:female, 425.6 vs 450.4, P = 0.34). When age is accounted for, the study and control groups did not differ significantly in their mean opacification in the main (436.8 vs 437.9, P = 0.48), left (416.6 vs 419.8, P = 0.45) or the right pulmonary arteries (417.3 vs 423.5, P = 0.40). The number of sub-optimally opacified scans (the mean opacification in the main pulmonary artery < 250 HU) did not differ significantly between the study and control groups (7 vs 10). CONCLUSION A lower dose of iodine contrast at 60 mL can be feasibly used in CTPA without resulting in a higher number of sub-optimally opacified scans.

  15. Barium sulfate aspiration: Severe chemical pneumonia induced by a massive reflux of contrast medium during small bowel barium enema.

    PubMed

    Zhang, Lin; Yang, Yi; Zhang, Ji; Zhou, Xiaowei; Dong, Hongmei; Zhou, Yiwu

    2015-08-01

    Barium contrast radiography is a conventional procedure aimed at revealing lesions of the alimentary tract using barium sulfate on X-ray irradiation. Although it is widely used in clinics, adverse effects and complications are observed, such as anaphylaxis, granuloma, fecalithes, abdomen-leaking, embolism, bacterial contamination, and aspiration. We report a case of death due to a massive barium sulfate aspiration resulted from an air-barium double contrast enema radiography. A 25-year-old female patient was hospitalized with symptoms of abdominal distention, nausea, vomiting and diarrhea for three days. A progressive respiratory distress presented only 1h after a small bowel air-barium double contrast enema. The patient died 11h later. The result of autopsy revealed the cause of death to be severe chemical pneumonitis induced by gastric fluid which was aspirated into her lungs. Barium sulfate is generally recognized to be chemically inert for the respiratory system, but a mixture of barium sulfate with gastric contents is fatal. Here we intend to suggest that, when determining the potential cause of death, medical examiners should consider a patient's status quo as well as the possible adverse effects and complications caused by the barium sulfate preparation during gastrointestinal radiography.

  16. The growth of filaments under macromolecular confinement using scaling theory.

    PubMed

    Zhu, Lin; Pan, Wei; Lu, Xi; Li, Desheng; Zhao, Jiang; Liang, Dehai

    2015-11-14

    Quantitatively describing macromolecular confinement is still a challenge. Using the assembly of DNA tiles in a polyacrylamide network as a model, we studied the effect of macromolecular confinement on the growth of the filament by scaling theory. The results show that the confinement regulates the morphology, the initial growth rate v, and the eventual length of the filament Nm. The initial growth rate is dependent on the medium viscosity η as ν∝η(-0.94), and the filament adjusts its length in the given confined space as Nm∝ (ξ/Rg)(1.8), with ξ being the mesh size of the polyacrylamide solution and Rg being the radius of gyration of polyacrylamide.

  17. Myelography in achondroplasia: value of a lateral C1-2 puncture and non-ionic, water-soluble contrast medium

    SciTech Connect

    Suss, R.A.; Udvarhelyi, G.B.; Wang, H.; Kumar, A.J.; Zinreich, S.J.; Rosenbaum, A.E.

    1983-10-01

    Because of technical difficulties and diagnostic limitations encountered with other myelographic techniques in patients with achondroplasia, the authors employed a lateral C1-2 puncture and non-ionic, water-soluble contrast medium in 18 achondroplastic patients with spinal compression (21 procedures). This technique proved most appropriate for identifying the upper limit of degenerative osteophytes causing exacerbation of congenital spinal stenosis, which is crucial in planning decompressive surgery. A potentially important additional finding was the presence of degenerative lower cervical spine disease in the majority of patients. There were no serious complications. The authors recommend this technique as safe and effective in achondroplastic patients with severe congenital spinal stenosis.

  18. Electromagnetic induction in a conductive strip in a medium of contrasting conductivity: application to VLF and MT above molten dykes

    NASA Astrophysics Data System (ADS)

    Davis, Paul M.

    2014-11-01

    Very low frequency (VLF) electromagnetic waves that penetrate conductive magma-filled dykes generate secondary fields on the surface that can be used to invert for dyke properties. The model used for the interpretation calculates currents induced in a conductive strip by an inducing field that decays exponentially with depth due to the conductivity of the surrounding medium. The differential equations are integrated to give an inhomogeneous Fredholm equation of the second kind with a kernel consisting of a modified Bessel function of the second kind. Numerical methods are typically used to solve for the induced currents in the strip. In this paper, we apply a modified Galerkin-Chebyshev method, which involves separating the kernel into source and field spectra and integrating the source terms to obtain a matrix equation for the unknown coefficients. The incident wave is expressed as a Chebyshev series. The modified Bessel function is separated into a logarithmic singularity and a non-singular remainder, both of which are expanded in complex Chebyshev polynomials. The Chebyshev coefficients for the remainder are evaluated using a fast Fourier transform, while the logarithmic term and incident field have analytic series. The deconvolution then involves a matrix inversion. The results depend on the ratio of strip-size to skin-depth. For infinite skin-depth and a singular conductivity distribution given by τ_0 a/√{a^2 - z^2 } (where τ0 is the conductance, a is the half-length and z the distance from the centre), Parker gives an analytic solution. We present a similar analytic series solution for the finite skin-depth case, where the size to skin depth ratio is small. Results are presented for different ratios of size to skin depth that can be compared with numerical solutions. We compare full-space and half-space solutions. A fit of the model to VLF data taken above a magma filled dykes in Hawaii and Mt Etna demonstrates that while properties such as depth to top

  19. Macromolecular Diffusion in Polymer Nanocomposites

    NASA Astrophysics Data System (ADS)

    Gam, Sangah; Meth, Jeff; Zane, Steve; Winey, Karen; Clarke, Nigel; Composto, Russell

    2011-03-01

    Macromolecular diffusion in crowded systems is important in biological and engineered systems. We have studied macromolecular diffusion through a model polymer nanocomposite (PNC) containing phenyl grafted silica nanoparticles (NPs), randomly distributed in a polystyrene matrix. Over a wide range of NP loading and tracer molecular weight (M), the scaling of the diffusion coefficient with M is in excellent agreement with the entropic barrier model (EBM) previously used to describe diffusion of DNA through confined media (e.g., gels and nanopores). To investigate the effect of NP size, diffusion was measured in PNC's with silica NPs having diameters of 28 and 12 nm. The normalized diffusion coefficients (D / D0) plotted against the interparticle separation relative to probe size (i.e., ID/ 2 Rg) collapse on a master curve. Diffusion in a poly(methyl methacrylate):silica NP system was also investigated to understand how attractive interactions (i.e., enthalpy) perturb motion relative to the polystyrene and phenyl-silica NP system which is athermal. Finally, a flux-based model is proposed and compared with experimental results.

  20. Data Mining of Macromolecular Structures.

    PubMed

    van Beusekom, Bart; Perrakis, Anastassis; Joosten, Robbie P

    2016-01-01

    The use of macromolecular structures is widespread for a variety of applications, from teaching protein structure principles all the way to ligand optimization in drug development. Applying data mining techniques on these experimentally determined structures requires a highly uniform, standardized structural data source. The Protein Data Bank (PDB) has evolved over the years toward becoming the standard resource for macromolecular structures. However, the process selecting the data most suitable for specific applications is still very much based on personal preferences and understanding of the experimental techniques used to obtain these models. In this chapter, we will first explain the challenges with data standardization, annotation, and uniformity in the PDB entries determined by X-ray crystallography. We then discuss the specific effect that crystallographic data quality and model optimization methods have on structural models and how validation tools can be used to make informed choices. We also discuss specific advantages of using the PDB_REDO databank as a resource for structural data. Finally, we will provide guidelines on how to select the most suitable protein structure models for detailed analysis and how to select a set of structure models suitable for data mining.

  1. Visual automated macromolecular model building.

    PubMed

    Langer, Gerrit G; Hazledine, Saul; Wiegels, Tim; Carolan, Ciaran; Lamzin, Victor S

    2013-04-01

    Automated model-building software aims at the objective interpretation of crystallographic diffraction data by means of the construction or completion of macromolecular models. Automated methods have rapidly gained in popularity as they are easy to use and generate reproducible and consistent results. However, the process of model building has become increasingly hidden and the user is often left to decide on how to proceed further with little feedback on what has preceded the output of the built model. Here, ArpNavigator, a molecular viewer tightly integrated into the ARP/wARP automated model-building package, is presented that directly controls model building and displays the evolving output in real time in order to make the procedure transparent to the user.

  2. Phase Sensitive X-Ray Diffraction Imaging of Defects in Biological Macromolecular Crystals

    NASA Technical Reports Server (NTRS)

    Hu, Z. W.; Lai, B.; Chu, Y. S.; Cai, Z.; Mancini, D. C.; Thomas, B. R.; Chernov, A. A.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Characterization of defects and/or disorder in biological macromolecular crystals presents much greater challenges than in conventional small-molecule crystals. The lack of sufficient contrast of defects is often a limiting factor in x-ray diffraction topography of protein crystals. This has seriously hampered efforts to understand mechanisms and origins of formation of imperfections, and the role of defects as essential entities in the bulk of macromolecular crystals. In this report, we employ a phase sensitive x-ray diffraction imaging approach for augmenting the contrast of defects in protein crystals.

  3. Automated data collection for macromolecular crystallography.

    PubMed

    Winter, Graeme; McAuley, Katherine E

    2011-09-01

    An overview, together with some practical advice, is presented of the current status of the automation of macromolecular crystallography (MX) data collection, with a focus on MX beamlines at Diamond Light Source, UK.

  4. Ordered macromolecular structures in ferrofluid mixtures

    SciTech Connect

    Hayter, J.B.; Pynn, R.; Charles, S.; Skjeltorp, A.T.; Trewhella, J.; Stubbs, G.; Timmins, P.

    1989-04-03

    We have observed ordering of dilute dispersions of spherical and cylindrical macromolecules in magnetized ferrofluids. The order results from structural correlations between macromolecular and ferrofluid particles rather than from macroscopic magnetostatic effects. We have aligned elongated macromolecules by this technique and have obtained anisotropic neutron-diffraction patterns, which reflect the internal structure of the macromolecules. The method provides a tool for orienting suspended macromolecular assemblies which are not amenable to conventional alignment techniques.

  5. A database of macromolecular motions.

    PubMed Central

    Gerstein, M; Krebs, W

    1998-01-01

    We describe a database of macromolecular motions meant to be of general use to the structural community. The database, which is accessible on the World Wide Web with an entry point at http://bioinfo.mbb.yale.edu/MolMovDB , attempts to systematize all instances of protein and nucleic acid movement for which there is at least some structural information. At present it contains >120 motions, most of which are of proteins. Protein motions are further classified hierarchically into a limited number of categories, first on the basis of size (distinguishing between fragment, domain and subunit motions) and then on the basis of packing. Our packing classification divides motions into various categories (shear, hinge, other) depending on whether or not they involve sliding over a continuously maintained and tightly packed interface. In addition, the database provides some indication about the evidence behind each motion (i.e. the type of experimental information or whether the motion is inferred based on structural similarity) and attempts to describe many aspects of a motion in terms of a standardized nomenclature (e.g. the maximum rotation, the residue selection of a fixed core, etc.). Currently, we use a standard relational design to implement the database. However, the complexity and heterogeneity of the information kept in the database makes it an ideal application for an object-relational approach, and we are moving it in this direction. Specifically, in terms of storing complex information, the database contains plausible representations for motion pathways, derived from restrained 3D interpolation between known endpoint conformations. These pathways can be viewed in a variety of movie formats, and the database is associated with a server that can automatically generate these movies from submitted coordinates. PMID:9722650

  6. Molecular Control of Macromolecular Properties

    NASA Astrophysics Data System (ADS)

    Holcombe, Thomas Wesley, III

    Molecular level control over macromolecules has been at the heart of human advancement, long before Hermann Staudinger coined the term Makromolekule. From the development of primitive pharmaceuticals to the advanced materials that sent Man into outer-space, We have been tinkering with God's paint since our inception. The work described herein primarily involves advances concerning poly-aromatic macromolecules for use in future electronic applications, particularly that of organic photovoltaics. There is a final chapter, however, that gives the reader a taste of how some molecular level changes can be directly visualized with modern microscopy techniques. Chapter 1 provides a very brief introduction to conjugated polymers and molecular level control over macromolecular properties. Chapters 2--4 introduces the concept of polymer substitution as a means by which to control and improve charge generation in organic photovoltaic devices. Chapters 5 and 6 show how these polymers can take on larger, defined structures, yet are still beholden to intrinsic molecular properties---such as regioregularity, a fancy word for the regularity of the position in which two aromatic rings are joined together. Chapter 7 re-examines the role of polymer substitution on photovoltaic performance, this time with an emphasis on homo-polymer packing rather than electron transfer at the donor/acceptor interface. Finally, Chapter 8 visualizes how controlling the environment about a single metal atom can lead directly to a cyclic polyolefin. Individually, these advances do not yield any breakthroughs noticeable to a general audience; collectively, they sit atop a mountain of human knowledge, waiting to provide a stepping stone for the next generation.

  7. Effects of macromolecular crowding on genetic networks.

    PubMed

    Morelli, Marco J; Allen, Rosalind J; Wolde, Pieter Rein ten

    2011-12-21

    The intracellular environment is crowded with proteins, DNA, and other macromolecules. Under physiological conditions, macromolecular crowding can alter both molecular diffusion and the equilibria of bimolecular reactions and therefore is likely to have a significant effect on the function of biochemical networks. We propose a simple way to model the effects of macromolecular crowding on biochemical networks via an appropriate scaling of bimolecular association and dissociation rates. We use this approach, in combination with kinetic Monte Carlo simulations, to analyze the effects of crowding on a constitutively expressed gene, a repressed gene, and a model for the bacteriophage λ genetic switch, in the presence and absence of nonspecific binding of transcription factors to genomic DNA. Our results show that the effects of crowding are mainly caused by the shift of association-dissociation equilibria rather than the slowing down of protein diffusion, and that macromolecular crowding can have relevant and counterintuitive effects on biochemical network performance.

  8. Protein stabilization by macromolecular crowding through enthalpy rather than entropy.

    PubMed

    Senske, Michael; Törk, Lisa; Born, Benjamin; Havenith, Martina; Herrmann, Christian; Ebbinghaus, Simon

    2014-06-25

    The interior of the cell is a densely crowded environment in which protein stability is affected differently than in dilute solution. Macromolecular crowding is commonly understood in terms of an entropic volume exclusion effect based on hardcore repulsions among the macromolecules. We studied the thermal unfolding of ubiquitin in the presence of different cosolutes (glucose, dextran, poly(ethylene glycol), KCl, urea). Our results show that for a correct dissection of the cosolute-induced changes of the free energy into its enthalpic and entropic contributions, the temperature dependence of the heat capacity change needs to be explicitly taken into account. In contrast to the prediction by the excluded volume theory, we observed an enthalpic stabilization and an entropic destabilization for glucose, dextran, and poly(ethylene glycol). The enthalpic stabilization mechanism induced by the macromolecular crowder dextran was similar to the enthalpic stabilization mechanism of its monomeric building block glucose. In the case of poly(ethylene glycol), entropy is dominating over enthalpy leading to an overall destabilization. We propose a new model to classify cosolute effects in terms of their enthalpic contributions to protein stability.

  9. A comparison of ionic versus nonionic contrast medium during primary percutaneous transluminal coronary angioplasty for acute myocardial infarction (GUSTO IIb). Global Use of Strategies to Open Occluded Coronary Arteries in Acute Coronary Syndromes.

    PubMed

    Batchelor, W B; Granger, C B; Kleiman, N S; Phillips, H R; Ellis, S G; Betriu, A; Criger, D A; Stebbins, A L; Topol, E J; Califf, R M

    2000-03-15

    The clinical impact of contrast medium selection during primary percutaneous transluminal coronary angioplasty for acute myocardial infarction (AMI) has not been studied. We compared the clinical outcomes of patients who received ionic versus nonionic low osmolar contrast medium in the setting of primary percutaneous transluminal coronary angioplasty for AMI in the second Global Use of Strategies to Open Occluded Coronary Arteries in Acute Coronary Syndromes (GUSTO IIb) trial. Univariable and multivariable analyses were performed to assess the relation between contrast medium selection and clinical outcome (death, reinfarction, or refractory ischemia) at 30 days. Although baseline clinical and angiographic characteristics were generally similar between the 2 groups, patients who received ionic, low osmolar contrast were less likely to have been enrolled at a US site (23% vs 43%, p = 0.001) and less likely to have occlusion of the left anterior descending coronary artery (34% vs 47%, p = 0.03) or a history of prior AMI (8% vs 16%, p = 0.02). The triple composite end point of death, reinfarction, or refractory ischemia occurred less frequently in the ionic group, both in the hospital (4.4% vs 11%, p = 0.018) and at 30 days (5.5% vs 11%, p = 0.044). Although the trend favoring ionic contrast persisted, the differences were no longer statistically significant after adjustment for imbalances in baseline characteristics using a risk model developed from the study sample (n = 454, adjusted odds ratio for ionic contrast 0.48 [0.22 to 1.02], p = 0.055), and using a model developed from the entire GUSTO IIb study cohort (n = 12,142, adjusted odds ratio for ionic contrast 0.50 [0.23 to 1.06], p = 0.072). The results of this observational study warrant further elucidation by a randomized study design in this setting.

  10. In situ macromolecular crystallography using microbeams.

    PubMed

    Axford, Danny; Owen, Robin L; Aishima, Jun; Foadi, James; Morgan, Ann W; Robinson, James I; Nettleship, Joanne E; Owens, Raymond J; Moraes, Isabel; Fry, Elizabeth E; Grimes, Jonathan M; Harlos, Karl; Kotecha, Abhay; Ren, Jingshan; Sutton, Geoff; Walter, Thomas S; Stuart, David I; Evans, Gwyndaf

    2012-05-01

    Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.

  11. Selection and evolution in macromolecular systems.

    PubMed

    Demetrius, L

    1983-08-21

    The notion of a quasi-species represents the ensemble of macromolecular sequences derived by the mechanism of mutation and replication from a single wild type. In Eigen (1971) and Eigen & Schuster (1979), the deterministic evolution of this ensemble under constant environmental conditions is given in terms of continuous models which describe the dynamics of the distribution of polynucleotides. This paper starts from a discrete model of macromolecular evolution and introduces the notion of a genealogy in order to study the dynamics of the quasi-species in constant and variable environments. We introduce, in terms of these genealogies, the notions of entropy and adaptive value of a quasi-species and the notion of capacity of the environment. We discuss the significance of these indices as measures of selective value and we analyse the conditions under which these measures coincide with the growth rate of the quasi-species.

  12. Growth and Dissolution of Macromolecular Markov Chains

    NASA Astrophysics Data System (ADS)

    Gaspard, Pierre

    2016-07-01

    The kinetics and thermodynamics of free living copolymerization are studied for processes with rates depending on k monomeric units of the macromolecular chain behind the unit that is attached or detached. In this case, the sequence of monomeric units in the growing copolymer is a kth-order Markov chain. In the regime of steady growth, the statistical properties of the sequence are determined analytically in terms of the attachment and detachment rates. In this way, the mean growth velocity as well as the thermodynamic entropy production and the sequence disorder can be calculated systematically. These different properties are also investigated in the regime of depolymerization where the macromolecular chain is dissolved by the surrounding solution. In this regime, the entropy production is shown to satisfy Landauer's principle.

  13. The structural dynamics of macromolecular processes

    PubMed Central

    Russel, Daniel; Lasker, Keren; Phillips, Jeremy; Schneidman-Duhovny, Dina; Velázquez-Muriel, Javier A.; Sali, Andrej

    2009-01-01

    Summary Dynamic processes involving macromolecular complexes are essential to cell function. These processes take place over a wide variety of length scales from nanometers to micrometers, and over time scales from nanoseconds to many minutes. As a result, information from a variety of different experimental and computational approaches is required. We review the relevant sources of information and introduce a framework for integrating the data to produce representations of dynamic processes. PMID:19223165

  14. Macromolecular extraction based on contour evolution

    NASA Astrophysics Data System (ADS)

    Wang, Zhaobin; Guo, Miao; Zhu, Ying; Yang, Lizhen; Ma, Yi-de

    2013-03-01

    Detecting the region of interest plays an important role in the field of image processing and analysis. For the microscopic image of plant embryo slice, region of interest usually indicates various cells or macromolecules. Combining contour evolution theory and pulse coupled neural network, we propose a new method of macromolecular detection and extraction for biological microscopic image. Some existing methods are compared with the proposed method. Experimental results show the proposed method has the better performance than existing methods.

  15. Protein conformational studies for macromolecularly imprinted polymers.

    PubMed

    Kryscio, David R; Fleming, Michael Q; Peppas, Nicholas A

    2012-08-01

    CD is used to clearly show the negative impact of common ligands on the overall conformation of BSA, a typical protein template in macromolecularly imprinted polymers. This change occurs at concentrations far lower than those generally used in the literature. These findings are important as they offer insight into a potential fundamental reason for the lack of success in protein imprinting to date despite significant interest from the scientific community.

  16. Stochastic dynamics of macromolecular-assembly networks.

    NASA Astrophysics Data System (ADS)

    Saiz, Leonor; Vilar, Jose

    2006-03-01

    The formation and regulation of macromolecular complexes provides the backbone of most cellular processes, including gene regulation and signal transduction. The inherent complexity of assembling macromolecular structures makes current computational methods strongly limited for understanding how the physical interactions between cellular components give rise to systemic properties of cells. Here we present a stochastic approach to study the dynamics of networks formed by macromolecular complexes in terms of the molecular interactions of their components [1]. Exploiting key thermodynamic concepts, this approach makes it possible to both estimate reaction rates and incorporate the resulting assembly dynamics into the stochastic kinetics of cellular networks. As prototype systems, we consider the lac operon and phage λ induction switches, which rely on the formation of DNA loops by proteins [2] and on the integration of these protein-DNA complexes into intracellular networks. This cross-scale approach offers an effective starting point to move forward from network diagrams, such as those of protein-protein and DNA-protein interaction networks, to the actual dynamics of cellular processes. [1] L. Saiz and J.M.G. Vilar, submitted (2005). [2] J.M.G. Vilar and L. Saiz, Current Opinion in Genetics & Development, 15, 136-144 (2005).

  17. Surface oxidation of gold nanoparticles supported on a glassy carbon electrode in sulphuric acid medium: contrasts with the behaviour of 'macro' gold.

    PubMed

    Wang, Ying; Laborda, Eduardo; Crossley, Alison; Compton, Richard G

    2013-03-07

    Consecutive electro-oxidation and reduction cycling of gold macroelectrodes in sulphuric acid medium is a widely-used cleaning and calibration procedure. In this paper this method is applied to electrodeposited nanoparticles revealing significant differences in the electro-oxidation process and the cleaning effectiveness. This suggests a higher density of surface defects on the nanoparticles.

  18. Coal chemistry for mechanical engineers: from macromolecular thermodynamics to reservoir simulation

    SciTech Connect

    Vyacheslav Romanov

    2007-06-15

    In pilot trials and commercial scale field demonstrations of CO{sub 2} storage in coal seams, quite often unexpected problems with coal swelling around injector and reducing injection efficiency (e.g., Allison unit in the San Juan Basin, RECOPOL in Poland, Hokkaido project in Japan, etc.) can stall or even terminate the site development. To avoid the costly mistakes with the prospective site evaluation, the state of the art in reservoir modeling needs to be improved by taking into account coal properties at the macromolecular level. The current models are based on the rock mechanics, which ignores decades of experimental and theoretical studies of interaction between coal and injected fluids. A pseudopolymer approach is introduced to the modelers as a viable alternative, especially, at medium to high fluid pressures. Further, it is discussed how the thermodynamics of CO{sub 2} dissolution in the macromolecular network of the coal matrix can be incorporated into geomechanical models. 96 refs., 4 figs.

  19. Coal Chemistry for Mechanical Engineers: From Macromolecular Thermodynamics to Reservoir Simulation

    SciTech Connect

    Romanov, V.

    2007-05-01

    In pilot trials and commercial scale field demonstrations of CO2 storage in coal seams, quite often unexpected problems with coal swelling around injector and reducing injection efficiency (e.g., Allison unit in the San Juan Basin, RECOPOL in Poland, Hokkaido project in Japan, etc.) can stall or even terminate the site development. To avoid the costly mistakes with the prospective site evaluation, the state of the art in reservoir modeling needs to be improved by taking into account coal properties at the macromolecular level. The current models are based on the rock mechanics, which ignores decades of experimental and theoretical studies of interaction between coal and injected fluids. A pseudopolymer approach is introduced to the modelers as a viable alternative, especially, at medium to high fluid pressures. Further, it is discussed how the thermodynamics of CO2 dissolution in the macromolecular network of the coal matrix can be incorporated into geomechanical models.

  20. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    PubMed Central

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; Ravelli, Raimond B. G.; Carmichael, Ian; Kneale, Geoff; McGeehan, John E.

    2015-01-01

    Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses. PMID:25723923

  1. A public database of macromolecular diffraction experiments.

    PubMed

    Grabowski, Marek; Langner, Karol M; Cymborowski, Marcin; Porebski, Przemyslaw J; Sroka, Piotr; Zheng, Heping; Cooper, David R; Zimmerman, Matthew D; Elsliger, Marc André; Burley, Stephen K; Minor, Wladek

    2016-11-01

    The low reproducibility of published experimental results in many scientific disciplines has recently garnered negative attention in scientific journals and the general media. Public transparency, including the availability of `raw' experimental data, will help to address growing concerns regarding scientific integrity. Macromolecular X-ray crystallography has led the way in requiring the public dissemination of atomic coordinates and a wealth of experimental data, making the field one of the most reproducible in the biological sciences. However, there remains no mandate for public disclosure of the original diffraction data. The Integrated Resource for Reproducibility in Macromolecular Crystallography (IRRMC) has been developed to archive raw data from diffraction experiments and, equally importantly, to provide related metadata. Currently, the database of our resource contains data from 2920 macromolecular diffraction experiments (5767 data sets), accounting for around 3% of all depositions in the Protein Data Bank (PDB), with their corresponding partially curated metadata. IRRMC utilizes distributed storage implemented using a federated architecture of many independent storage servers, which provides both scalability and sustainability. The resource, which is accessible via the web portal at http://www.proteindiffraction.org, can be searched using various criteria. All data are available for unrestricted access and download. The resource serves as a proof of concept and demonstrates the feasibility of archiving raw diffraction data and associated metadata from X-ray crystallographic studies of biological macromolecules. The goal is to expand this resource and include data sets that failed to yield X-ray structures in order to facilitate collaborative efforts that will improve protein structure-determination methods and to ensure the availability of `orphan' data left behind for various reasons by individual investigators and/or extinct structural genomics

  2. Development of macromolecular prodrug for rheumatoid arthritis☆

    PubMed Central

    Yuan, Fang; Quan, Ling-dong; Cui, Liao; Goldring, Steven R.; Wang, Dong

    2012-01-01

    Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases. PMID:22433784

  3. Crystallization of macromolecular complexes:. stoichiometric variation screening

    NASA Astrophysics Data System (ADS)

    Stura, Enrico A.; Graille, Marc; Taussig, Michael J.; Sutton, Brian; Gore, Michael G.; Silverman, Gregg J.; Charbonnier, Jean-Baptiste

    2001-11-01

    Theoretically a crystal may contain both complexed and uncomplexed molecules simultaneously in the same lattice. Since we seldom screen for such possibilities, such occurrences are only rarely reported. Here we propose that stoichiometry should be one of the parameters to be screened in the crystallization of macromolecular complexes. By allowing for non-biologically significant stoichiometries, we may increase the chances of crystallizing a macromolecular complex and of selecting arrangements which crystallize better or yield more ordered crystals. Although biological forces tend to be stronger than lattice-building interactions, in the crystal the latter will dominate numerically. By allowing for a varied stoichiometry we permit a wider selection of lattice-building contacts and increase the probability of crystallization. From these theoretical considerations we have developed methodology compatible with classical solubility screening and other well-established crystallization principles. We discuss this technique, stoichiometric variation screening (SVS), as part of a multicomponent system for the enhancement of crystallization of macromolecular complexes. We present this technique as an extension of reverse screening and illustrate the complementarity in the methodology. We present two examples of the use of SVS: the complexes between an immunoglobulin Fab fragment and two bacterial proteins, namely the D domain of protein A from Staphylococcus aureus (SpA) and a single domain of protein L from Peptostreptococcus magnus (PpL). In the first example there are 3 Fab molecules and only 2 SpA D domains (domD) (2 complexed and 1 unliganded Fab), in the second 2 Fabs and only 1 PpL domain (1 complexed and 1 unliganded Fab). SVS has the added and unique advantage that in the same crystal we have information on both the unliganded and complexed states under precisely identical conditions: one structure, two answers. Together with a combinatorial method for complex

  4. Automated macromolecular crystal detection system and method

    DOEpatents

    Christian, Allen T.; Segelke, Brent; Rupp, Bernard; Toppani, Dominique

    2007-06-05

    An automated macromolecular method and system for detecting crystals in two-dimensional images, such as light microscopy images obtained from an array of crystallization screens. Edges are detected from the images by identifying local maxima of a phase congruency-based function associated with each image. The detected edges are segmented into discrete line segments, which are subsequently geometrically evaluated with respect to each other to identify any crystal-like qualities such as, for example, parallel lines, facing each other, similarity in length, and relative proximity. And from the evaluation a determination is made as to whether crystals are present in each image.

  5. Dominance hierarchies, diversity and species richness of vascular plants in an alpine meadow: contrasting short and medium term responses to simulated global change

    PubMed Central

    Little, Chelsea J.; Jägerbrand, Annika K.; Molau, Ulf

    2014-01-01

    We studied the impact of simulated global change on a high alpine meadow plant community. Specifically, we examined whether short-term (5 years) responses are good predictors for medium-term (7 years) changes in the system by applying a factorial warming and nutrient manipulation to 20 plots in Latnjajaure, subarctic Sweden. Seven years of experimental warming and nutrient enhancement caused dramatic shifts in dominance hierarchies in response to the nutrient and the combined warming and nutrient enhancement treatments. Dominance hierarchies in the meadow moved from a community being dominated by cushion plants, deciduous, and evergreen shrubs to a community being dominated by grasses, sedges, and forbs. Short-term responses were shown to be inconsistent in their ability to predict medium-term responses for most functional groups, however, grasses showed a consistent and very substantial increase in response to nutrient addition over the seven years. The non-linear responses over time point out the importance of longer-term studies with repeated measurements to be able to better predict future changes. Forecasted changes to temperature and nutrient availability have implications for trophic interactions, and may ultimately influence the access to and palatability of the forage for grazers. Depending on what anthropogenic change will be most pronounced in the future (increase in nutrient deposits, warming, or a combination of them both), different shifts in community dominance hierarchies may occur. Generally, this study supports the productivity–diversity relationship found across arctic habitats, with community diversity peaking in mid-productivity systems and degrading as nutrient availability increases further. This is likely due the increasing competition in plant–plant interactions and the shifting dominance structure with grasses taking over the experimental plots, suggesting that global change could have high costs to biodiversity in the Arctic. PMID

  6. Dominance hierarchies, diversity and species richness of vascular plants in an alpine meadow: contrasting short and medium term responses to simulated global change.

    PubMed

    Alatalo, Juha M; Little, Chelsea J; Jägerbrand, Annika K; Molau, Ulf

    2014-01-01

    We studied the impact of simulated global change on a high alpine meadow plant community. Specifically, we examined whether short-term (5 years) responses are good predictors for medium-term (7 years) changes in the system by applying a factorial warming and nutrient manipulation to 20 plots in Latnjajaure, subarctic Sweden. Seven years of experimental warming and nutrient enhancement caused dramatic shifts in dominance hierarchies in response to the nutrient and the combined warming and nutrient enhancement treatments. Dominance hierarchies in the meadow moved from a community being dominated by cushion plants, deciduous, and evergreen shrubs to a community being dominated by grasses, sedges, and forbs. Short-term responses were shown to be inconsistent in their ability to predict medium-term responses for most functional groups, however, grasses showed a consistent and very substantial increase in response to nutrient addition over the seven years. The non-linear responses over time point out the importance of longer-term studies with repeated measurements to be able to better predict future changes. Forecasted changes to temperature and nutrient availability have implications for trophic interactions, and may ultimately influence the access to and palatability of the forage for grazers. Depending on what anthropogenic change will be most pronounced in the future (increase in nutrient deposits, warming, or a combination of them both), different shifts in community dominance hierarchies may occur. Generally, this study supports the productivity-diversity relationship found across arctic habitats, with community diversity peaking in mid-productivity systems and degrading as nutrient availability increases further. This is likely due the increasing competition in plant-plant interactions and the shifting dominance structure with grasses taking over the experimental plots, suggesting that global change could have high costs to biodiversity in the Arctic.

  7. [Comparative microangiographic and histological study of hepatic metastases. Possible implications in the phenomena of contrast medium uptake in x-ray computed tomography of the neoplastic liver].

    PubMed

    Tshibwabwa-Tumba, E; Marchal, G; Pylyser, K; Verbeken, E; Goddeeris, P; Baert, A L; Lauweryns, J

    1984-10-01

    A comparative study using microangiographic and histologic techniques was realized in 43 metastatic livers, totaling 109 lesions. Three different types of tumor vascularization could be recognized. In 30 lesions, residual vessels could be identified. They constitute the only vascular elements in most of the hypervascular lesions studied. On histology, these vessels corresponded to preserved hepatic arterial branches and portal radicles. 79 lesions appeared hypervascular. In 51 lesions, hypervascularity was due to tortuous irregular vessels, corresponding at microscopy to dysplastic capillaries clearly lined with endothelial cells. On the contrary, in 28 metastases, microangiographies showed amorphous contrast uptake. In these cases the contrast was found in large intercellular spaces without endothelial lining, suggesting free interstitial circulation. These observations suggest that the morphology and the dynamic of CT in liver metastases must be influenced by the arterial or portal venous nature of the different vessels. Furthermore, the extravascular diffusion should be function of the type of tumor circulation. If this circulation is confined to real vessels, diffusion will be function of the structure of the walls of these vessels. On the contrary, if the intratumoral circulation is of the free interstitial type, diffusion will be absent and mixing will occur because of the continuity of vascular and interstitial spaces.

  8. Effects of macromolecular crowding on nuclear size.

    PubMed

    Rosania, G R; Swanson, J A

    1995-05-01

    The concentration of macromolecules inside cells is high, and the resultant crowding of cytoplasm can be expected to affect many interactions involving macromolecular assemblies. Here, we have examined the effect of solute size and concentration on nuclear volume in saponin-permeabilized macrophages. Nuclei swelled in the presence of small solutes and shrank reversibly in the presence of larger permeant solutes. Remarkably, the smallest solutes capable of shrinking the nucleus were not excluded by the pores in the nuclear envelope. Indeed, nuclei shrank in the presence of such solutes even after the nuclear envelope had been sheared mechanically or permeabilized with detergent. Nuclei extracted with 1% Triton X-100 shrank in the presence of very high concentrations of small solute molecules (30% w/v) as well as in lower concentrations of larger solutes. Consistent with a macromolecular crowding effect, changes in nuclear volume were dependent on solute size and not simply dependent on the colligative properties of solutes or the exclusion of solutes by the nuclear envelope. Solute size-dependent changes in nuclear volume were independent of the chemical nature of the solutes and of the activity of the ions in the buffer. Together, these observations indicate that high concentrations of macromolecules such as those found inside cells can influence the size of the nucleus by directly affecting nuclear structure.

  9. Multiscale macromolecular simulation: role of evolving ensembles.

    PubMed

    Singharoy, A; Joshi, H; Ortoleva, P J

    2012-10-22

    Multiscale analysis provides an algorithm for the efficient simulation of macromolecular assemblies. This algorithm involves the coevolution of a quasiequilibrium probability density of atomic configurations and the Langevin dynamics of spatial coarse-grained variables denoted order parameters (OPs) characterizing nanoscale system features. In practice, implementation of the probability density involves the generation of constant OP ensembles of atomic configurations. Such ensembles are used to construct thermal forces and diffusion factors that mediate the stochastic OP dynamics. Generation of all-atom ensembles at every Langevin time step is computationally expensive. Here, multiscale computation for macromolecular systems is made more efficient by a method that self-consistently folds in ensembles of all-atom configurations constructed in an earlier step, history, of the Langevin evolution. This procedure accounts for the temporal evolution of these ensembles, accurately providing thermal forces and diffusions. It is shown that efficiency and accuracy of the OP-based simulations is increased via the integration of this historical information. Accuracy improves with the square root of the number of historical timesteps included in the calculation. As a result, CPU usage can be decreased by a factor of 3-8 without loss of accuracy. The algorithm is implemented into our existing force-field based multiscale simulation platform and demonstrated via the structural dynamics of viral capsomers.

  10. Macromolecular recognition in the Protein Data Bank

    PubMed Central

    Janin, Joël; Rodier, Francis; Chakrabarti, Pinak; Bahadur, Ranjit P.

    2007-01-01

    Crystal structures deposited in the Protein Data Bank illustrate the diversity of biological macromolecular recognition: transient interactions in protein–protein and protein–DNA complexes and permanent assemblies in homodimeric proteins. The geometric and physical chemical properties of the macromolecular interfaces that may govern the stability and specificity of recognition are explored in complexes and homodimers compared with crystal-packing interactions. It is found that crystal-packing interfaces are usually much smaller; they bury fewer atoms and are less tightly packed than in specific assemblies. Standard-size interfaces burying 1200–2000 Å2 of protein surface occur in protease–inhibitor and antigen–antibody complexes that assemble with little or no conformation changes. Short-lived electron-transfer complexes have small interfaces; the larger size of the interfaces observed in complexes involved in signal transduction and homodimers correlates with the presence of conformation changes, often implicated in biological function. Results of the CAPRI (critical assessment of predicted interactions) blind prediction experiment show that docking algorithms efficiently and accurately predict the mode of assembly of proteins that do not change conformation when they associate. They perform less well in the presence of large conformation changes and the experiment stimulates the development of novel procedures that can handle such changes. PMID:17164520

  11. EIGER detector: application in macromolecular crystallography

    PubMed Central

    Casanas, Arnau; Warshamanage, Rangana; Finke, Aaron D.; Panepucci, Ezequiel; Olieric, Vincent; Nöll, Anne; Tampé, Robert; Brandstetter, Stefan; Förster, Andreas; Mueller, Marcus; Schulze-Briese, Clemens; Bunk, Oliver; Wang, Meitian

    2016-01-01

    The development of single-photon-counting detectors, such as the PILATUS, has been a major recent breakthrough in macromolecular crystallography, enabling noise-free detection and novel data-acquisition modes. The new EIGER detector features a pixel size of 75 × 75 µm, frame rates of up to 3000 Hz and a dead time as low as 3.8 µs. An EIGER 1M and EIGER 16M were tested on Swiss Light Source beamlines X10SA and X06SA for their application in macromolecular crystallography. The combination of fast frame rates and a very short dead time allows high-quality data acquisition in a shorter time. The ultrafine φ-slicing data-collection method is introduced and validated and its application in finding the optimal rotation angle, a suitable rotation speed and a sufficient X-ray dose are presented. An improvement of the data quality up to slicing at one tenth of the mosaicity has been observed, which is much finer than expected based on previous findings. The influence of key data-collection parameters on data quality is discussed. PMID:27599736

  12. Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase.

    PubMed

    Nadar, Shamraja S; Rathod, Virendra K

    2016-02-01

    This work illustrates the preparation of magnetic macromolecular glucoamylase CLEAs using dialdehydic pectin, as a cross linker instead of traditional glutaraldehyde. The effect of precipitators type and amount, cross linker concentration, cross linking time and amount of amino functionalized magnetic nanoparticles (AFMNs) on glucoamylase activity was studied. Glucoamylase magnetic macromolecular CLEAs prepared by precipitation in presence of AFMNs by ammonium sulfate were subsequently cross linked by dialdehydic pectin. After cross-linked by pectin, 95.4% activity recovery was achieved in magnetic macromolecular CLEAs, whereas in case of glutaraldehyde cross linker, 85.3% activity recovery was achieved. Magnetic macromolecular CLEAs showed 2.91 and 1.27 folds higher thermal stability as compared to free and magnetic glutaraldehyde CLEAs. In kinetics study, magnetic macromolecular CLEAs retained same Km values, whereas magnetic glutaraldehyde CLEAs showed higher Km value than free enzyme. The porous structure of magnetic macromolecular CLEAs was not only enhanced mass transfer toward macromolecular substrates, but also showed compression resistance for 5 consecutive cycles which was checked in terms of effectiveness factor. At the end, in reusability study; magnetic macromolecular CLEAs were retained 84% activity after 10(th) cycle without leaching of enzyme which is 22% higher than traditional magnetic CLEAs.

  13. Macromolecular Synthesis and Thymineless Death in Mycoplasma laidlawii B1

    PubMed Central

    Smith, Douglas W.; Hanawalt, Philip C.

    1968-01-01

    The relationships between macromolecular synthesis and viability have been studied in the pleuropneumonia-like organism Mycoplasma laidlawii B adapted to a semidefined grwoth medium. This organism exhibited an absolute growth requirement for the nucleosides uridine and thymidine, a partial requirement for guanosine and deoxyguanosine, but no requirement for adenosine, deoxyadenosine, cytosine, and deoxycytosine. Cytosine and deoxycytosine partially satisfied the requirement for uridine. Loss in viability resulted from thymidine deprivation, but not from a deficiency in other growth requirements. This phenomenon of thymineless death in a mycoplasma is similar in many respects to that reported in other bacterial systems. Chloramphenicol specifically inhibited protein synthesis and allowed deoxyribonucleic acid synthesis to proceed to only about 40% of that normally produced per generation period, while causing less inhibition of ribonucleic acid synthesis. Protein synthesis inhibition permitted thymineless death to a survival level of less than 0.5%, but ribonucleic acid synthesis inhibition resulted in a higher (10%) survival level. These results are consistent with previously noted aspects of thymineless death in Escherichia coli strains, which suggest that thymineless death is coupled to ribonucleic acid synthesis. PMID:4881702

  14. Nitric Oxide Release Part I. Macromolecular Scaffolds

    PubMed Central

    Riccio, Daniel A.; Schoenfisch, Mark H.

    2012-01-01

    Summary The roles of nitric oxide (NO) in physiology and pathophysiology merit the use of NO as a therapeutic for certain biomedical applications. Unfortunately, limited NO payloads, too rapid NO release, and the lack of targeted NO delivery have hindered the clinical utility of NO gas and low molecular weight NO donor compounds. A wide-variety of NO-releasing macromolecular scaffolds has thus been developed to improve NO’s pharmacological potential. In this tutorial review, we provide an overview of the most promising NO release scaffolds including protein, organic, inorganic, and hybrid organic-inorganic systems. The NO release vehicles selected for discussion were chosen based on their enhanced NO storage, tunable NO release characteristics, and potential as therapeutics. PMID:22362355

  15. Macromolecular crowding explains overflow metabolism in cells

    PubMed Central

    Vazquez, Alexei; Oltvai, Zoltán N.

    2016-01-01

    Overflow metabolism is a metabolic phenotype of cells characterized by mixed oxidative phosphorylation (OxPhos) and fermentative glycolysis in the presence of oxygen. Recently, it was proposed that a combination of a protein allocation constraint and a higher proteome fraction cost of energy generation by OxPhos relative to fermentation form the basis of overflow metabolism in the bacterium, Escherichia coli. However, we argue that the existence of a maximum or optimal macromolecular density is another essential requirement. Here we re-evaluate our previous theory of overflow metabolism based on molecular crowding following the proteomic fractions formulation. We show that molecular crowding is a key factor in explaining the switch from OxPhos to overflow metabolism. PMID:27484619

  16. Simulation and display of macromolecular complexes

    NASA Technical Reports Server (NTRS)

    Nir, S.; Garduno, R.; Rein, R.; Macelroy, R. D.

    1977-01-01

    In association with an investigation of the interaction of proteins with DNA and RNA, an interactive computer program for building, manipulating, and displaying macromolecular complexes has been designed. The system provides perspective, planar, and stereoscopic views on the computer terminal display, as well as views for standard and nonstandard observer locations. The molecule or its parts may be rotated and/or translated in any direction; bond connections may be added or removed by the viewer. Molecular fragments may be juxtaposed in such a way that given bonds are aligned, and given planes and points coincide. Another subroutine provides for the duplication of a given unit such as a DNA or amino-acid base.

  17. Macromolecular recognition and macroscopic interactions by cyclodextrins.

    PubMed

    Harada, Akira; Takashima, Yoshinori

    2013-10-01

    Herein macromolecular recognition by cyclodextrins (CDs) is summarized. Recognition of macromolecules by CDs is classified as main-chain recognition or side-chain recognition. We found that CDs form inclusion complexes with various polymers with high selectivity. Polyrotaxanes in which many CDs are entrapped in a polymer chain were prepared. Tubular polymers were prepared from the polyrotaxanes. CDs were found to recognize side-chains of polymers selectively. CD host polymers were found to form gels with guest polymers in water. These gels showed self-healing properties. When azobenzene was used as a guest, the gel showed sol-gel transition by photoirradiation. When ferrocene was used, redox-responsive gels were obtained. Macroscopic self-assembly through molecular recognition has been discovered. Photoswitchable gel association and dissociation have been observed.

  18. Macromolecular diffractive imaging using imperfect crystals

    PubMed Central

    Ayyer, Kartik; Yefanov, Oleksandr; Oberthür, Dominik; Roy-Chowdhury, Shatabdi; Galli, Lorenzo; Mariani, Valerio; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Schaffer, Alexander; Dörner, Katerina; James, Daniel; Kupitz, Christopher; Metz, Markus; Nelson, Garrett; Lourdu Xavier, Paulraj; Beyerlein, Kenneth R.; Schmidt, Marius; Sarrou, Iosifina; Spence, John C. H.; Weierstall, Uwe; White, Thomas A.; Yang, Jay-How; Zhao, Yun; Liang, Mengning; Aquila, Andrew; Hunter, Mark S.; Robinson, Joseph S.; Koglin, Jason E.; Boutet, Sébastien; Fromme, Petra; Barty, Anton; Chapman, Henry N.

    2016-01-01

    The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7. PMID:26863980

  19. Macromolecular diffractive imaging using imperfect crystals

    NASA Astrophysics Data System (ADS)

    Ayyer, Kartik; Yefanov, Oleksandr M.; Oberthür, Dominik; Roy-Chowdhury, Shatabdi; Galli, Lorenzo; Mariani, Valerio; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Schaffer, Alexander; Dörner, Katerina; James, Daniel; Kupitz, Christopher; Metz, Markus; Nelson, Garrett; Xavier, Paulraj Lourdu; Beyerlein, Kenneth R.; Schmidt, Marius; Sarrou, Iosifina; Spence, John C. H.; Weierstall, Uwe; White, Thomas A.; Yang, Jay-How; Zhao, Yun; Liang, Mengning; Aquila, Andrew; Hunter, Mark S.; Robinson, Joseph S.; Koglin, Jason E.; Boutet, Sébastien; Fromme, Petra; Barty, Anton; Chapman, Henry N.

    2016-02-01

    The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing.

  20. Phylogenetic Diversity in the Macromolecular Composition of Microalgae

    PubMed Central

    Finkel, Zoe V.; Follows, Mick J.; Liefer, Justin D.; Brown, Chris M.; Benner, Ina; Irwin, Andrew J.

    2016-01-01

    The elemental stoichiometry of microalgae reflects their underlying macromolecular composition and influences competitive interactions among species and their role in the food web and biogeochemistry. Here we provide a new estimate of the macromolecular composition of microalgae using a hierarchical Bayesian analysis of data compiled from the literature. The median macromolecular composition of nutrient-sufficient exponentially growing microalgae is 32.2% protein, 17.3% lipid, 15.0% carbohydrate, 17.3% ash, 5.7% RNA, 1.1% chlorophyll-a and 1.0% DNA as percent dry weight. Our analysis identifies significant phylogenetic differences in macromolecular composition undetected by previous studies due to small sample sizes and the large inherent variability in macromolecular pools. The phylogenetic differences in macromolecular composition lead to variations in carbon-to-nitrogen ratios that are consistent with independent observations. These phylogenetic differences in macromolecular and elemental composition reflect adaptations in cellular architecture and biochemistry; specifically in the cell wall, the light harvesting apparatus, and storage pools. PMID:27228080

  1. Phylogenetic Diversity in the Macromolecular Composition of Microalgae.

    PubMed

    Finkel, Zoe V; Follows, Mick J; Liefer, Justin D; Brown, Chris M; Benner, Ina; Irwin, Andrew J

    2016-01-01

    The elemental stoichiometry of microalgae reflects their underlying macromolecular composition and influences competitive interactions among species and their role in the food web and biogeochemistry. Here we provide a new estimate of the macromolecular composition of microalgae using a hierarchical Bayesian analysis of data compiled from the literature. The median macromolecular composition of nutrient-sufficient exponentially growing microalgae is 32.2% protein, 17.3% lipid, 15.0% carbohydrate, 17.3% ash, 5.7% RNA, 1.1% chlorophyll-a and 1.0% DNA as percent dry weight. Our analysis identifies significant phylogenetic differences in macromolecular composition undetected by previous studies due to small sample sizes and the large inherent variability in macromolecular pools. The phylogenetic differences in macromolecular composition lead to variations in carbon-to-nitrogen ratios that are consistent with independent observations. These phylogenetic differences in macromolecular and elemental composition reflect adaptations in cellular architecture and biochemistry; specifically in the cell wall, the light harvesting apparatus, and storage pools.

  2. The use of a mini-κ goniometer head in macromolecular crystallography diffraction experiments

    SciTech Connect

    Brockhauser, Sandor; Ravelli, Raimond B. G.; McCarthy, Andrew A.

    2013-07-01

    Hardware and software solutions for MX data-collection strategies using the EMBL/ESRF miniaturized multi-axis goniometer head are presented. Most macromolecular crystallography (MX) diffraction experiments at synchrotrons use a single-axis goniometer. This markedly contrasts with small-molecule crystallography, in which the majority of the diffraction data are collected using multi-axis goniometers. A novel miniaturized κ-goniometer head, the MK3, has been developed to allow macromolecular crystals to be aligned. It is available on the majority of the structural biology beamlines at the ESRF, as well as elsewhere. In addition, the Strategy for the Alignment of Crystals (STAC) software package has been developed to facilitate the use of the MK3 and other similar devices. Use of the MK3 and STAC is streamlined by their incorporation into online analysis tools such as EDNA. The current use of STAC and MK3 on the MX beamlines at the ESRF is discussed. It is shown that the alignment of macromolecular crystals can result in improved diffraction data quality compared with data obtained from randomly aligned crystals.

  3. Macromolecular Prodrugs of Ribavirin: Structure-Function Correlation as Inhibitors of Influenza Infectivity.

    PubMed

    Riber, Camilla Frich; Hinton, Tracey M; Gajda, Paulina; Zuwala, Kaja; Tolstrup, Martin; Stewart, Cameron; Zelikin, Alexander N

    2017-01-03

    The requirement for new antiviral therapeutics is an ever present need. Particularly lacking are broad spectrum antivirals that have low toxicity. We develop such agents based on macromolecular prodrugs whereby both the polymer chain and the drug released from the polymer upon cell entry have antiviral effects. Specifically, macromolecular prodrugs were designed herein based on poly(methacrylic acid) and ribavirin. Structure-function parameter space was analyzed via the synthesis of 10 polymer compositions varied by molar mass and drug content. Antiviral activity was tested in cell culture against both low and high pathogenic strains of influenza. Lead compounds were successfully used to counter infectivity of influenza in chicken embryos. The lead composition with the highest activity against influenza was also active against another respiratory pathogen, respiratory syncytial virus, providing opportunity to potentially treat infection by the two pathogens with one antiviral agent. In contrast, structure-function activity against the herpes simplex virus was drastically different, revealing limitations of the broad spectrum antiviral agents based on macromolecular prodrugs.

  4. Fluid Physics and Macromolecular Crystal Growth in Microgravity

    NASA Technical Reports Server (NTRS)

    Helliwell, John R.; Snell, Edward H.; Chayen, Naomi E.; Judge, Russell A.; Boggon, Titus J.; Pusey, M. L.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The first protein crystallization experiment in microgravity was launched in April, 1981 and used Germany's Technologische Experimente unter Schwerelosigkeit (TEXUS 3) sounding rocket. The protein P-galactosidase (molecular weight 465Kda) was chosen as the sample with a liquid-liquid diffusion growth method. A sliding device brought the protein, buffer and salt solution into contact when microgravity was reached. The sounding rocket gave six minutes of microgravity time with a cine camera and schlieren optics used to monitor the experiment, a single growth cell. In microgravity a strictly laminar diffusion process was observed in contrast to the turbulent convection seen on the ground. Several single crystals, approx 100micron in length, were formed in the flight which were of inferior but of comparable visual quality to those grown on the ground over several days. A second experiment using the same protocol but with solutions cooled to -8C (kept liquid with glycerol antifreeze) again showed laminar diffusion. The science of macromolecular structural crystallography involves crystallization of the macromolecule followed by use of the crystal for X-ray diffraction experiments to determine the three dimensional structure of the macromolecule. Neutron protein crystallography is employed for elucidation of H/D exchange and for improved definition of the bound solvent (D20). The structural information enables an understanding of how the molecule functions with important potential for rational drug design, improved efficiency of industrial enzymes and agricultural chemical development. The removal of turbulent convection and sedimentation in microgravity, and the assumption that higher quality crystals will be produced, has given rise to the growing number of crystallization experiments now flown. Many experiments can be flown in a small volume with simple, largely automated, equipment - an ideal combination for a microgravity experiment. The term "protein crystal growth

  5. Phase sensitive x-ray diffraction imaging of defects in biological macromolecular crystals

    NASA Technical Reports Server (NTRS)

    Hu, Z. W.; Lai, B.; Chu, Y. S.; Cai, Z.; Mancini, D. C.; Thomas, B. R.; Chernov, A. A.

    2001-01-01

    Conventional x-ray diffraction topography is currently used to map defects in the bulk of protein crystals, but the lack of sufficient contrast is frequently a limiting factor. We experimentally demonstrate that this barrier can be circumvented using a method that combines phase sensitive and diffraction imaging principles. Details of defects revealed in tetragonal lysozyme and cubic ferritin crystals are presented and discussed. The approach enabling the detection of the phase changes of diffracted x rays should prove to be useful in the study of defect structures in a broad range of biological macromolecular crystals.

  6. Macromolecular Crystal Growth by Means of Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark; Ferree, Darren; Spearing, Scott; Monaco, Lisa; Molho, Josh; Spaid, Michael; Brasseur, Mike; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    We have performed a feasibility study in which we show that chip-based, microfluidic (LabChip(TM)) technology is suitable for protein crystal growth. This technology allows for accurate and reliable dispensing and mixing of very small volumes while minimizing bubble formation in the crystallization mixture. The amount of (protein) solution remaining after completion of an experiment is minimal, which makes this technique efficient and attractive for use with proteins, which are difficult or expensive to obtain. The nature of LabChip(TM) technology renders it highly amenable to automation. Protein crystals obtained in our initial feasibility studies were of excellent quality as determined by X-ray diffraction. Subsequent to the feasibility study, we designed and produced the first LabChip(TM) device specifically for protein crystallization in batch mode. It can reliably dispense and mix from a range of solution constituents into two independent growth wells. We are currently testing this design to prove its efficacy for protein crystallization optimization experiments. In the near future we will expand our design to incorporate up to 10 growth wells per LabChip(TM) device. Upon completion, additional crystallization techniques such as vapor diffusion and liquid-liquid diffusion will be accommodated. Macromolecular crystallization using microfluidic technology is envisioned as a fully automated system, which will use the 'tele-science' concept of remote operation and will be developed into a research facility for the International Space Station as well as on the ground.

  7. The solvent component of macromolecular crystals

    SciTech Connect

    Weichenberger, Christian X.; Kantardjieff, Katherine; Rupp, Bernhard

    2015-04-30

    On average, the mother liquor or solvent and its constituents occupy about 50% of a macromolecular crystal. Ordered as well as disordered solvent components need to be accurately accounted for in modelling and refinement, often with considerable complexity. The mother liquor from which a biomolecular crystal is grown will contain water, buffer molecules, native ligands and cofactors, crystallization precipitants and additives, various metal ions, and often small-molecule ligands or inhibitors. On average, about half the volume of a biomolecular crystal consists of this mother liquor, whose components form the disordered bulk solvent. Its scattering contributions can be exploited in initial phasing and must be included in crystal structure refinement as a bulk-solvent model. Concomitantly, distinct electron density originating from ordered solvent components must be correctly identified and represented as part of the atomic crystal structure model. Herein, are reviewed (i) probabilistic bulk-solvent content estimates, (ii) the use of bulk-solvent density modification in phase improvement, (iii) bulk-solvent models and refinement of bulk-solvent contributions and (iv) modelling and validation of ordered solvent constituents. A brief summary is provided of current tools for bulk-solvent analysis and refinement, as well as of modelling, refinement and analysis of ordered solvent components, including small-molecule ligands.

  8. Macromolecular components of tomato fruit pectin.

    PubMed

    Fishman, M L; Gross, K C; Gillespie, D T; Sondey, S M

    1989-10-01

    Chelate and alkaline-soluble pectin extracted from cell walls of pericarp tissue from mature green, turning, and red ripe (Lycopersicon esculentum Mill.) fruit (cv. Rutgers), were studied by high-performance size-exclusion chromatography. Computer-aided curve fitting of the chromatograms to a series of Gaussian-shaped components revealed that pectin from all fractions was composed of a linear combination of five macromolecular-sized species. The relative sizes of these macromolecules as obtained from their radii of gyration were 1:2:4:8:16. Dialysis against 0.05 M NaCl induced partial dissociation of the biopolymers. Apparently, the weight fraction of smaller sized species increased at the expense of larger ones. Also, the dissociation produced low-molecular-weight fragments. Behavior in the presence of 0.05 M NaCl led to the conclusion that cell wall pectin acted as if it were an aggregated mosaic, held together at least partially through noncovalent interactions.

  9. Benefits and Limitations of Low-kV Macromolecular Imaging of Frozen-Hydrated Biological Samples

    PubMed Central

    Majorovits, Endre; Angert, Isabel; Kaiser, Ute; Schröder, Rasmus R.

    2016-01-01

    Object contrast is one of the most important parameters of macromolecular imaging. Low-voltage transmission electron microscopy has shown an increased atom contrast for carbon materials, indicating that amplitude contrast contributions increase at a higher rate than phase contrast and inelastic scattering. Here, we studied image contrast using ice-embedded tobacco mosaic virus particles as test samples at 20–80 keV electron energy. The particles showed the expected increase in contrast for lower energies, but at the same time the 2.3-nm-resolution measure decayed more rapidly. We found a pronounced signal loss below 60 keV, and therefore we conclude that increased inelastic scattering counteracts increased amplitude contrast. This model also implies that as long as the amplitude contrast does not increase with resolution, beam damage and multiple scattering will always win over increased contrast at the lowest energies. Therefore, we cannot expect that low-energy imaging of conventionally prepared samples would provide better data than state-of-the-art 200–300 keV imaging. PMID:26910420

  10. Macromolecular networks and intelligence in microorganisms

    PubMed Central

    Westerhoff, Hans V.; Brooks, Aaron N.; Simeonidis, Evangelos; García-Contreras, Rodolfo; He, Fei; Boogerd, Fred C.; Jackson, Victoria J.; Goncharuk, Valeri; Kolodkin, Alexey

    2014-01-01

    Living organisms persist by virtue of complex interactions among many components organized into dynamic, environment-responsive networks that span multiple scales and dimensions. Biological networks constitute a type of information and communication technology (ICT): they receive information from the outside and inside of cells, integrate and interpret this information, and then activate a response. Biological networks enable molecules within cells, and even cells themselves, to communicate with each other and their environment. We have become accustomed to associating brain activity – particularly activity of the human brain – with a phenomenon we call “intelligence.” Yet, four billion years of evolution could have selected networks with topologies and dynamics that confer traits analogous to this intelligence, even though they were outside the intercellular networks of the brain. Here, we explore how macromolecular networks in microbes confer intelligent characteristics, such as memory, anticipation, adaptation and reflection and we review current understanding of how network organization reflects the type of intelligence required for the environments in which they were selected. We propose that, if we were to leave terms such as “human” and “brain” out of the defining features of “intelligence,” all forms of life – from microbes to humans – exhibit some or all characteristics consistent with “intelligence.” We then review advances in genome-wide data production and analysis, especially in microbes, that provide a lens into microbial intelligence and propose how the insights derived from quantitatively characterizing biomolecular networks may enable synthetic biologists to create intelligent molecular networks for biotechnology, possibly generating new forms of intelligence, first in silico and then in vivo. PMID:25101076

  11. Macromolecular Topography Leaps into the Digital Age

    NASA Technical Reports Server (NTRS)

    Lovelace, J.; Bellamy, H.; Snell, E. H.; Borgstahl, G.

    2003-01-01

    A low-cost, real-time digital topography system is under development which will replace x-ray film and nuclear emulsion plates. The imaging system is based on an inexpensive surveillance camera that offers a 1000x1000 array of 8 im square pixels, anti-blooming circuitry, and very quick read out. Currently, the system directly converts x-rays to an image with no phosphor. The system is small and light and can be easily adapted to work with other crystallographic equipment. Preliminary images have been acquired of cubic insulin at the NSLS x26c beam line. NSLS x26c was configured for unfocused monochromatic radiation. Six reflections were collected with stills spaced from 0.002 to 0.001 degrees apart across the entire oscillation range that the reflections were in diffracting condition. All of the reflections were rotated to the vertical to reduce Lorentz and beam related effects. This particular CCD is designed for short exposure applications (much less than 1 sec) and so has a relatively high dark current leading to noisy raw images. The images are processed to remove background and other system noise with a multi-step approach including the use of wavelets, histogram, and mean window filtering. After processing, animations were constructed with the corresponding reflection profile to show the diffraction of the crystal volume vs. the oscillation angle as well as composite images showing the parts of the crystal with the strongest diffraction for each reflection. The final goal is to correlate features seen in reflection profiles captured with fine phi slicing to those seen in the topography images. With this development macromolecular topography finally comes into the digital age.

  12. Macromolecular Antioxidants and Dietary Fiber in Edible Seaweeds.

    PubMed

    Sanz-Pintos, Nerea; Pérez-Jiménez, Jara; Buschmann, Alejandro H; Vergara-Salinas, José Rodrigo; Pérez-Correa, José Ricardo; Saura-Calixto, Fulgencio

    2017-02-01

    Seaweeds are rich in different bioactive compounds with potential uses in drugs, cosmetics and the food industry. The objective of this study was to analyze macromolecular antioxidants or nonextractable polyphenols, in several edible seaweed species collected in Chile (Gracilaria chilensis, Callophyllis concepcionensis, Macrocystis pyrifera, Scytosyphon lomentaria, Ulva sp. and Enteromorpha compressa), including their 1st HPLC characterization. Macromolecular antioxidants are commonly ignored in studies of bioactive compounds. They are associated with insoluble dietary fiber and exhibit significant biological activity, with specific features that are different from those of both dietary fiber and extractable polyphenols. We also evaluated extractable polyphenols and dietary fiber, given their relationship with macromolecular antioxidants. Our results show that macromolecular antioxidants are a major polyphenol fraction (averaging 42% to total polyphenol content), with hydroxycinnamic acids, hydroxybenzoic acids and flavonols being the main constituents. This fraction also showed remarkable antioxidant capacity, as determined by 2 complementary assays. The dietary fiber content was over 50% of dry weight, with some samples exhibiting the target proportionality between soluble and insoluble dietary fiber for adequate nutrition. Overall, our data show that seaweed could be an important source of commonly ignored macromolecular antioxidants.

  13. RECENT ADVANCES IN MACROMOLECULAR HYDRODYNAMIC MODELING

    PubMed Central

    Aragon, Sergio R.

    2010-01-01

    The modern implementation of the boundary element method (S.R. Aragon, J. Comput. Chem. 25(2004)1191–12055) has ushered unprecedented accuracy and precision for the solution of the Stokes equations of hydrodynamics with stick boundary conditions. This article begins by reviewing computations with the program BEST of smooth surface objects such as ellipsoids, the dumbbell, and cylinders that demonstrate that the numerical solution of the integral equation formulation of hydrodynamics yields very high precision and accuracy. When BEST is used for macromolecular computations, the limiting factor becomes the definition of the molecular hydrodynamic surface and the implied effective solvation of the molecular surface. Studies on 49 different proteins, ranging in molecular weight from 9 to over 400 kDa, have shown that a model using a 1.1 A thick hydration layer describes all protein transport properties very well for the overwhelming majority of them. In addition, this data implies that the crystal structure is an excellent representation of the average solution structure for most of them. In order to investigate the origin of a handful of significant discrepancies in some multimeric proteins (over −20% observed in the intrinsic viscosity), the technique of Molecular Dynamics simulation (MD) has been incorporated into the research program. A preliminary study of dimeric α-chymotrypsin using approximate implicit water MD is presented. In addition I describe the successful validation of modern protein force fields, ff03 and ff99SB, for the accurate computation of solution structure in explicit water simulation by comparison of trajectory ensemble average computed transport properties with experimental measurements. This work includes small proteins such as lysozyme, ribonuclease and ubiquitin using trajectories around 10 ns duration. We have also studied a 150 kDa flexible monoclonal IgG antibody, trastuzumab, with multiple independent trajectories encompassing over

  14. Polysaccharide-based micro/nanohydrogels for delivering macromolecular therapeutics.

    PubMed

    Ganguly, Kuntal; Chaturvedi, Kiran; More, Uttam A; Nadagouda, Mallikarjuna N; Aminabhavi, Tejraj M

    2014-11-10

    Increased interest in developing novel micro/nanohydrogel based formulations for delivering macromolecular therapeutics has led to multiple choices of biodegradable and biocompatible natural polymers. This interest is largely due to the availability of large number of highly pure recombinant proteins and peptides with tunable properties as well as RNA interference technology that are used in treating some of the deadly diseases that were difficult to be treated by the conventional approaches. The majority of marketed drugs that are now available are in the form of injectables that pose limited patient compliance and convenience. On the other hand, micro/nanotechnology based macromolecular delivery formulations offer many alternative routes of administration and advantages with improved patient compliance and efficient or targeted delivery of intracellular therapeutics to the site of action. This review outlines and critically evaluates the research findings on micro and nano-carrier polymeric hydrogels for the delivery of macromolecular therapeutics.

  15. Electrostatics in the self-assembly of macromolecular surfactants

    NASA Astrophysics Data System (ADS)

    Mendes, E.; Schädler, V.; Marques, C. M.; Lindner, P.; Wiesner, U.

    1997-12-01

    We report on a small-angle neutron scattering (SANS) study of dilute solutions of neutral and charged polystyrene-polyisoprene (PS-b-PI) diblock copolymers in dimethyl acetamide (DMAc), a polar selective solvent for PS. This is a model macromolecular surfactant system: the low glass temperature of the PI block ensures that thermodynamic equilibrium can be attained; the ionic character of the copolymers is provided by a single sulfonate group at the free chain end of the PI block. The crossover from ionic to non-ionic behaviour is investigated by addition of salt. The results are compared to theoretical predictions for micellization of these model macromolecular systems.

  16. Crystallization of macromolecular complexes: combinatorial complex crystallization

    NASA Astrophysics Data System (ADS)

    Stura, Enrico A.; Graille, Marc; Charbonnier, Jean-Baptiste

    2001-11-01

    The usefulness of antibody complexation, as a way of increasing the chances of crystallization needs to be re-evaluated after many antibody complexes have been crystallized and their structure determined. It is somewhat striking that among these, only a small number is a complex with a large protein antigen. The problem is that the effort of raising, cleaving and purifying an Fab is rewarded only by an extra chance of getting crystals; depending on the relative likelihood of crystallization of the complexed and uncomplexed protein. The example of the complex between HIV gp120, CD4 and an Fab fragment from a neutralizing antibody suggests that further complexation of an antigen-antibody complex with a third protein could, by increasing the number of possible combinations, improve the likelihood of crystallization. We propose the use of Ig-binding proteins as a way of extending the method from HIV gp120 to all proteins for which there are monoclonal antibodies. We discuss this technique, combinatorial complex crystallization (CCC), as part of a multi-component system for the enhancement of crystallization of macromolecular complexes. The method makes use of single Ig-binding domains from Staphylococcus aureus protein A (SpA), Peptostreptococcus magnus protein L (PpL) and the streptococcal protein G (SpG). The generality of the method depends on the ability of these domains to interact with a large repertoire of antibodies without affecting antigen binding. There is strong evidence to suggest that these Ig-binding domains bind outside the antigen-combining site of the antibody without perturbing antigen binding. It is clear from the crystal structure of the single SpG domain complexed with an Fab that the interaction involves mainly the immunoglobulin CH1 domain, a region not involved in antigen recognition. We have recently determined the structure of the complex between a human Fab and the domain D from SpA and found that steric hindrance is unlikely even for large

  17. Contrast Materials

    MedlinePlus

    ... veins of the body, including vessels in the brain, neck, chest, abdomen, pelvis and legs soft tissues of the body, including the muscles, fat and skin brain breast Microbubble Contrast Materials Microbubble contrast materials are ...

  18. Macromolecular Crowding Regulates the Gene Expression Profile by Limiting Diffusion

    PubMed Central

    Golkaram, Mahdi; Hellander, Stefan; Drawert, Brian; Petzold, Linda R.

    2016-01-01

    We seek to elucidate the role of macromolecular crowding in transcription and translation. It is well known that stochasticity in gene expression can lead to differential gene expression and heterogeneity in a cell population. Recent experimental observations by Tan et al. have improved our understanding of the functional role of macromolecular crowding. It can be inferred from their observations that macromolecular crowding can lead to robustness in gene expression, resulting in a more homogeneous cell population. We introduce a spatial stochastic model to provide insight into this process. Our results show that macromolecular crowding reduces noise (as measured by the kurtosis of the mRNA distribution) in a cell population by limiting the diffusion of transcription factors (i.e. removing the unstable intermediate states), and that crowding by large molecules reduces noise more efficiently than crowding by small molecules. Finally, our simulation results provide evidence that the local variation in chromatin density as well as the total volume exclusion of the chromatin in the nucleus can induce a homogenous cell population. PMID:27893768

  19. Macromolecular Pt(IV) Prodrugs from Poly(organo)phosphazenes

    PubMed Central

    Banfić, Jelena; Theiner, Sarah; Körner, Wilfried; Brüggemann, Oliver; Berger, Walter; Keppler, Bernhard K.; Heffeter, Petra; Teasdale, Ian

    2016-01-01

    The preparation of novel macromolecular prodrugs via the conjugation of two platinum(IV) complexes to suitably functionalized poly(organo)phosphazenes is presented. The inorganic/organic polymers provide carriers with controlled dimensions due to the use of living cationic polymerization and allow the preparation of conjugates with excellent aqueous solubility but long-term hydrolytic degradability. The macromolecular Pt(IV) prodrugs are designed to undergo intracellular reduction and simultaneous release from the macromolecular carrier to present the active Pt(II) drug derivatives. In vitro investigations show a significantly enhanced intracellular uptake of Pt for the macromolecular prodrugs when compared to small molecule Pt complexes, which is also reflected in an increase in cytotoxicity. Interestingly, drug-resistant sublines also show a significantly smaller resistance against the conjugates compared to clinically established platinum drugs, indicating that an alternative uptake route of the Pt(IV) conjugates might also be able to overcome acquired resistance against Pt(II) drugs. In vivo studies of a selected conjugate show improved tumor shrinkage compared to the respective Pt(IV) complex. PMID:27169668

  20. Effects of macromolecular crowding and osmolyte on human Tau fibrillation.

    PubMed

    Wu, Yingying; Teng, Ningning; Li, Sen

    2016-09-01

    Tau fibrillation is reported to be involved in neurodegenerative disorders, such as Alzheimer's disease, in which the natural environment is very crowded in the cells. Understanding the role of crowding environments in regulating Tau fibrillation is of great importance for elucidating the etiology of these diseases. In this experiment, the effects of macromolecular crowding and osmolyte reagents in the crowding environment on Tau fibrillation were studied by thioflavin T binding, SDS-PAGE and TEM assays. Ficoll 70 and Dextran 70 of different concentrations were used as macromolecular crowding reagents inside the cells and showed a strong enhancing effect on the fibrillation of normal and hyperphosphorylated Tau. The enhancing effect of Dextran is stronger than that of Ficoll 70 at the same concentration. In addition, the cellular osmolyte sucrose was found to protect Tau against fibrillation, and inhibit the enhancing effect of macromolecular crowding on Tau fibrillation. A possible model for the fibrillation process of Tau and the effect of macromolecular crowding and osmolyte on this process was proposed based on these experimental results. The information obtained from our study can enhance the understanding of how proteins aggregate and avoid aggregation in crowded physiological environments and might lead to a better understanding of the molecular mechanisms of Alzheimer's disease in vivo.

  1. Combined Effects of Agitation, Macromolecular Crowding, and Interfaces on Amyloidogenesis*

    PubMed Central

    Lee, Chiu Fan; Bird, Sarah; Shaw, Michael; Jean, Létitia; Vaux, David J.

    2012-01-01

    Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimer's disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. α-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-β peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD. PMID:22988239

  2. Contrastive Lexicology.

    ERIC Educational Resources Information Center

    Hartmann, R. R. K.

    This paper deals with the relation between etymologically related words in different languages. A survey is made of seven stages in the development of contrastive lexicology. These are: prelinguistic word studies, semantics, lexicography, translation, foreign language learning, bilingualism, and finally contrastive analysis. Concerning contrastive…

  3. Contrastive Analysis.

    ERIC Educational Resources Information Center

    James, Carl

    Contrastive analysis is viewed as an interlinguistic, bidirectional phenomenon which is concerned with both the form and function of language. As such, contrastive analysis must view language psycholinguistically and sociolinguistically as a system to be both described and acquired. Due to the need for a psychological component in the analysis,…

  4. Temperature-dependent macromolecular X-ray crystallography

    SciTech Connect

    Weik, Martin Colletier, Jacques-Philippe

    2010-04-01

    The dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. X-ray crystallography provides structural details of biological macromolecules. Whereas routine data are collected close to 100 K in order to mitigate radiation damage, more exotic temperature-controlled experiments in a broader temperature range from 15 K to room temperature can provide both dynamical and structural insights. Here, the dynamical behaviour of crystalline macromolecules and their surrounding solvent as a function of cryo-temperature is reviewed. Experimental strategies of kinetic crystallography are discussed that have allowed the generation and trapping of macromolecular intermediate states by combining reaction initiation in the crystalline state with appropriate temperature profiles. A particular focus is on recruiting X-ray-induced changes for reaction initiation, thus unveiling useful aspects of radiation damage, which otherwise has to be minimized in macromolecular crystallography.

  5. Maintaining network security: how macromolecular structures cross the peptidoglycan layer.

    PubMed

    Scheurwater, Edie M; Burrows, Lori L

    2011-05-01

    Peptidoglycan plays a vital role in bacterial physiology, maintaining cell shape and resisting cellular lysis from high internal turgor pressures. Its integrity is carefully maintained by controlled remodeling during growth and division by the coordinated activities of penicillin-binding proteins, lytic transglycosylases, and N-acetylmuramyl-l-alanine amidases. However, its small pore size (∼2 nm) and covalently closed structure make it a formidable barrier to the assembly of large macromolecular cell-envelope-spanning complexes involved in motility and secretion. Here, we review the strategies used by Gram-negative bacteria to assemble such macromolecular complexes across the peptidoglycan layer, while preserving its essential structural role. In addition, we discuss evidence that suggests that peptidoglycan can be integrated into cell-envelope-spanning complexes as a structural and functional extension of their architecture.

  6. Controlled architecture for improved macromolecular memory within polymer networks.

    PubMed

    DiPasquale, Stephen A; Byrne, Mark E

    2016-08-01

    This brief review analyzes recent developments in the field of living/controlled polymerization and the potential of this technique for creating imprinted polymers with highly structured architecture with macromolecular memory. As a result, it is possible to engineer polymers at the molecular level with increased homogeneity relating to enhanced template binding and transport. Only recently has living/controlled polymerization been exploited to decrease heterogeneity and substantially improve the efficiency of the imprinting process for both highly and weakly crosslinked imprinted polymers. Living polymerization can be utilized to create imprinted networks that are vastly more efficient than similar polymers produced using conventional free radical polymerization, and these improvements increase the role that macromolecular memory can play in the design and engineering of new drug delivery and sensing platforms.

  7. Isotope labeling for NMR studies of macromolecular structure and interactions

    SciTech Connect

    Wright, P.E.

    1994-12-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.

  8. Branched Macromolecular Architectures for Degradable, Multifunctional Phosphorus-Based Polymers.

    PubMed

    Henke, Helena; Brüggemann, Oliver; Teasdale, Ian

    2017-02-01

    This feature article briefly highlights some of the recent advances in polymers in which phosphorus is an integral part of the backbone, with a focus on the preparation of functional, highly branched, soluble polymers. A comparison is made between the related families of materials polyphosphazenes, phosphazene/phosphorus-based dendrimers and polyphosphoesters. The work described herein shows this to be a rich and burgeoning field, rapidly catching up with organic chemistry in terms of the macromolecular synthetic control and variety of available macromolecular architectures, whilst offering unique property combinations not available with carbon backbones, such as tunable degradation rates, high multi-valency and facile post-polymerization functionalization. As an example of their use in advanced applications, we highlight some investigations into their use as water-soluble drug carriers, whereby in particular the degradability in combination with multivalent nature has made them useful materials, as underlined by some of the recent studies in this area.

  9. Stochastic reaction-diffusion algorithms for macromolecular crowding

    NASA Astrophysics Data System (ADS)

    Sturrock, Marc

    2016-06-01

    Compartment-based (lattice-based) reaction-diffusion algorithms are often used for studying complex stochastic spatio-temporal processes inside cells. In this paper the influence of macromolecular crowding on stochastic reaction-diffusion simulations is investigated. Reaction-diffusion processes are considered on two different kinds of compartmental lattice, a cubic lattice and a hexagonal close packed lattice, and solved using two different algorithms, the stochastic simulation algorithm and the spatiocyte algorithm (Arjunan and Tomita 2010 Syst. Synth. Biol. 4, 35-53). Obstacles (modelling macromolecular crowding) are shown to have substantial effects on the mean squared displacement and average number of molecules in the domain but the nature of these effects is dependent on the choice of lattice, with the cubic lattice being more susceptible to the effects of the obstacles. Finally, improvements for both algorithms are presented.

  10. Enhancement of scleral macromolecular permeability with prostaglandins.

    PubMed Central

    Weinreb, R N

    2001-01-01

    PURPOSE: It is proposed that the sclera is a metabolically active and pharmacologically responsive tissue. These studies were undertaken to determine whether prostaglandin exposure can enhance scleral permeability to high-molecular-weight substances. METHODS: Topical prostaglandin F2 alpha (PGF2 alpha) was administered to monkeys to determine if this altered the amount of scleral matrix metalloproteinases (MMPs). Experiments also were performed to determine whether the prostaglandin F (FP) receptor and gene transcripts are expressed in normal human sclera. Permeability of organ-cultured human sclera following prostaglandin exposure then was studied and the amount of MMP released into the medium measured. Finally, the permeability of human sclera to basic fibroblast growth factor (FGF-2) was determined following prostaglandin exposure. RESULTS: Topical prostaglandin administration that reduced scleral collagen also increased scleral MMP-1, MMP-2, and MMP-3 by 63 +/- 35%, 267 +/- 210%, and 729 +/- 500%, respectively. FP receptor protein was localized in scleral fibroblasts, and FP receptor gene transcript was identified in sclera. Exposure to prostaglandin F2 alpha, 17-phenyltrinor, PGF2 alpha, or latanoprost acid increased scleral permeability by up to 124%, 183%, or 213%, respectively. In these cultures, MMP-1, MMP-2, and MMP-3 were increased by up to 37%, 267%, and 96%, respectively. Finally, transscleral absorption of FGF-2 was increased by up to 126% with scleral exposure to latanoprost. CONCLUSIONS: These studies demonstrate that the sclera is metabolically active and pharmacologically responsive to prostaglandins. Further, they demonstrate the feasibility of cotreatment with prostaglandin to enhance transscleral delivery of peptides, such as growth factors and high-molecular-weight substances, to the posterior segment of the eye. PMID:11797317

  11. A 3D cellular context for the macromolecular world

    PubMed Central

    Patwardhan, Ardan; Ashton, Alun; Brandt, Robert; Butcher, Sarah; Carzaniga, Raffaella; Chiu, Wah; Collinson, Lucy; Doux, Pascal; Duke, Elizabeth; Ellisman, Mark H; Franken, Erik; Grünewald, Kay; Heriche, Jean-Karim; Koster, Abraham; Kühlbrandt, Werner; Lagerstedt, Ingvar; Larabell, Carolyn; Lawson, Catherine L; Saibil, Helen R; Sanz-García, Eduardo; Subramaniam, Sriram; Verkade, Paul; Swedlow, Jason R; Kleywegt, Gerard J

    2015-01-01

    We report the outcomes of the discussion initiated at the workshop entitled A 3D Cellular Context for the Macromolecular World and propose how data from emerging three-dimensional (3D) cellular imaging techniques—such as electron tomography, 3D scanning electron microscopy and soft X-ray tomography—should be archived, curated, validated and disseminated, to enable their interpretation and reuse by the biomedical community. PMID:25289590

  12. Ultrashort Laser Pulse Induced Electromagnetic Stress on Biological Macromolecular Systems.

    DTIC Science & Technology

    1979-11-01

    ULTRASHORT LASER PULSE INDUCED ~~~~~ ELECTROMAGNET IC STRESS ON BIOLOGICAL MACROMOLECULAR SYSTEMS Adam P. Bruckner , Ph.D. ( i~iiCJ. Michael ...AFSC, Brooks Air Force Base, Texas. Dr. John Taboada (RZL) was the Laboratory Project Scientjst..in...Charge When U.S. Goverrijie~t drawings...available to the general public , including foreignnations. Thi s technical report has been reviewed and is approved for publ i-cation. OHN TABOADA , Ph.D

  13. A strategy for dissecting the architectures of native macromolecular assemblies

    PubMed Central

    Shi, Yi; Pellarin, Riccardo; Fridy, Peter C.; Fernandez-Martinez, Javier; Thompson, Mary K.; Li, Yinyin; Wang, Qing Jun; Sali, Andrej; Rout, Michael P.; Chait, Brian T.

    2015-01-01

    Despite the central role of large multi-protein complexes in many biological processes, it remains challenging to elucidate their structures and particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here, we present a strategy for isolating such complexes and for extracting distance restraints that allow the determination of their molecular architectures. The method was optimized to allow facile use of the extensive global resources of GFP-tagged transgenic cells and animals. PMID:26436480

  14. Fast native-SAD phasing for routine macromolecular structure determination.

    PubMed

    Weinert, Tobias; Olieric, Vincent; Waltersperger, Sandro; Panepucci, Ezequiel; Chen, Lirong; Zhang, Hua; Zhou, Dayong; Rose, John; Ebihara, Akio; Kuramitsu, Seiki; Li, Dianfan; Howe, Nicole; Schnapp, Gisela; Pautsch, Alexander; Bargsten, Katja; Prota, Andrea E; Surana, Parag; Kottur, Jithesh; Nair, Deepak T; Basilico, Federica; Cecatiello, Valentina; Pasqualato, Sebastiano; Boland, Andreas; Weichenrieder, Oliver; Wang, Bi-Cheng; Steinmetz, Michel O; Caffrey, Martin; Wang, Meitian

    2015-02-01

    We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

  15. Impact of synchrotron radiation on macromolecular crystallography: a personal view

    PubMed Central

    Dauter, Zbigniew; Jaskolski, Mariusz; Wlodawer, Alexander

    2010-01-01

    The introduction of synchrotron radiation sources almost four decades ago has led to a revolutionary change in the way that diffraction data from macromolecular crystals are being collected. Here a brief history of the development of methodologies that took advantage of the availability of synchrotron sources are presented, and some personal experiences with the utilization of synchrotrons in the early days are recalled. PMID:20567074

  16. International summer school on macromolecular crystallographic computing. Final report

    SciTech Connect

    1998-08-01

    The School was the seventh in a series of International Union of Crystallography (IUCr) Crystallographic Symposia. The format of the School was formal lectures in the morning, tutorials in the afternoon, and software demonstrations and more lectures in the evening. The full program which left both the organizers and attendees exhausted, reflects the current state of excitement in the field of macromolecular structure determination using the technique of X-ray crystallography. The new and improved technologies and techniques described in these Proceedings are contributing to that growth and at the same time, as pointed out in the paper given by Sussman, creating challenges for the Protein Data Bank (PDB). As the School progressed, the authors were struck by the similarities to events which took place in small molecule crystallography beginning some 20 to 25 years ago. Growth then was fueled by the advent of new algorithms, affordable computer hardware, and good software. So it is today for macromolecular crystallography, but with the added bonus of the Internet which is changing how scientist conduct their research. Flack presented this view as part of his on-going contribution to how crystallographers use the Internet. After presentations discussing structures en masse they returned to the more traditional mode of presentation which parallels the determination of a single macromolecular structure: data collection -- phasing -- model building and visualization -- refinement.

  17. VQLM: A visual query language for macromolecular structural databases

    SciTech Connect

    Cohen, D.; Dickinson, B.; Salem, H.

    1994-12-31

    Databases of macromolecular structures allow researchers to identify general principles of molecular behavior. They do this by providing a variety of data obtained under a number of different experimental conditions. Many new tools have been developed recently to aid in exploratory analysis of structural data. However, some queries of interest still require considerable manual filtering of data. In particular, studies attempting to make generalizations about complex arrangements of atoms or building blocks in macro-molecular structures cannot be approached directly with existing tools. Such studies are frequently carried out on only a few structures or else require a labor-intensive process. To address this problem, we have developed a visual language, VQLM (Visual Query Language for Macromolecules). A query is formulated in this language by drawing an abstract picture of sub-structures to be searched for in the database and specifying constraints on the objects in them. To illustrate the usefulness of our language, we show how to encode a number of queries that were found scientifically interesting in the published literature in molecular biology. VQLM relies on VQL, a new database language, as its underlying engine for database retrieval and computation. We believe that VQLM will make macromolecular structural data more accessible to scientists, enabling faster and deeper data analysis.

  18. Macromolecular Assemblage in the Design of a Synthetic AIDS Vaccine

    NASA Astrophysics Data System (ADS)

    Defoort, Jean-Philippe; Nardelli, Bernardetta; Huang, Wolin; Ho, David D.; Tam, James P.

    1992-05-01

    We describe a peptide vaccine model based on the mimicry of surface coat protein of a pathogen. This model used a macromolecular assemblage approach to amplify peptide antigens in liposomes or micelles. The key components of the model consisted of an oligomeric lysine scaffolding to amplify peptide antigens covalently 4-fold and a lipophilic membrane-anchoring group to further amplify noncovalently the antigens many-fold in liposomal or micellar form. A peptide antigen derived from the third variable domain of glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1), consisting of neutralizing, T-helper, and T-cytotoxic epitopes, was used in a macromolecular assemblage model (HIV-1 linear peptide amino acid sequence 308-331 in a tetravalent multiple antigen peptide system linked to tripalmitoyl-S-glycerylcysteine). The latter complex, in liposome or micelle, was used to immunize mice and guinea pigs without any adjuvant and found to induce gp120-specific antibodies that neutralize virus infectivity in vitro, elicit cytokine production, and prime CD8^+ cytotoxic T lymphocytes in vivo. Our results show that the macromolecular assemblage approach bears immunological mimicry of the gp120 of HIV virus and may lead to useful vaccines against HIV infection.

  19. Cryo-electron tomography for structural characterization of macromolecular complexes.

    PubMed

    Cope, Julia; Heumann, John; Hoenger, Andreas

    2011-08-01

    Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions.

  20. Macromolecular amplification of binding response in superaptamer hydrogels.

    PubMed

    Bai, Wei; Gariano, Nicholas A; Spivak, David A

    2013-05-08

    It is becoming more important to detect ultralow concentrations of analytes for biomedical, environmental, and national security applications. Equally important is that new methods should be easy to use, inexpensive, portable, and if possible allow detection by the naked eye. By and large, detection of low concentrations of analytes cannot be achieved directly but requires signal amplification by catalysts, macromolecules, metal surfaces, or supramolecular aggregates. The rapidly progressing field of macromolecular signal amplification has been advanced using conjugated polymers, chirality in polymers, solvating polymers, and polymerization/depolymerization strategies. A new type of aptamer-based hydrogel with specific response to target proteins presented in this report demonstrates an additional category of macromolecular signal amplification. This superaptamer assembly provides the first example of using protein-specific aptamers to create volume-changing hydrogels with amplified response to the target protein. A remarkable aspect of these superaptamer hydrogels is that volume shrinking is visible to the naked eye down to femtomolar concentrations of protein. This extraordinary macromolecular amplification is attributed to a complex interplay between protein-aptamer supramolecular cross-links and the consequential reduction of excluded volume in the hydrogel. Specific recognition is even maintained in biological matrices such as urine and tears. Furthermore, the gels can be dried for long-term storage and regenerated for use without loss of activity. In practice, the ease of this biomarker detection method offers an alternative to traditional analytical techniques that require sophisticated instrumentation and highly trained personnel.

  1. Polybivalency and disordered proteins in ordering macromolecular assemblies.

    PubMed

    Barbar, Elisar; Nyarko, Afua

    2015-01-01

    Intrinsically disordered proteins (IDPs) are prevalent in macromolecular assemblies and are thought to mediate protein recognition in complex regulatory processes and signaling pathways. The formation of a polybivalent scaffold is a key process by which IDPs drive early steps in macromolecular assemblies. Three intrinsically disordered proteins, IC, Swallow and Nup159, are core components, respectively, of cytoplasmic dynein, bicoid mRNA localization apparatus, and nuclear pore complexes. In all three systems, the hub protein LC8 recognizes on the IDP, short linear motifs that are fully disordered in the apo form, but adopt a β-strand when bound to LC8. The IDP/LC8 complex forms a bivalent scaffold primed to bind additional bivalent ligands. Scaffold formation also promotes self-association and/or higher order organization of the IDP components at a site distant from LC8 binding. Rigorous thermodynamic analyses imply that association of additional bivalent ligands is driven by entropic effects where the first binding event is weak but subsequent binding of additional ligands occurs with higher affinity. Here, we review specific examples of macromolecular assemblies in which polybivalency of aligned IDP duplexes not only enhances binding affinity and results in formation of a stable complex but also compensates unfavorable steric and enthalpic interactions. We propose that polybivalent scaffold assembly involving IDPs and LC8-like proteins is a general process in the cell biology of a class of multi-protein structures that are stable yet fine-tuned for diverse cellular requirements.

  2. Workshop on algorithms for macromolecular modeling. Final project report, June 1, 1994--May 31, 1995

    SciTech Connect

    Leimkuhler, B.; Hermans, J.; Skeel, R.D.

    1995-07-01

    A workshop was held on algorithms and parallel implementations for macromolecular dynamics, protein folding, and structural refinement. This document contains abstracts and brief reports from that workshop.

  3. Contrast lipocryolysis

    PubMed Central

    Pinto, Hernán; Melamed, Graciela

    2014-01-01

    Alternative crystal structures are possible for all lipids and each different crystal structure is called a polymorphic form. Inter-conversion between polymorphisms would imply the possibility of leaning crystal formation toward the most effective polymorphism for adipocyte destruction. Food industry has been tempering lipids for decades. Tempering technology applied to lipocryolysis gave birth to “contrast lipocryolysis”, which involves pre- and post-lipocryolysis fat layer heating as part of a specific tempering protocol. In this study, we evaluated the skinfold thickness of 10 subjects after a single contrast lipocryolysis session and witnessed important and fast reductions. PMID:25068088

  4. A General Method for Modeling Macromolecular Shape in Solution

    PubMed Central

    Harding, Stephen E.

    1987-01-01

    A general method for modeling macromolecular shape in solution is described involving measurements of viscosity, radius of gyration, and the second thermodynamic virial coefficient. The method, which should be relatively straightforward to apply, does not suffer from uniqueness problems, involves shape functions that are independent of hydration, and models the gross conformation of the macromolecule in solution as a general triaxial ellipsoid. The method is illustrated by application to myosin, and the relevance and applicability of ellipsoid modeling to biological structures is discussed. PMID:19431695

  5. Bringing macromolecular machinery to life using 3D animation.

    PubMed

    Iwasa, Janet H

    2015-04-01

    Over the past decade, there has been a rapid rise in the use of three-dimensional (3D) animation to depict molecular and cellular processes. Much of the growth in molecular animation has been in the educational arena, but increasingly, 3D animation software is finding its way into research laboratories. In this review, I will discuss a number of ways in which 3d animation software can play a valuable role in visualizing and communicating macromolecular structures and dynamics. I will also consider the challenges of using animation tools within the research sphere.

  6. Macromolecular therapeutics in cancer treatment: the EPR effect and beyond.

    PubMed

    Maeda, Hiroshi

    2012-12-10

    In this review, I have discussed various issues of the cancer drug targeting primarily related to the EPR (enhanced permeability and retention) effect, which utilized nanomedicine or macromolecular drugs. The content goes back to the development of the first polymer-protein conjugate anticancer agent SMANCS and development of the arterial infusion in Lipiodol formulation into the tumor feeding artery (hepatic artery for hepatoma). The brief account on the EPR effect and its definition, factors involved, heterogeneity, and various methods of augmentation of the EPR effect, which showed remarkably improved clinical outcomes are also discussed. Various obstacles involved in drug developments and commercialization are also discussed through my personal experience and recollections.

  7. Cryosalts: suppression of ice formation in macromolecular crystallography.

    PubMed

    Rubinson, K A; Ladner, J E; Tordova, M; Gilliland, G L

    2000-08-01

    Quality data collection for macromolecular cryocrystallography requires suppressing the formation of crystalline or microcrystalline ice that may result from flash-freezing crystals. Described here is the use of lithium formate, lithium chloride and other highly soluble salts for forming ice-ring-free aqueous glasses upon cooling from ambient temperature to 100 K. These cryosalts are a new class of cryoprotectants that are shown to be effective with a variety of commonly used crystallization solutions and with proteins crystallized under different conditions. The influence of cryosalts on crystal mosaicity and diffraction resolution is comparable with or superior to traditional organic cryoprotectants.

  8. Thermodynamic signatures in macromolecular interactions involving conformational flexibility.

    PubMed

    Menzel, Anja; Neumann, Piotr; Schwieger, Christian; Stubbs, Milton T

    2014-07-01

    The energetics of macromolecular interactions are complex, particularly where protein flexibility is involved. Exploiting serendipitous differences in the plasticity of a series of closely related trypsin variants, we analyzed the enthalpic and entropic contributions accompanying interaction with L45K-eglin C. Binding of the four variants show significant differences in released heat, although the affinities vary little, in accordance with the principle of enthalpy-entropy compensation. Binding of the most disordered variant is almost entirely enthalpically driven, with practically no entropy change. As structures of the complexes reveal negligible differences in protein-inhibitor contacts, we conclude that solvent effects contribute significantly to binding affinities.

  9. Comparison of two self-assembled macromolecular prodrug micelles with different conjugate positions of SN38 for enhancing antitumor activity.

    PubMed

    Liu, Yi; Piao, Hongyu; Gao, Ying; Xu, Caihong; Tian, Ye; Wang, Lihong; Liu, Jinwen; Tang, Bo; Zou, Meijuan; Cheng, Gang

    2015-01-01

    7-Ethyl-10-hydroxycamptothecin (SN38), an active metabolite of irinotecan (CPT-11), is a remarkably potent antitumor agent. The clinical application of SN38 has been extremely restricted by its insolubility in water. In this study, we successfully synthesized two macromolecular prodrugs of SN38 with different conjugate positions (chitosan-(C10-OH)SN38 and chitosan-(C20-OH)SN38) to improve the water solubility and antitumor activity of SN38. These prodrugs can self-assemble into micelles in aqueous medium. The particle size, morphology, zeta potential, and in vitro drug release of SN38 and its derivatives, as well as their cytotoxicity, pharmacokinetics, and in vivo antitumor activity in a xenograft BALB/c mouse model were studied. In vitro, chitosan-(C10-OH)SN38 (CS-(10s)SN38) and chitosan-(C20-OH) SN38 (CS-(20s)SN38) were 13.3- and 25.9-fold more potent than CPT-11 in the murine colon adenocarcinoma cell line CT26, respectively. The area under the curve (AUC)0-24 of SN38 after intravenously administering CS-(10s)SN38 and CS-(20s)SN38 to Sprague Dawley rats was greatly improved when compared with CPT-11 (both P<0.01). A larger AUC0-24 of CS-(20s)SN38 was observed when compared to CS-(10s)SN38 (P<0.05). Both of the novel self-assembled chitosan-SN38 prodrugs demonstrated superior anticancer activity to CPT-11 in the CT26 xenograft BALB/c mouse model. We have also investigated the differences between these macromolecular prodrug micelles with regards to enhancing the antitumor activity of SN38. CS-(20s)SN38 exhibited better in vivo antitumor activity than CS-(10s)SN38 at a dose of 2.5 mg/kg (P<0.05). In conclusion, both macromolecular prodrug micelles improved the in vivo conversion rate and antitumor activity of SN38, but the prodrug in which C20-OH was conjugated to macromolecular materials could be a more promising platform for SN38 delivery.

  10. Ab initio solution of macromolecular crystal structures without direct methods.

    PubMed

    McCoy, Airlie J; Oeffner, Robert D; Wrobel, Antoni G; Ojala, Juha R M; Tryggvason, Karl; Lohkamp, Bernhard; Read, Randy J

    2017-04-04

    The majority of macromolecular crystal structures are determined using the method of molecular replacement, in which known related structures are rotated and translated to provide an initial atomic model for the new structure. A theoretical understanding of the signal-to-noise ratio in likelihood-based molecular replacement searches has been developed to account for the influence of model quality and completeness, as well as the resolution of the diffraction data. Here we show that, contrary to current belief, molecular replacement need not be restricted to the use of models comprising a substantial fraction of the unknown structure. Instead, likelihood-based methods allow a continuum of applications depending predictably on the quality of the model and the resolution of the data. Unexpectedly, our understanding of the signal-to-noise ratio in molecular replacement leads to the finding that, with data to sufficiently high resolution, fragments as small as single atoms of elements usually found in proteins can yield ab initio solutions of macromolecular structures, including some that elude traditional direct methods.

  11. Outrunning free radicals in room-temperature macromolecular crystallography

    SciTech Connect

    Owen, Robin L. Axford, Danny; Nettleship, Joanne E.; Owens, Raymond J.; Robinson, James I.; Morgan, Ann W.; Doré, Andrew S.; Lebon, Guillaume; Tate, Christopher G.; Fry, Elizabeth E.; Ren, Jingshan; Stuart, David I.; Evans, Gwyndaf

    2012-06-15

    A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector. A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A{sub 2A} adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.

  12. Production of macromolecular chloramines by chlorine-transfer reactions.

    PubMed

    Bedner, Mary; MacCrehan, William A; Helz, George R

    2004-03-15

    Chlorination of treated wastewaters is undertaken to prevent dispersal of human pathogens into the environment. Except in well-nitrified effluents, the primary agents in chlorination, Cl2(g) or NaOCl(aq), are short-lived and quickly transfer oxidative chlorine to secondary agents (N-chloramines), which then participate in the disinfection process. Maturation of residual chlorine resulting from chlorine-transfer reactions is still poorly characterized. Using gel permeation and reversed-phase liquid chromatography combined with a novel, oxidant-specific detector, unanticipated trends during the maturation of residual chlorine in wastewater are identified. Within 2 min after addition of NaOCl, and continuing for several hours at least, significant amounts of oxidative chlorine are transferred to secondary agents that are moderately to strongly hydrophobic and to agents that have high relative molecular masses (Mr 1300-25000). It is hypothesized that hydrophobic stabilization of organic chloramines (RNHCl(o)) thermodynamically drives these transfers, making macromolecular chloramines the ultimate oxidative chlorine carriers. Macromolecular chloramines are expected to be sluggish oxidants, as observed in their reduction by sulfite, and are expected to be poor disinfectants. If transfer of oxidative chlorine to high Mr components occurs widely at treatment plants, then this phenomenon offers a new, physicochemical explanation for the well-known impotency of organic chloramines in wastewater disinfection.

  13. Preparation of phenylboronate affinity rigid monolith with macromolecular porogen.

    PubMed

    Li, Xiang-Jie; Jia, Man; Zhao, Yong-Xin; Liu, Zhao-Sheng; Akber Aisa, Haji

    2016-03-18

    Boronate-affinity monolithic column was first prepared via polystyrene (PS) as porogen in this work. The monolithic polymer was synthetized using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. Isoquercitrin (ISO) and hyperoside (HYP), isomer diol flavonoid glycosides, can be baseline separated on the poly(VPBA-co-EDMA) monolith. The effect of polymerization variables on the selectivity factor, e.g., the ratio of monomer to crosslinker (M/C), the amount of PS and the molecular weight of macromolecular porogen was investigated. The surface properties of the monolithic polymer were characterized by scanning electron microscopy and nitrogen adsorption. The best polymerization condition was the M/C ratio of 7:3, and the PS concentration of 40 mg/ml. The poly(VPBA-co-EDMA) polymer was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. After treated by poly(VPBA-co-EDMA) for solid phase extraction, high purity ISO and HYP (>99.96%) can be obtained with recovery of 83.7% and 78.6%, respectively.

  14. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics

    PubMed Central

    Lönn, Peter; Kacsinta, Apollo D.; Cui, Xian-Shu; Hamil, Alexander S.; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F.

    2016-01-01

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells. PMID:27604151

  15. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.

    PubMed

    Lönn, Peter; Kacsinta, Apollo D; Cui, Xian-Shu; Hamil, Alexander S; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F

    2016-09-08

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.

  16. Identifying and Visualizing Macromolecular Flexibility in Structural Biology

    PubMed Central

    Palamini, Martina; Canciani, Anselmo; Forneris, Federico

    2016-01-01

    Structural biology comprises a variety of tools to obtain atomic resolution data for the investigation of macromolecules. Conventional structural methodologies including crystallography, NMR and electron microscopy often do not provide sufficient details concerning flexibility and dynamics, even though these aspects are critical for the physiological functions of the systems under investigation. However, the increasing complexity of the molecules studied by structural biology (including large macromolecular assemblies, integral membrane proteins, intrinsically disordered systems, and folding intermediates) continuously demands in-depth analyses of the roles of flexibility and conformational specificity involved in interactions with ligands and inhibitors. The intrinsic difficulties in capturing often subtle but critical molecular motions in biological systems have restrained the investigation of flexible molecules into a small niche of structural biology. Introduction of massive technological developments over the recent years, which include time-resolved studies, solution X-ray scattering, and new detectors for cryo-electron microscopy, have pushed the limits of structural investigation of flexible systems far beyond traditional approaches of NMR analysis. By integrating these modern methods with powerful biophysical and computational approaches such as generation of ensembles of molecular models and selective particle picking in electron microscopy, more feasible investigations of dynamic systems are now possible. Using some prominent examples from recent literature, we review how current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes. PMID:27668215

  17. The Phenix software for automated determination of macromolecular structures.

    PubMed

    Adams, Paul D; Afonine, Pavel V; Bunkóczi, Gábor; Chen, Vincent B; Echols, Nathaniel; Headd, Jeffrey J; Hung, Li-Wei; Jain, Swati; Kapral, Gary J; Grosse Kunstleve, Ralf W; McCoy, Airlie J; Moriarty, Nigel W; Oeffner, Robert D; Read, Randy J; Richardson, David C; Richardson, Jane S; Terwilliger, Thomas C; Zwart, Peter H

    2011-09-01

    X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favor of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.

  18. On the calculation of absolute macromolecular binding free energies

    PubMed Central

    Luo, Hengbin; Sharp, Kim

    2002-01-01

    The standard framework for calculating the absolute binding free energy of a macromolecular association reaction A + B → AB with an association constant KAB is to equate chemical potentials of the species on the left- and right-hand sides of this reaction and evaluate the chemical potentials from theory. This theory involves (usually hidden) assumptions about what constitutes the bound species, AB, and where the contribution of the solvent appears. We present here an alternative derivation that can be traced back to Bjerrum, in which the expectation value of KAB is obtained directly through the statistical mechanical method of evaluating its ensemble (Boltzmann-weighted) average. The generalized Bjerrum approach more clearly delineates: (i) the different contributions to binding; (ii) the origin of the much-discussed and somewhat controversial association entropy term; and (iii) where the solvent contribution appears. This approach also allows approximations required for practical evaluation of the binding constant in complex macromolecular systems, to be introduced in a well defined way. We provide an example, with application to test cases that illustrate a range of binding behavior. PMID:12149474

  19. Preparation of macromolecular complexes for cryo-electron microscopy.

    PubMed

    Grassucci, Robert A; Taylor, Derek J; Frank, Joachim

    2007-01-01

    This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.

  20. Critical review and perspective of macromolecularly imprinted polymers.

    PubMed

    Kryscio, David R; Peppas, Nicholas A

    2012-02-01

    Molecular recognition is a fundamental and ubiquitous process that is the driving force behind life. Natural recognition elements - including antibodies, enzymes, nucleic acids, and cells - exploit non-covalent interactions to bind to their targets with exceptionally strong affinities. Due to this unparalleled proficiency, scientists have long sought to mimic natural recognition pathways. One promising approach is molecularly imprinted polymers (MIPs), which are fully synthetic systems formed via the crosslinking of organic polymers in the presence of a template molecule, which results in stereo-specific binding sites for this analyte of interest. Macromolecularly imprinted polymers, those synthesized in the presence of macromolecule templates (>1500 Da), are of particular importance because they open up the field for a whole new set of robust diagnostic tools. Although the specific recognition of small-molecular-weight analytes is now considered routine, extension of these efficacious procedures to the protein regime has, thus far, proved challenging. This paper reviews the main approaches employed, highlights studies of interest with an emphasis on recent work, and offers suggestions for future success in the field of macromolecularly imprinted polymers.

  1. Contrast cystography.

    PubMed

    Essman, Stephanie C

    2005-02-01

    Cystography is a radiographic study performed to aid in evaluation of the urinary bladder for extramural, mural, or intraluminal lesions. These lesions may primarily involve the urinary bladder or may be an extension of disease from adjacent organs. Cystography is easy to perform with relatively few complications. Different types of cystography (positive versus negative contrast) may be used depending on the type of information that the clinician hopes to obtain. Although a valuable technique, it is important to correlate the findings on cystography with other clinical information to arrive at the final diagnosis.

  2. Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.

    PubMed

    Zeiger, Adam S; Loe, Felicia C; Li, Ran; Raghunath, Michael; Van Vliet, Krystyn J

    2012-01-01

    Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro.

  3. Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior

    PubMed Central

    Zeiger, Adam S.; Loe, Felicia C.; Li, Ran; Raghunath, Michael; Van Vliet, Krystyn J.

    2012-01-01

    Microenvironments of biological cells are dominated in vivo by macromolecular crowding and resultant excluded volume effects. This feature is absent in dilute in vitro cell culture. Here, we induced macromolecular crowding in vitro by using synthetic macromolecular globules of nm-scale radius at physiological levels of fractional volume occupancy. We quantified the impact of induced crowding on the extracellular and intracellular protein organization of human mesenchymal stem cells (MSCs) via immunocytochemistry, atomic force microscopy (AFM), and AFM-enabled nanoindentation. Macromolecular crowding in extracellular culture media directly induced supramolecular assembly and alignment of extracellular matrix proteins deposited by cells, which in turn increased alignment of the intracellular actin cytoskeleton. The resulting cell-matrix reciprocity further affected adhesion, proliferation, and migration behavior of MSCs. Macromolecular crowding can thus aid the design of more physiologically relevant in vitro studies and devices for MSCs and other cells, by increasing the fidelity between materials synthesized by cells in vivo and in vitro. PMID:22649562

  4. Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

    PubMed

    Farley, Christopher; Burks, Geoffry; Siegert, Thomas; Juers, Douglas H

    2014-08-01

    In macromolecular cryocrystallography unit-cell parameters can have low reproducibility, limiting the effectiveness of combining data sets from multiple crystals and inhibiting the development of defined repeatable cooling protocols. Here, potential sources of unit-cell variation are investigated and crystal dehydration during loop-mounting is found to be an important factor. The amount of water lost by the unit cell depends on the crystal size, the loop size, the ambient relative humidity and the transfer distance to the cooling medium. To limit water loss during crystal mounting, a threefold strategy has been implemented. Firstly, crystal manipulations are performed in a humid environment similar to the humidity of the crystal-growth or soaking solution. Secondly, the looped crystal is transferred to a vial containing a small amount of the crystal soaking solution. Upon loop transfer, the vial is sealed, which allows transport of the crystal at its equilibrated humidity. Thirdly, the crystal loop is directly mounted from the vial into the cold gas stream. This strategy minimizes the exposure of the crystal to relatively low humidity ambient air, improves the reproducibility of low-temperature unit-cell parameters and offers some new approaches to crystal handling and cryoprotection.

  5. Multiphoton excitation and photodynamic activity of macromolecular derivatized mTHPC

    NASA Astrophysics Data System (ADS)

    Schneider, Marc; Graschew, Georgi; Roelofs, Theo A.; Balanos, Evangelos; Rakowsky, Stefan; Sinn, Hanns-joerg; Schlag, Peter M.

    2000-03-01

    Multiphoton excitation of photosensitizers in photodynamic therapy constitutes a promising approach, because of the increasing tissue penetration for longer wavelength of illumination. In this contribution the photodynamic activity of polyethylene glycol macromolecular derivatized mTHPC upon two-photon excitation is established. To test the photo- activity of the photosensitizer, human colon carcinoma cells, HCT-116, were incubated with 2 (mu) g/ml of mTHPC- CMPEG4 in the nutrition medium. Subsequent pulsed laser irradiation at 784 nm focused down on growing cell monolayers restricts cell vitality clearly within 24 hours after irradiation. To investigate whether an anoxic or euoxic energy transfer mechanism is involved, a uric acid assay was performed to test for the generation of singlet oxygen. Upon single-photon excitation mTHPC-CMPEG4 in TriPEG decomposed uric acid via the generation of singlet oxygen. Using femtosecond pulsed laser irradiation no decomposition of the uric acid was found, implying an anoxic energy transfer mechanism after tow-photon excitation. However, at present, we cannot exclude local hyperthermic effects in the cells containing the photosensitizer to contribute to the photodynamic activity upon two-photon excitation.

  6. The Effect of Attractive Interactions and Macromolecular Crowding on Crystallins Association

    PubMed Central

    Wei, Jiachen; Dobnikar, Jure; Curk, Tine; Song, Fan

    2016-01-01

    In living systems proteins are typically found in crowded environments where their effective interactions strongly depend on the surrounding medium. Yet, their association and dissociation needs to be robustly controlled in order to enable biological function. Uncontrolled protein aggregation often causes disease. For instance, cataract is caused by the clustering of lens proteins, i.e., crystallins, resulting in enhanced light scattering and impaired vision or blindness. To investigate the molecular origins of cataract formation and to design efficient treatments, a better understanding of crystallin association in macromolecular crowded environment is needed. Here we present a theoretical study of simple coarse grained colloidal models to characterize the general features of how the association equilibrium of proteins depends on the magnitude of intermolecular attraction. By comparing the analytic results to the available experimental data on the osmotic pressure in crystallin solutions, we identify the effective parameters regimes applicable to crystallins. Moreover, the combination of two models allows us to predict that the number of binding sites on crystallin is small, i.e. one to three per protein, which is different from previous estimates. We further observe that the crowding factor is sensitive to the size asymmetry between the reactants and crowding agents, the shape of the protein clusters, and to small variations of intermolecular attraction. Our work may provide general guidelines on how to steer the protein interactions in order to control their association. PMID:26954357

  7. High-resolution three-dimensional quantitative map of the macromolecular proton fraction distribution in the normal rat brain.

    PubMed

    Naumova, Anna V; Akulov, Andrey E; Khodanovich, Marina Yu; Yarnykh, Vasily L

    2017-02-01

    The presented dataset provides a normative high-resolution three-dimensional (3D) macromolecular proton fraction (MPF) map of the healthy rat brain in vivo and source images used for its reconstruction. The images were acquired using the protocol described elsewhere (Naumova, et al. High-resolution three-dimensional macromolecular proton fraction mapping for quantitative neuroanatomical imaging of the rodent brain in ultra-high magnetic fields. Neuroimage (2016) doi: 10.1016/j.neuroimage.2016.09.036). The map was reconstructed from three source images with different contrast weightings (proton density, T1, and magnetization transfer) using the single-point algorithm with a synthetic reference image. Source images were acquired from a living animal on an 11.7 T small animal MRI scanner with isotropic spatial resolution of 170 µm(3) and total acquisition time about 1.5 h. The 3D dataset can be used for multiple purposes including interactive viewing of rat brain anatomy, measurements of reference MPF values in various brain structures, and development of image processing techniques for the rodent brain segmentation. It also can serve as a gold standard image for implementation and optimization of rodent brain MRI protocols.

  8. The promise of macromolecular crystallization in microfluidic chips

    NASA Technical Reports Server (NTRS)

    van der Woerd, Mark; Ferree, Darren; Pusey, Marc

    2003-01-01

    Microfluidics, or lab-on-a-chip technology, is proving to be a powerful, rapid, and efficient approach to a wide variety of bioanalytical and microscale biopreparative needs. The low materials consumption, combined with the potential for packing a large number of experiments in a few cubic centimeters, makes it an attractive technique for both initial screening and subsequent optimization of macromolecular crystallization conditions. Screening operations, which require a macromolecule solution with a standard set of premixed solutions, are relatively straightforward and have been successfully demonstrated in a microfluidics platform. Optimization methods, in which crystallization solutions are independently formulated from a range of stock solutions, are considerably more complex and have yet to be demonstrated. To be competitive with either approach, a microfluidics system must offer ease of operation, be able to maintain a sealed environment over several weeks to months, and give ready access for the observation and harvesting of crystals as they are grown.

  9. In-vacuum long-wavelength macromolecular crystallography.

    PubMed

    Wagner, Armin; Duman, Ramona; Henderson, Keith; Mykhaylyk, Vitaliy

    2016-03-01

    Structure solution based on the weak anomalous signal from native (protein and DNA) crystals is increasingly being attempted as part of synchrotron experiments. Maximizing the measurable anomalous signal by collecting diffraction data at longer wavelengths presents a series of technical challenges caused by the increased absorption of X-rays and larger diffraction angles. A new beamline at Diamond Light Source has been built specifically for collecting data at wavelengths beyond the capability of other synchrotron macromolecular crystallography beamlines. Here, the theoretical considerations in support of the long-wavelength beamline are outlined and the in-vacuum design of the endstation is discussed, as well as other hardware features aimed at enhancing the accuracy of the diffraction data. The first commissioning results, representing the first in-vacuum protein structure solution, demonstrate the promising potential of the beamline.

  10. Macromolecular Crystallization in Microfluidics for the International Space Station

    NASA Technical Reports Server (NTRS)

    Monaco, Lisa A.; Spearing, Scott

    2003-01-01

    At NASA's Marshall Space Flight Center, the Iterative Biological Crystallization (IBC) project has begun development on scientific hardware for macromolecular crystallization on the International Space Station (ISS). Currently ISS crystallization research is limited to solution recipes that were prepared on the ground prior to launch. The proposed hardware will conduct solution mixing and dispensing on board the ISS, be fully automated, and have imaging functions via remote commanding from the ground. Utilizing microfluidic technology, IBC will allow for on orbit iterations. The microfluidics LabChip(R) devices that have been developed, along with Caliper Technologies, will greatly benefit researchers by allowing for precise fluid handling of nano/pico liter sized volumes. IBC will maximize the amount of science return by utilizing the microfluidic approach and be a valuable tool to structural biologists investigating medically relevant projects.

  11. Detecting stoichiometry of macromolecular complexes in live cells using FRET

    PubMed Central

    Ben-Johny, Manu; Yue, Daniel N.; Yue, David T.

    2016-01-01

    The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca2+-induced switch in calmodulin stoichiometry with Ca2+ channels—one calmodulin binds at basal cytosolic Ca2+ levels while two calmodulins interact following Ca2+ elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells. PMID:27922011

  12. Large-volume protein crystal growth for neutron macromolecular crystallography

    SciTech Connect

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; Garcia-Ruiz, Juan M.; Hodge, Teresa A.; Huang, Sijay

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.

  13. Large-volume protein crystal growth for neutron macromolecular crystallography

    DOE PAGES

    Ng, Joseph D.; Baird, James K.; Coates, Leighton; ...

    2015-03-30

    Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for themore » growth of crystals to significant dimensions that are now relevant to NMC are revisited. We report that these include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.« less

  14. E-MSD: the European Bioinformatics Institute Macromolecular Structure Database

    PubMed Central

    Boutselakis, H.; Dimitropoulos, D.; Fillon, J.; Golovin, A.; Henrick, K.; Hussain, A.; Ionides, J.; John, M.; Keller, P. A.; Krissinel, E.; McNeil, P.; Naim, A.; Newman, R.; Oldfield, T.; Pineda, J.; Rachedi, A.; Copeland, J.; Sitnov, A.; Sobhany, S.; Suarez-Uruena, A.; Swaminathan, J.; Tagari, M.; Tate, J.; Tromm, S.; Velankar, S.; Vranken, W.

    2003-01-01

    The E-MSD macromolecular structure relational database (http://www.ebi.ac.uk/msd) is designed to be a single access point for protein and nucleic acid structures and related information. The database is derived from Protein Data Bank (PDB) entries. Relational database technologies are used in a comprehensive cleaning procedure to ensure data uniformity across the whole archive. The search database contains an extensive set of derived properties, goodness-of-fit indicators, and links to other EBI databases including InterPro, GO, and SWISS-PROT, together with links to SCOP, CATH, PFAM and PROSITE. A generic search interface is available, coupled with a fast secondary structure domain search tool. PMID:12520052

  15. Macromolecularly "Caged" Carbon Nanoparticles for Intracellular Trafficking via Switchable Photoluminescence.

    PubMed

    Misra, Santosh K; Srivastava, Indrajit; Tripathi, Indu; Daza, Enrique; Ostadhossein, Fatemeh; Pan, Dipanjan

    2017-02-08

    Reversible switching of photoluminescence (PL) of carbon nanoparticles (CNP) can be achieved with counterionic macromolecular caging and decaging at the nanoscale. A negatively charged uncoated, "bare" CNP with high luminescence loses its PL when positively charged macromolecules are wrapped around its surface. Prepared caged carbons could regain their emission only through interaction with anionic surfactant molecules, representing anionic amphiphiles of endocytic membranes. This process could be verified by gel electrophoresis, spectroscopically and in vitro confocal imaging studies. Results indicated for the first time that luminescence switchable CNPs can be synthesized for efficient intracellular tracking. This study further supports the origin of photoluminescence in CNP as a surface phenomenon correlated a function of characteristic charged macromolecules.

  16. Enhancement of Biological Reactions on Cell Surfaces via Macromolecular Crowding

    PubMed Central

    Chapanian, Rafi; Kwan, David H.; Constantinescu, Iren; Shaikh, Fathima A.; Rossi, Nicholas A.A.; Withers, Stephen G.; Kizhakkedathu, Jayachandran N.

    2016-01-01

    The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of Type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors. PMID:25140641

  17. In-vacuum long-wavelength macromolecular crystallography

    PubMed Central

    Wagner, Armin; Duman, Ramona; Henderson, Keith; Mykhaylyk, Vitaliy

    2016-01-01

    Structure solution based on the weak anomalous signal from native (protein and DNA) crystals is increasingly being attempted as part of synchrotron experiments. Maximizing the measurable anomalous signal by collecting diffraction data at longer wavelengths presents a series of technical challenges caused by the increased absorption of X-rays and larger diffraction angles. A new beamline at Diamond Light Source has been built specifically for collecting data at wavelengths beyond the capability of other synchrotron macromolecular crystallography beamlines. Here, the theoretical considerations in support of the long-wavelength beamline are outlined and the in-vacuum design of the endstation is discussed, as well as other hardware features aimed at enhancing the accuracy of the diffraction data. The first commissioning results, representing the first in-vacuum protein structure solution, demonstrate the promising potential of the beamline. PMID:26960130

  18. Bulk-solvent correction in large macromolecular structures.

    PubMed

    Rees, Bernard; Jenner, Lasse; Yusupov, Marat

    2005-09-01

    The estimation of the bulk-solvent contribution to the diffraction of a macromolecular crystal makes use of a solvent mask which delimits the bulk-solvent regions in the crystal. It is shown that the way this mask is usually defined in CNS contains a bias which can lead to absurd results in the case of very large structures, where the calculations can only be made on relatively coarse grids. A modified procedure is described and applied to 70S ribosome data at 5.5 A resolution. The B factor affecting the bulk solvent is also discussed. Even in this case of very high and widely variable atomic B factors, it seems sufficient to consider a constant and isotropic B factor for the bulk solvent. This is initially set to the average value of the atomic B factor, but can be refined.

  19. Revealing the macromolecular targets of complex natural products

    NASA Astrophysics Data System (ADS)

    Reker, Daniel; Perna, Anna M.; Rodrigues, Tiago; Schneider, Petra; Reutlinger, Michael; Mönch, Bettina; Koeberle, Andreas; Lamers, Christina; Gabler, Matthias; Steinmetz, Heinrich; Müller, Rolf; Schubert-Zsilavecz, Manfred; Werz, Oliver; Schneider, Gisbert

    2014-12-01

    Natural products have long been a source of useful biological activity for the development of new drugs. Their macromolecular targets are, however, largely unknown, which hampers rational drug design and optimization. Here we present the development and experimental validation of a computational method for the discovery of such targets. The technique does not require three-dimensional target models and may be applied to structurally complex natural products. The algorithm dissects the natural products into fragments and infers potential pharmacological targets by comparing the fragments to synthetic reference drugs with known targets. We demonstrate that this approach results in confident predictions. In a prospective validation, we show that fragments of the potent antitumour agent archazolid A, a macrolide from the myxobacterium Archangium gephyra, contain relevant information regarding its polypharmacology. Biochemical and biophysical evaluation confirmed the predictions. The results obtained corroborate the practical applicability of the computational approach to natural product ‘de-orphaning’.

  20. Metabolomics reveals elevated macromolecular degradation in periodontal disease.

    PubMed

    Barnes, V M; Ciancio, S G; Shibly, O; Xu, T; Devizio, W; Trivedi, H M; Guo, L; Jönsson, T J

    2011-11-01

    Periodontitis is a chronic inflammatory disease characterized by tissue destruction. In the diseased oral environment, saliva has primarily been considered to act as a protectant by lubricating the tissue, mineralizing the bones, neutralizing the pH, and combating microbes. To understand the metabolic role that saliva plays in the diseased state, we performed untargeted metabolomic profiling of saliva from healthy and periodontitic individuals. Several classes of biochemicals, including dipeptide, amino acid, carbohydrate, lipids, and nucleotide metabolites, were altered, consistent with increased macromolecular degradation of proteins, triacylglycerol, glycerolphospholipids, polysaccharides, and polynucleotides in the individuals with periodontal disease. These changes partially reflected the enhanced host-bacterial interactions in the diseased state as supported by increased levels of bacterially modified amino acids and creatine metabolite. More importantly, the increased lipase, protease, and glycosidase activities associated with periodontitis generated a more favorable energy environment for oral bacteria, potentially exacerbating the disease state.

  1. Scientific Benchmarks for Guiding Macromolecular Energy Function Improvement

    PubMed Central

    Leaver-Fay, Andrew; O’Meara, Matthew J.; Tyka, Mike; Jacak, Ron; Song, Yifan; Kellogg, Elizabeth H.; Thompson, James; Davis, Ian W.; Pache, Roland A.; Lyskov, Sergey; Gray, Jeffrey J.; Kortemme, Tanja; Richardson, Jane S.; Havranek, James J.; Snoeyink, Jack; Baker, David; Kuhlman, Brian

    2013-01-01

    Accurate energy functions are critical to macromolecular modeling and design. We describe new tools for identifying inaccuracies in energy functions and guiding their improvement, and illustrate the application of these tools to improvement of the Rosetta energy function. The feature analysis tool identifies discrepancies between structures deposited in the PDB and low energy structures generated by Rosetta; these likely arise from inaccuracies in the energy function. The optE tool optimizes the weights on the different components of the energy function by maximizing the recapitulation of a wide range of experimental observations. We use the tools to examine three proposed modifications to the Rosetta energy function: improving the unfolded state energy model (reference energies), using bicubic spline interpolation to generate knowledge based torisonal potentials, and incorporating the recently developed Dunbrack 2010 rotamer library (Shapovalov and Dunbrack, 2011). PMID:23422428

  2. On macromolecular refinement at subatomic resolution withinteratomic scatterers

    SciTech Connect

    Afonine, Pavel V.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Lunin, Vladimir Y.; Urzhumtsev, Alexandre

    2007-11-09

    A study of the accurate electron density distribution in molecular crystals at subatomic resolution, better than {approx} 1.0 {angstrom}, requires more detailed models than those based on independent spherical atoms. A tool conventionally used in small-molecule crystallography is the multipolar model. Even at upper resolution limits of 0.8-1.0 {angstrom}, the number of experimental data is insufficient for the full multipolar model refinement. As an alternative, a simpler model composed of conventional independent spherical atoms augmented by additional scatterers to model bonding effects has been proposed. Refinement of these mixed models for several benchmark datasets gave results comparable in quality with results of multipolar refinement and superior of those for conventional models. Applications to several datasets of both small- and macro-molecules are shown. These refinements were performed using the general-purpose macromolecular refinement module phenix.refine of the PHENIX package.

  3. Size-exclusion chromatography system for macromolecular interaction analysis

    DOEpatents

    Stevens, Fred J.

    1988-01-01

    A low pressure, microcomputer controlled system employing high performance liquid chromatography (HPLC) allows for precise analysis of the interaction of two reversibly associating macromolecules such as proteins. Since a macromolecular complex migrates faster than its components during size-exclusion chromatography, the difference between the elution profile of a mixture of two macromolecules and the summation of the elution profiles of the two components provides a quantifiable indication of the degree of molecular interaction. This delta profile is used to qualitatively reveal the presence or absence of significant interaction or to rank the relative degree of interaction in comparing samples and, in combination with a computer simulation, is further used to quantify the magnitude of the interaction in an arrangement wherein a microcomputer is coupled to analytical instrumentation in a novel manner.

  4. Outrunning free radicals in room-temperature macromolecular crystallography

    PubMed Central

    Owen, Robin L.; Axford, Danny; Nettleship, Joanne E.; Owens, Raymond J.; Robinson, James I.; Morgan, Ann W.; Doré, Andrew S.; Lebon, Guillaume; Tate, Christopher G.; Fry, Elizabeth E.; Ren, Jingshan; Stuart, David I.; Evans, Gwyndaf

    2012-01-01

    A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A2A adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macro­molecular crystallography. PMID:22751666

  5. Extracting trends from two decades of microgravity macromolecular crystallization history

    NASA Technical Reports Server (NTRS)

    Judge, Russell A.; Snell, Edward H.; van der Woerd, Mark J.

    2005-01-01

    Since the 1980s hundreds of macromolecular crystal growth experiments have been performed in the reduced acceleration environment of an orbiting spacecraft. Significant enhancements in structural knowledge have resulted from X-ray diffraction of the crystals grown. Similarly, many samples have shown no improvement or degradation in comparison to those grown on the ground. A complex series of interrelated factors affect these experiments and by building a comprehensive archive of the results it was aimed to identify factors that result in success and those that result in failure. Specifically, it was found that dedicated microgravity missions increase the chance of success when compared with those where crystallization took place as a parasitic aspect of the mission. It was also found that the chance of success could not be predicted based on any discernible property of the macromolecule available to us.

  6. Reciprocal Space Mapping of Macromolecular Crystals in the Home Laboratory

    NASA Technical Reports Server (NTRS)

    Snell, Edward H.; Fewster, P. F.; Andrew, Norman; Boggon, T. J.; Judge, Russell A.; Pusey, Marc A.

    1999-01-01

    Reciprocal space mapping techniques are used widely by the materials science community to provide physical information about their crystal samples. We have used similar methods at synchrotron sources to look at the quality of macromolecular crystals produced both on the ground and under microgravity conditions. The limited nature of synchrotron time has led us to explore the use of a high resolution materials research diffractometer to perform similar measurements in the home laboratory. Although the available intensity is much reduced due to the beam conditioning necessary for high reciprocal space resolution, lower resolution data can be collected in the same detail as the synchrotron source. Experiments can be optimized at home to make most benefit from the synchrotron time available. Preliminary results including information on the mosaicity and the internal strains from reciprocal space maps will be presented.

  7. Femtosecond light-induced macromolecular self-assembly

    NASA Astrophysics Data System (ADS)

    Rebane, Aleksander; Mikhaylov, Alexander

    2016-09-01

    We report femtosecond light-induced macromolecular self-assembly (FLIMSA), which is observed when a high peak intensity femtosecond laser beam propagates through aqueous solution of pseudoisocyanine iodide (PIC) J-aggregates and induces the formation of 0.1 - 1.0 mm-size tube-like structure surrounding the laser beam, while at the same time allowing the beam to continue propagating without obstruction or scattering. The FLIMSA material is morphologically heterogeneous and gel-like and is formed at the margins rather than at the center of the beam. As a potential explanation of this effect we assume that the FLIMSA is induced by the high photon flux gradient characteristic of the femtosecond laser beam periphery. This hypothesis is corroborated by control experiments, where J-aggregate samples were illuminated with nanosecond laser sources with a varying pulse duration, power- and beam shape characteristics, but where no FLIMSA formation was observed.

  8. Studying macromolecular complex stoichiometries by peptide‐based mass spectrometry

    PubMed Central

    Wohlgemuth, Ingo; Lenz, Christof

    2015-01-01

    A majority of cellular functions are carried out by macromolecular complexes. A host of biochemical and spectroscopic methods exists to characterize especially protein/protein complexes, however there has been a lack of a universal method to determine protein stoichiometries. Peptide‐based MS, especially as a complementary method to the MS analysis of intact protein complexes, has now been developed to a point where it can be employed to assay protein stoichiometries in a routine manner. While the experimental demands are still significant, peptide‐based MS has been successfully applied to analyze stoichiometries for a variety of protein complexes from very different biological backgrounds. In this review, we discuss the requirements especially for targeted MS acquisition strategies to be used in this context, with a special focus on the interconnected experimental aspects of sample preparation, protein digestion, and peptide stability. In addition, different strategies for the introduction of quantitative peptide standards and their suitability for different scenarios are compared. PMID:25546807

  9. Detecting stoichiometry of macromolecular complexes in live cells using FRET.

    PubMed

    Ben-Johny, Manu; Yue, Daniel N; Yue, David T

    2016-12-06

    The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor's perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca(2+)-induced switch in calmodulin stoichiometry with Ca(2+) channels-one calmodulin binds at basal cytosolic Ca(2+) levels while two calmodulins interact following Ca(2+) elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells.

  10. Conformational States of macromolecular assemblies explored by integrative structure calculation.

    PubMed

    Thalassinos, Konstantinos; Pandurangan, Arun Prasad; Xu, Min; Alber, Frank; Topf, Maya

    2013-09-03

    A detailed description of macromolecular assemblies in multiple conformational states can be very valuable for understanding cellular processes. At present, structural determination of most assemblies in different biologically relevant conformations cannot be achieved by a single technique and thus requires an integrative approach that combines information from multiple sources. Different techniques require different computational methods to allow efficient and accurate data processing and analysis. Here, we summarize the latest advances and future challenges in computational methods that help the interpretation of data from two techniques-mass spectrometry and three-dimensional cryo-electron microscopy (with focus on alignment and classification of heterogeneous subtomograms from cryo-electron tomography). We evaluate how new developments in these two broad fields will lead to further integration with atomic structures to broaden our picture of the dynamic behavior of assemblies in their native environment.

  11. Spontaneous and specific activation of chemical bonds in macromolecular fluids.

    PubMed

    Park, Insun; Shirvanyants, David; Nese, Alper; Matyjaszewski, Krzysztof; Rubinstein, Michael; Sheiko, Sergei S

    2010-09-08

    Mechanical activation of chemical bonds typically involves the application of external forces, which implies a broad distribution of bond tensions. We demonstrate that controlling the flow profile of a macromolecular fluid generates and delineates mechanical force concentration, enabling a hierarchical activation of chemical bonds on different length scales from the macroscopic to the molecular. Bond tension is spontaneously generated within brushlike macromolecules as they spread on a solid substrate. The molecular architecture creates an uneven distribution of tension in the covalent bonds, leading to spatially controlled bond scission. By controlling the flow rate and the gradient of the film pressure, one can sever the flowing macromolecules with high precision. Specific chemical bonds are activated within distinct macromolecules located in a defined area of a thin film. Furthermore, the flow-controlled loading rate enables quantitative analysis of the bond activation parameters.

  12. Macromolecular Crystallization with Microfluidic Free-Interface Diffusion

    SciTech Connect

    Segelke, B

    2005-02-24

    Fluidigm released the Topaz 1.96 and 4.96 crystallization chips in the fall of 2004. Topaz 1.96 and 4.96 are the latest evolution of Fluidigm's microfluidics crystallization technologies that enable ultra low volume rapid screening for macromolecular crystallization. Topaz 1.96 and 4.96 are similar to each other but represent a major redesign of the Topaz system and have of substantially improved ease of automation and ease of use, improved efficiency and even further reduced amount of material needed. With the release of the new Topaz system, Fluidigm continues to set the standard in low volume crystallization screening which is having an increasing impact in the field of structural genomics, and structural biology more generally. In to the future we are likely to see further optimization and increased utility of the Topaz crystallization system, but we are also likely to see further innovation and the emergence of competing technologies.

  13. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    DOE PAGES

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; ...

    2015-01-30

    Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. Despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under themore » same controlled conditions. A model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.« less

  14. Macromolecular structure analysis and effective liquefaction pretreatment. Final report

    SciTech Connect

    Suuberg, E.M.; Yun, Y.; Lilly, W.D.; Leung, K.; Gates, T.; Otake, Y.; Deevi, S.C.

    1994-07-01

    This project was concerned with characterizing the changes in coal macromolecular structure, that are of significance for liquefaction pretreatments of coal. The macromolecular structure of the insoluble portion of coal is difficult to characterize. Techniques that do so indirectly (based upon, for example, NMR and FTIR characterizations of atomic linkages) are not particularly sensitive for this purpose. Techniques that characterize the elastic structure (such as solvent swelling) are much more sensitive to subtle changes in the network structure. It is for this reason that we focused upon these techniques. The overall objective involved identifying pretreatments that reduce the crosslinking (physical or chemical) of the network structure, and thus lead to materials that can be handled to a greater extent by traditional liquid-phase processing techniques. These techniques tend to be inherently more efficient at producing desirable products. This report is divided into seven chapters. Chapter II summarizes the main experimental approaches used throughout the project, and summarizes the main findings on the Argonne Premium coal samples. Chapter III considers synergistic effects of solvent pairs. It is divided into two subsections. The first is concerned with mixtures of CS{sub 2} with electron donor solvents. The second subsection is concerned with aromatic hydrocarbon - alcohol or hydrocarbon - alcohol mixtures, as might be of interest for preliquefaction delivery of catalysts into bituminous coals. Chapter IV deals with questions of how oxidation might influence the results that are obtained. Chapter V briefly details what conclusions may be drawn concerning the elastic behavior of coals, and the effects of thermal treatments on this behavior. Chapter VI is concerned with theories to describe the action of solvents that are capable of dissociating non-covalent crosslinks. Finally, Chapter VII discusses the practical implications of the study.

  15. Macromolecular crystal growth experiments on International Microgravity Laboratory--1.

    PubMed Central

    Day, J.; McPherson, A.

    1992-01-01

    Macromolecular crystal growth experiments, using satellite tobacco mosaic virus (STMV) and canavalin from jack beans as samples, were conducted on a US Space Shuttle mission designated International Microgravity Laboratory--1 (IML-1), flown January 22-29, 1992. Parallel experiments using identical samples were carried out in both a vapor diffusion-based device (PCG) and a liquid-liquid diffusion-based instrument (CRYOSTAT). The experiments in each device were run at 20-22 degrees C and at colder temperatures. Crystals were grown in virtually every trial, but the characteristics of the crystals were highly dependent on the crystallization technique employed and the temperature experience of the sample. In general, very good results, based on visual inspection of the crystals, were obtained in both PCG and CRYOSTAT. Unusually impressive results were, however, achieved for STMV in the CRYOSTAT instrument. STMV crystals grown in microgravity by liquid-liquid diffusion were more than 10-fold greater in total volume than any STMV crystals previously grown in the laboratory. X-ray diffraction data collected from eight STMV crystals grown in CRYOSTAT demonstrated a substantial improvement in diffraction quality over the entire resolution range when compared to data from crystals grown on Earth. In addition, the extent of the diffraction pattern for the STMV crystals grown in space extended to 1.8 A resolution, whereas the best crystals that were ever grown under conditions of Earth's gravity produced data limited to 2.3 A resolution. Other observations indicate that the growth of macromolecular crystals is indeed influenced by the presence or absence of gravity. These observations further suggest, consistent with earlier results, that the elimination of gravity provides a more favorable environment for such processes. PMID:1303744

  16. Radiation damage to nucleoprotein complexes in macromolecular crystallography

    SciTech Connect

    Bury, Charles; Garman, Elspeth F.; Ginn, Helen Mary; Ravelli, Raimond B. G.; Carmichael, Ian; Kneale, Geoff; McGeehan, John E.

    2015-01-30

    Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. Despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. A model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. We observed the protein at low doses and found that they were susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.

  17. Neutron Spectroscopy as a Probe of Macromolecular Structure and Dynamics under Extreme Spatial Confinement

    NASA Astrophysics Data System (ADS)

    Barroso-Bujans, F.; Fernandez-Alonso, F.; Colmenero, J.

    2014-11-01

    We illustrate the use of high-resolution neutron spectroscopy to explore the extreme spatial confinement of soft matter in nanostructured materials. Two well-defined limits are considered, involving either intercalation or interfacial adsorption of the ubiquitous polymer poly(ethylene oxide) in graphite-oxide-based hosts. Vibrational modes associated with the confined macromolecular phase undergo dramatic changes over a broad range of energy transfers, from those associated with intermolecular modes in the Terahertz frequency range (1 THz = 33 cm-1), to those characteristic of strong chemical bonds above 2000 cm-1. We also consider the effects of polymer chain size and chemical composition of the host material. Variation of the degree of oxidation and exfoliation of graphite oxide leads to two distinct cases, namely: (i) subnanometer two-dimensional confinement; and (ii) surface immobilization. Case (i) is characterised by significant changes to conformational and collective vibrational modes of the polymer as a consequence of a preferentially planar trans-trans-trans chain conformation, whereas case (ii) leads to a substantial increase in the population of gauche conformers. Macroscopically, case (i) translates into the complete suppression of crystallization and glassy behaviour. In contrast, case (ii) exhibits well-defined glass and melting transitions associated with the confined phase, yet at significantly lower temperatures than those of the bulk.

  18. Towards an efficient compression of 3D coordinates of macromolecular structures

    PubMed Central

    Valasatava, Yana; Bradley, Anthony R.; Rose, Alexander S.; Duarte, Jose M.; Prlić, Andreas

    2017-01-01

    The size and complexity of 3D macromolecular structures available in the Protein Data Bank is constantly growing. Current tools and file formats have reached limits of scalability. New compression approaches are required to support the visualization of large molecular complexes and enable new and scalable means for data analysis. We evaluated a series of compression techniques for coordinates of 3D macromolecular structures and identified the best performing approaches. By balancing compression efficiency in terms of the decompression speed and compression ratio, and code complexity, our results provide the foundation for a novel standard to represent macromolecular coordinates in a compact and useful file format. PMID:28362865

  19. Towards an efficient compression of 3D coordinates of macromolecular structures.

    PubMed

    Valasatava, Yana; Bradley, Anthony R; Rose, Alexander S; Duarte, Jose M; Prlić, Andreas; Rose, Peter W

    2017-01-01

    The size and complexity of 3D macromolecular structures available in the Protein Data Bank is constantly growing. Current tools and file formats have reached limits of scalability. New compression approaches are required to support the visualization of large molecular complexes and enable new and scalable means for data analysis. We evaluated a series of compression techniques for coordinates of 3D macromolecular structures and identified the best performing approaches. By balancing compression efficiency in terms of the decompression speed and compression ratio, and code complexity, our results provide the foundation for a novel standard to represent macromolecular coordinates in a compact and useful file format.

  20. Macromolecular composition of terrestrial and marine organic matter in sediments across the East Siberian Arctic Shelf

    NASA Astrophysics Data System (ADS)

    Sparkes, Robert B.; Doğrul Selver, Ayça; Gustafsson, Örjan; Semiletov, Igor P.; Haghipour, Negar; Wacker, Lukas; Eglinton, Timothy I.; Talbot, Helen M.; van Dongen, Bart E.

    2016-10-01

    Mobilisation of terrestrial organic carbon (terrOC) from permafrost environments in eastern Siberia has the potential to deliver significant amounts of carbon to the Arctic Ocean, via both fluvial and coastal erosion. Eroded terrOC can be degraded during offshore transport or deposited across the wide East Siberian Arctic Shelf (ESAS). Most studies of terrOC on the ESAS have concentrated on solvent-extractable organic matter, but this represents only a small proportion of the total terrOC load. In this study we have used pyrolysis-gas chromatography-mass spectrometry (py-GCMS) to study all major groups of macromolecular components of the terrOC; this is the first time that this technique has been applied to the ESAS. This has shown that there is a strong offshore trend from terrestrial phenols, aromatics and cyclopentenones to marine pyridines. There is good agreement between proportion phenols measured using py-GCMS and independent quantification of lignin phenol concentrations (r2 = 0.67, p < 0.01, n = 24). Furfurals, thought to represent carbohydrates, show no offshore trend and are likely found in both marine and terrestrial organic matter. We have also collected new radiocarbon data for bulk OC (14COC) which, when coupled with previous measurements, allows us to produce the most comprehensive 14COC map of the ESAS to date. Combining the 14COC and py-GCMS data suggests that the aromatics group of compounds is likely sourced from old, aged terrOC, in contrast to the phenols group, which is likely sourced from modern woody material. We propose that an index of the relative proportions of phenols and pyridines can be used as a novel terrestrial vs. marine proxy measurement for macromolecular organic matter. Principal component analysis found that various terrestrial vs. marine proxies show different patterns across the ESAS, and it shows that multiple river-ocean transects of surface sediments transition from river-dominated to coastal-erosion-dominated to marine

  1. Macromolecular Crowding Enhances Catalytic Efficiency and Stability of α-Amylase

    PubMed Central

    Yadav, Jay Kant

    2013-01-01

    In the present study an attempt was made to investigate the macromolecular crowding effect on functional attributes of α-amylase. High concentrations of sugar based cosolvents, (e.g., trehalose, sucrose, sorbitol, and glycerol) were used to mimic the macromolecular crowding environment (of cellular milieu) under in vitro conditions. To assess the effect of macromolecular crowding, the activity and structural properties of the enzyme were evaluated in the presence of different concentrations of the above cosolvents. Based on the results it is suggested that the macromolecular crowding significantly improves the catalytic efficiency of the enzyme with marginal change in the structure. Out of four cosolvents examined, trehalose was found to be the most effective in consistently enhancing thermal stability of the enzyme. Moreover, the relative effectiveness of the above cosolvents was found to be dependent on their concentration used. PMID:25969780

  2. The Macromolecular Neutron Diffractometer MaNDi at the Spallation Neutron Source

    DOE PAGES

    Coates, Leighton; Cuneo, Matthew J.; Frost, Matthew J.; ...

    2015-07-18

    The Macromolecular Neutron Diffractometer (MaNDi) is located on beamline 11B of the Spallation Neutron Source at Oak Ridge National Laboratory. Moreover, the instrument is a neutron time-of-flight wavelength-resolved Laue diffractometer optimized to collect diffraction data from single crystals. Finally, the instrument has been designed to provide flexibility in several instrumental parameters, such as beam divergence and wavelength bandwidth, to allow data collection from a range of macromolecular systems.

  3. The Macromolecular Neutron Diffractometer MaNDi at the Spallation Neutron Source

    SciTech Connect

    Coates, Leighton; Cuneo, Matthew J.; Frost, Matthew J.; He, Junhong; Weiss, Kevin L.; McFeeters, Hana; Tomanicek, Stephen J.; Vandavasi, Venu Gopal; Langan, Paul; Iverson, Erik B.

    2015-07-18

    The Macromolecular Neutron Diffractometer (MaNDi) is located on beamline 11B of the Spallation Neutron Source at Oak Ridge National Laboratory. Moreover, the instrument is a neutron time-of-flight wavelength-resolved Laue diffractometer optimized to collect diffraction data from single crystals. Finally, the instrument has been designed to provide flexibility in several instrumental parameters, such as beam divergence and wavelength bandwidth, to allow data collection from a range of macromolecular systems.

  4. Contrast-Enhanced Digital Mammography and Angiogenesis

    SciTech Connect

    Rosado-Mendez, I.; Palma, B. A.; Villasenor, Y.; Benitez-Bribiesca, L.; Brandan, M. E.

    2007-11-26

    Angiogenesis could be a means for pouring contrast media around tumors. In this work, optimization of radiological parameters for contrast-enhanced subtraction techniques in mammography has been performed. A modification of Lemacks' analytical formalism was implemented to model the X-ray absorption in the breast with contrast medium and detection by a digital image receptor. Preliminary results of signal-to-noise ratio analysis show the advantage of subtracting two images taken at different energies, one prior and one posterior to the injection of contrast medium. Preliminary experimental results using a custom-made phantom have shown good agreement with calculations. A proposal is presented for the clinical application of the optimized technique, which aims at finding correlations between angiogenesis indicators and dynamic variables of contrast medium uptake.

  5. Automated identification of elemental ions in macromolecular crystal structures

    SciTech Connect

    Echols, Nathaniel Morshed, Nader; Afonine, Pavel V.; McCoy, Airlie J.; Read, Randy J.; Terwilliger, Thomas C.; Adams, Paul D.

    2014-04-01

    The solvent-picking procedure in phenix.refine has been extended and combined with Phaser anomalous substructure completion and analysis of coordination geometry to identify and place elemental ions. Many macromolecular model-building and refinement programs can automatically place solvent atoms in electron density at moderate-to-high resolution. This process frequently builds water molecules in place of elemental ions, the identification of which must be performed manually. The solvent-picking algorithms in phenix.refine have been extended to build common ions based on an analysis of the chemical environment as well as physical properties such as occupancy, B factor and anomalous scattering. The method is most effective for heavier elements such as calcium and zinc, for which a majority of sites can be placed with few false positives in a diverse test set of structures. At atomic resolution, it is observed that it can also be possible to identify tightly bound sodium and magnesium ions. A number of challenges that contribute to the difficulty of completely automating the process of structure completion are discussed.

  6. The Promise of Macromolecular Crystallization in Micro-fluidic Chips

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark; Ferree, Darren; Pusey, Marc

    2003-01-01

    Micro-fluidics, or lab on a chip technology, is proving to be a powerful, rapid, and efficient approach to a wide variety of bio-analytical and microscale bio-preparative needs. The low materials consumption, combined with the potential for packing a large number of experiments in a few cubic centimeters, makes it an attractive technique for both initial screening and subsequent optimization of macromolecular crystallization conditions. Screening operations, which require equilibrating macromolecule solution with a standard set of premixed solutions, are relatively straightforward and have been successfully demonstrated in a micro-fluidics platform. More complex optimization methods, where crystallization solutions are independently formulated from a range of stock solutions, are considerably more complex and have yet to be demonstrated. To be competitive with either approach, a micro-fluidics system must offer ease of operation, be able to maintain a sealed environment over several weeks to months, and give ready access for the observation of crystals as they are grown.

  7. Specific ion effects on macromolecular interactions in Escherichia coli extracts.

    PubMed

    Kyne, Ciara; Ruhle, Brian; Gautier, Virginie W; Crowley, Peter B

    2015-03-01

    Protein characterization in situ remains a major challenge for protein science. Here, the interactions of ΔTat-GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). ΔTat-GB1 was found to participate in high molecular weight complexes that remain intact at physiologically-relevant ionic strength. This observation helps to explain why ΔTat-GB1 was not detected by in-cell NMR spectroscopy. Extracts pre-treated with RNase A had a different SEC elution profile indicating that ΔTat-GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of ΔTat-GB1 could be disrupted by biologically-relevant multivalent ions. The most effective shielding of interactions occurred in Mg(2+) -containing buffers. Moreover, a combination of RNA digestion and Mg(2+) greatly enhanced the NMR detection of ΔTat-GB1 in cell extracts.

  8. Macromolecular crystallization in microgravity generated by a superconducting magnet.

    PubMed

    Wakayama, N I; Yin, D C; Harata, K; Kiyoshi, T; Fujiwara, M; Tanimoto, Y

    2006-09-01

    About 30% of the protein crystals grown in space yield better X-ray diffraction data than the best crystals grown on the earth. The microgravity environments provided by the application of an upward magnetic force constitute excellent candidates for simulating the microgravity conditions in space. Here, we describe a method to control effective gravity and formation of protein crystals in various levels of effective gravity. Since 2002, the stable and long-time durable microgravity generated by a convenient type of superconducting magnet has been available for protein crystal growth. For the first time, protein crystals, orthorhombic lysozyme, were grown at microgravity on the earth, and it was proved that this microgravity improved the crystal quality effectively and reproducibly. The present method always accompanies a strong magnetic field, and the magnetic field itself seems to improve crystal quality. Microgravity is not always effective for improving crystal quality. When we applied this microgravity to the formation of cubic porcine insulin and tetragonal lysozyme crystals, we observed no dependence of effective gravity on crystal quality. Thus, this kind of test will be useful for selecting promising proteins prior to the space experiments. Finally, the microgravity generated by the magnet is compared with that in space, considering the cost, the quality of microgravity, experimental convenience, etc., and the future use of this microgravity for macromolecular crystal growth is discussed.

  9. Synchrotron radiation macromolecular crystallography: science and spin-offs

    PubMed Central

    Helliwell, John R.; Mitchell, Edward P.

    2015-01-01

    A current overview of synchrotron radiation (SR) in macromolecular crystallography (MX) instrumentation, methods and applications is presented. Automation has been and remains a central development in the last decade, as have the rise of remote access and of industrial service provision. Results include a high number of Protein Data Bank depositions, with an increasing emphasis on the successful use of microcrystals. One future emphasis involves pushing the frontiers of using higher and lower photon energies. With the advent of X-ray free-electron lasers, closely linked to SR developments, the use of ever smaller samples such as nanocrystals, nanoclusters and single molecules is anticipated, as well as the opening up of femtosecond time-resolved diffraction structural studies. At SR sources, a very high-throughput assessment for the best crystal samples and the ability to tackle just a few micron and sub-micron crystals will become widespread. With higher speeds and larger detectors, diffraction data volumes are becoming long-term storage and archiving issues; the implications for today and the future are discussed. Together with the rise of the storage ring to its current pre-eminence in MX data provision, the growing tendency of central facility sites to offer other centralized facilities complementary to crystallography, such as cryo-electron microscopy and NMR, is a welcome development. PMID:25866664

  10. JBluIce-EPICS control system for macromolecular crystallography.

    PubMed

    Stepanov, Sergey; Makarov, Oleg; Hilgart, Mark; Pothineni, Sudhir Babu; Urakhchin, Alex; Devarapalli, Satish; Yoder, Derek; Becker, Michael; Ogata, Craig; Sanishvili, Ruslan; Venugopalan, Nagarajan; Smith, Janet L; Fischetti, Robert F

    2011-03-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.

  11. Macromolecular metallurgy of binary mesocrystals via designed multiblock terpolymers.

    PubMed

    Xie, Nan; Liu, Meijiao; Deng, Hanlin; Li, Weihua; Qiu, Feng; Shi, An-Chang

    2014-02-26

    Self-assembling block copolymers provide access to the fabrication of various ordered phases. In particular, the ordered spherical phases can be used to engineer soft mesocrystals with domain size at the 5-100 nm scales. Simple block copolymers, such as diblock copolymers, form a limited number of mesocrystals. However multiblock copolymers are capable to form more complex mesocrystals. We demonstrate that designed B1AB2CB3 multiblock terpolymers, in which the A- and C-blocks form spherical domains and the packing of these spheres can be controlled by changing the lengths of the middle and terminal B-blocks, self-assemble into various binary mesocrystals with space group symmetries of a large number of binary ionic crystals, including NaCl, CsCl, ZnS, α-BN, AlB2, CaF2, TiO2, ReO3, Li3Bi, Nb3Sn(A15), and α-Al2O3. This approach can be generalized to other terpolymers as well as to tetrapolymers to obtain ternary mesocrystals. Our study provides a new concept of macromolecular metallurgy for producing crystal phases in a mesoscale and thus makes multiblock copolymers a robust platform for the engineering of functional materials.

  12. Use of Capillaries for Macromolecular Crystallization in a Cryogenic Dewar

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Hammons, Aaron S.; Hong, Young Soo

    2002-01-01

    The enhanced gaseous nitrogen (EGN) dewar is a cryogenic dry shipper with a sealed cylinder inserted inside along with a temperature monitoring device, and is intended for macromolecular crystallization experiments on the International Space Station. Within the dewar, each crystallization experiment is contained as a solution within a plastic capillary tube. The standard procedure for loading samples in these tubes has involved rapid freezing of the precipitant and biomolecular solution, e.g., protein, directly in liquid nitrogen; this method, however, often resulted in uncontrolled formation of air voids, These air pockets, or bubbles, can lead to irreproducible crystallization results. A novel protocol has been developed to prevent formation of bubbles, and this has been tested in the laboratory as well as aboard the International Space Station during a 42-day long mission of July/August 2001. The gain or loss of mass from solutions within the plastic capillaries revealed that mass transport occurred among separated tubes, and that this mass transport was dependent upon the hygroscopic character of the solution contained in any given tube. The surface area of the plastic capillary tube also related to the observed mass transport. Furthermore, the decreased mass of solutions of-protein correlated to observed formation of protein crystals.

  13. Use of Capillaries for Macromolecular Crystallization in a Cryogenic Dewar

    NASA Technical Reports Server (NTRS)

    Ciszak, Ewa; Hammons, Aaron S.; Hong, Young Soo; Curreri, Peter A. (Technical Monitor)

    2001-01-01

    The Enhanced Gaseous Nitrogen (EGN) Dewar is a cryogenic dry shipper with a sealed cylinder inserted inside along with a temperature-monitoring device, and is intended for macromolecular crystallization experiments on the International Space Station. Within the Dewar, each crystallization experiment is contained as a solution within a plastic capillary. The standard procedure for loading samples in these tubes has involved rapid freezing of the precipitant and biomolecule solution directly in liquid nitrogen; this method, however, often results in uncontrolled formation of air voids. These air pockets, or bubbles, then can lead to irreproducible crystallization results. A novel protocol has been developed to prevent formation of bubbles, and this has been tested in the laboratory as well as aboard the International Space Station during a 42-day long mission of July/August of 2001. Furthermore, gain or loss of mass from solutions within the capillaries revealed that mass transport amongst separated tubes occurred, and that this mass transport was determined by the hygroscopic character of a solution contained in any given tube. The sample volume and the surface area of the plastic capillary tube also related to the observed mass transport.

  14. Macromolecular Crystallography and Structural Biology Databases at NIST

    PubMed Central

    Gilliland, Gary L.

    2001-01-01

    In the late 1970s, macromolecular crystallography at NIST began with collaboration between NIST and NIH to establish a single-crystal neutron diffractometer. This instrument was constructed and employed to solve a number of crystal structures: bovine ribonuclease A, bovine-ribonuclease-uridine vanadate complex, and porcine insulin. In the mid 1980s a Biomolecular Structure Group was created establishing NIST capabilities in biomolecular singe-crystal x-ray diffraction. The group worked on a variety of structural problems until joining the NIST/UMBI Center for Advanced Research in Biotechnology (CARB) in 1987. Crystallographic studies at CARB were then focused on protein engineering efforts that included among others chymosin, subtilisin BPN', interleukin 1β, and glutathione S-transferase. Recently, the structural biology efforts have centered on enzymes in the chorismate metabolic pathways involved in amino acid biosynthesis and in structural genomics that involves determining the structures of “hypothetical” proteins to aid in assigning function. In addition to crystallographic studies, structural biology database activities began with the formal establishment of the Biological Macro-molecule Crystallization Database in 1989. Later, in 1997, NIST in partnership with Rutgers and UCSD formed the Research Collaboratory for Structural Bioinformatics that successfully acquired the Protein Data Bank. The NIST efforts in these activities have focused on data uniformity, establishing and maintaining the physical archive, and working with the NMR community. PMID:27500071

  15. JBluIce-EPICS control system for macromolecular crystallography.

    SciTech Connect

    Stepanov, S.; Makarov, O.; Hilgart, M.; Pothineni, S.; Urakhchin, A.; Devarapalli, S.; Yoder, D.; Becker, M.; Ogata, C.; Sanishvili, R.; Nagarajan, V.; Smith, J. L.; Fischetti, R. F.

    2011-01-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline component. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallographic experiments, especially in the field of microcrystallography.

  16. Reciprocal Space Mapping of Macromolecular Crystals in the Laboratory

    NASA Technical Reports Server (NTRS)

    Snell, Edward H.; Boggon, T. J.; Fewster, P. F.; Siddons, D. P.; Stojanof, V.; Pusey, M. L.

    1998-01-01

    The technique of reciprocal space mapping applied to the physical measurement of macromolecular crystals will be described. This technique uses a triple axis diffractometer setup whereby the monochromator is the first crystal, the sample is the second and the third crystal (of the same material as the monochromator) analyzes the diffracted beam. The geometry is such that it is possible to separate mosaic volume effects from lattice strain effects. The deconvolution of the instrument parameters will also be addressed. Results from measurements at Brookhaven National Synchrotron Radiation Source carried out on microgravity and ground-grown crystals will be presented. The required beam characteristics for reciprocal space mapping are also ideal for topographic studies and the first topographs ever recorded from microgravity protein crystal samples will be shown. We are now working on a system which will enable reciprocal space mapping, mosaicity and topography studies to be carried out in the home laboratory. This system uses a rotating anode X-ray source to provide an intense beam then a Bartels double crystal, four reflection monochromator to provide the spectral and geometric beam conditioning necessary such that the instrument characteristics do not mask the measurement. This is coupled to a high precision diffractometer and sensitive detector. Commissioning data and first results from the system will be presented.

  17. Macromolecular Crystallography and Structural Biology Databases at NIST.

    PubMed

    Gilliland, G L

    2001-01-01

    In the late 1970s, macromolecular crystallography at NIST began with collaboration between NIST and NIH to establish a single-crystal neutron diffractometer. This instrument was constructed and employed to solve a number of crystal structures: bovine ribonuclease A, bovine-ribonuclease-uridine vanadate complex, and porcine insulin. In the mid 1980s a Biomolecular Structure Group was created establishing NIST capabilities in biomolecular singe-crystal x-ray diffraction. The group worked on a variety of structural problems until joining the NIST/UMBI Center for Advanced Research in Biotechnology (CARB) in 1987. Crystallographic studies at CARB were then focused on protein engineering efforts that included among others chymosin, subtilisin BPN', interleukin 1β, and glutathione S-transferase. Recently, the structural biology efforts have centered on enzymes in the chorismate metabolic pathways involved in amino acid biosynthesis and in structural genomics that involves determining the structures of "hypothetical" proteins to aid in assigning function. In addition to crystallographic studies, structural biology database activities began with the formal establishment of the Biological Macro-molecule Crystallization Database in 1989. Later, in 1997, NIST in partnership with Rutgers and UCSD formed the Research Collaboratory for Structural Bioinformatics that successfully acquired the Protein Data Bank. The NIST efforts in these activities have focused on data uniformity, establishing and maintaining the physical archive, and working with the NMR community.

  18. Quantitative influence of macromolecular crowding on gene regulation kinetics

    PubMed Central

    Tabaka, Marcin; Kalwarczyk, Tomasz; Hołyst, Robert

    2014-01-01

    We introduce macromolecular crowding quantitatively into the model for kinetics of gene regulation in Escherichia coli. We analyse and compute the specific-site searching time for 180 known transcription factors (TFs) regulating 1300 operons. The time is between 160 s (e.g. for SoxS Mw = 12.91 kDa) and 1550 s (e.g. for PepA6 of Mw = 329.28 kDa). Diffusion coefficients for one-dimensional sliding are between for large proteins up to for small monomers or dimers. Three-dimensional diffusion coefficients in the cytoplasm are 2 orders of magnitude larger than 1D sliding coefficients, nevertheless the sliding enhances the binding rates of TF to specific sites by 1–2 orders of magnitude. The latter effect is due to ubiquitous non-specific binding. We compare the model to experimental data for LacI repressor and find that non-specific binding of the protein to DNA is activation- and not diffusion-limited. We show that the target location rate by LacI repressor is optimized with respect to microscopic rate constant for association to non-specific sites on DNA. We analyse the effect of oligomerization of TFs and DNA looping effects on searching kinetics. We show that optimal searching strategy depends on TF abundance. PMID:24121687

  19. Macromolecular Powder Diffraction: Ready for genuine biological problems.

    PubMed

    Karavassili, Fotini; Margiolaki, Irene

    2016-01-01

    Knowledge of 3D structures of biological molecules plays a major role in both understanding important processes of life and developing pharmaceuticals. Among several methods available for structure determination, macromolecular X-ray powder diffraction (XRPD) has transformed over the past decade from an impossible dream to a respectable method. XRPD can be employed in biosciences for various purposes such as observing phase transitions, characterizing bulk pharmaceuticals, determining structures via the molecular replacement method, detecting ligands in protein-ligand complexes, as well as combining micro-sized single crystal crystallographic data and powder diffraction data. Studies using synchrotron and laboratory sources in some standard configuration setups are reported in this review, including their respective advantages and disadvantages. Methods presented here provide an alternative, complementary set of tools to resolve structural problems. A variety of already existing software packages for powder diffraction data processing and analysis, some of which have been adapted to large unit cell studies, are briefly described. This review aims to provide necessary elements of theory and current methods, along with practical explanations, available software packages and highlighted case studies.

  20. Timely deposition of macromolecular structures is necessary for peer review

    SciTech Connect

    Joosten, Robbie P.; Soueidan, Hayssam; Wessels, Lodewyk F. A.; Perrakis, Anastassis

    2013-12-01

    Deposition of crystallographic structures should be concurrent with or prior to manuscript submission for peer review, enabling validation and increasing reliability of the PDB. Most of the macromolecular structures in the Protein Data Bank (PDB), which are used daily by thousands of educators and scientists alike, are determined by X-ray crystallography. It was examined whether the crystallographic models and data were deposited to the PDB at the same time as the publications that describe them were submitted for peer review. This condition is necessary to ensure pre-publication validation and the quality of the PDB public archive. It was found that a significant proportion of PDB entries were submitted to the PDB after peer review of the corresponding publication started, and many were only submitted after peer review had ended. It is argued that clear description of journal policies and effective policing is important for pre-publication validation, which is key in ensuring the quality of the PDB and of peer-reviewed literature.

  1. Polyion complex micelle MRI contrast agents from poly(ethylene glycol)-b-poly(l-lysine) block copolymers having Gd-DOTA; preparations and their control of T(1)-relaxivities and blood circulation characteristics.

    PubMed

    Shiraishi, Kouichi; Kawano, Kumi; Maitani, Yoshie; Yokoyama, Masayuki

    2010-12-01

    The current study synthesized macromolecular magnetic resonance imaging (MRI) contrast agents constituted of the poly(ethylene glycol)-b-poly(L-lysine) block copolymer (PEG-P(Lys)). A chelate group, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), was attached to the primary amino group of the block copolymer in desired contents. Gd-DOTA-based macromolecular contrast agents were prepared from PEG-P(Lys) having DOTA (PEG-P(Lys-DOTA) and Gd(III) ions. All of the PEG-P(Lys) block copolymers having gadolinium ions (PEG-P(Lys-DOTA-Gd)) showed higher T(1) relaxivity (per gadolinium), r(1)=5.6-7.3mM(-1)s(-1), than that of a low-molecular-weight gadolinium-chelate, diethylenetriaminepentaacetic acid-gadolinium(III) (Gd-DTPA) at 9.4T. The study prepared the polyion complex (PIC) micelles from the amino groups of the lysine units and an oppositely charged polyanion, poly(methacrylic acid) or dextran sulfate, in an aqueous medium. In contrast, the fully DOTA-attached PEG-P(Lys-DOTA-Gd) formed a PIC with a polycation. Compared with partially DOTA-attached cationic PEG-P(Lys-DOTA-Gd), this PIC micelle yielded a forty percent decrease of r(1). This r(1) decrease was considered to result from a change in the accessibility of water molecules to gadolinium ions in the micelles' inner core. The r(1) was decreased upon formation of the PIC micelle, and this change proved that our concept worked in vitro. Blood-circulation characteristics of PIC micelles were controlled by means of changing the molecular weight of the counter anion. The PIC micelles accumulated in tumor tissues, and MRI study showed T1W image of axial slice of tumor area was significantly enhanced at 24h after the injection.

  2. Contrast Adaptation Contributes to Contrast-Invariance of Orientation Tuning of Primate V1 Cells

    PubMed Central

    Nowak, Lionel G.; Barone, Pascal

    2009-01-01

    Background Studies in rodents and carnivores have shown that orientation tuning width of single neurons does not change when stimulus contrast is modified. However, in these studies, stimuli were presented for a relatively long duration (e. g., 4 seconds), making it possible that contrast adaptation contributed to contrast-invariance of orientation tuning. Our first purpose was to determine, in marmoset area V1, whether orientation tuning is still contrast-invariant with the stimulation duration is comparable to that of a visual fixation. Methodology/Principal Findings We performed extracellular recordings and examined orientation tuning of single-units using static sine-wave gratings that were flashed for 200 msec. Sixteen orientations and three contrast levels, representing low, medium and high values in the range of effective contrasts for each neuron, were randomly intermixed. Contrast adaptation being a slow phenomenon, cells did not have enough time to adapt to each contrast individually. With this stimulation protocol, we found that the tuning width obtained at intermediate contrast was reduced to 89% (median), and that at low contrast to 76%, of that obtained at high contrast. Therefore, when probed with briefly flashed stimuli, orientation tuning is not contrast-invariant in marmoset V1. Our second purpose was to determine whether contrast adaptation contributes to contrast-invariance of orientation tuning. Stationary gratings were presented, as previously, for 200 msec with randomly varying orientations, but the contrast was kept constant within stimulation blocks lasting >20 sec, allowing for adaptation to the single contrast in use. In these conditions, tuning widths obtained at low contrast were still significantly less than at high contrast (median 85%). However, tuning widths obtained with medium and high contrast stimuli no longer differed significantly. Conclusions/Significance Orientation tuning does not appear to be contrast-invariant when

  3. Macromolecular compositions of phytoplankton in the Amundsen Sea, Antarctica

    NASA Astrophysics Data System (ADS)

    Kim, Bo Kyung; Lee, Jang Han; Joo, HuiTae; Song, Ho Jung; Yang, Eun Jin; Lee, Sang Hoon; Lee, Sang H.

    2016-01-01

    The biochemical compositions (proteins, carbohydrates, and lipids) of phytoplankton provide useful information for their environmental growth conditions and nutritional status as a basic food source for upper trophic consumers. Concentrations of these compositions were assessed at 100, 30, and 1% light penetration depths within the euphotic zone in the Amundsen Sea, Antarctica, using colorimetric techniques. The major inorganic nutrients were generally abundant throughout the study area. The average chlorophyll a (chl-a) concentration was 49.2 mg m-2 (S.D.=±27.6 mg m-2) and large phytoplankton (>20 μm) accounted for 64.1% of the total chl-a concentration. The biochemical compositions of the phytoplankton were not significantly different among different light depths or productivity stations. The overall compositions of proteins, carbohydrates, and lipids from all stations averaged 65.9% (S.D.=±12.5%), 22.4% (S.D.=±10.9%), and 11.7% (S.D.=±6.5%), respectively. Regardless of dominant phytoplankton species, nitrogen-abundant conditions sustained high protein compositions of phytoplankton in the Amundsen Sea during the cruise period. Based on the macromolecular compositions, the average food material (FM) concentration was 219.4 μg L-1 (S.D.=±151.1 μg L-1) and correlated positively with the primary productivity in the Amundsen Sea. High protein/carbohydrate ratios (>1) and large proportions of proteins suggest that phytoplankton provide nitrogen-sufficient foods to higher trophic consumers through a higher efficiency of protein carbon incorporated into herbivores.

  4. A MACROMOLECULAR REPEATING UNIT OF MITOCHONDRIAL STRUCTURE AND FUNCTION

    PubMed Central

    Fernández-Morán, H.; Oda, T.; Blair, P. V.; Green, D. E.

    1964-01-01

    A repeating particle associated with the cristae and the inner membrane of the external envelope has been recognized and characterized in beef heart mitochondria by correlated electron microscopic and biochemical studies. Many thousands (ca. 104 to 105) of these particles, disposed in regular arrays, are present in a single mitochondrion. The repeating particle, called the elementary particle (EP), consists of three parts: (1) a spherical or polyhedral head piece (80 to 100 A in diameter); (2) a cylindrical stalk (about 50 A long and 30 to 40 A wide); and (3) a base piece (40 x 110 A). The base pieces of the elementary particles form an integral part of the outer dense layers of the cristae. The elementary particles can be seen in electron micrographs of mitochondria in situ, of isolated mitochondria, and of submitochondrial particles with a complete electron transfer chain. Negative staining with phosphotungstate is only one of several techniques that can be used for reproducible demonstration of the repeating particles and underlying subunit organization of mitochondrial membranes. A particulate unit containing a complete electron transfer chain can be isolated from beef heart mitochondria. The isolated unit approximates in size that of the elementary particle in situ. The molecular weight of the particle in situ is calculated to be 1.3 x 106. Evidence is presented for identifying the isolated unit with the elementary particle visualized in situ. The elementary particle of the mitochondrion is believed to be a prototype of a class of functional particles or macromolecular assemblies of similar size found in association with membranes generally. PMID:14195622

  5. JBluIce–EPICS control system for macromolecular crystallography

    PubMed Central

    Stepanov, Sergey; Makarov, Oleg; Hilgart, Mark; Pothineni, Sudhir Babu; Urakhchin, Alex; Devarapalli, Satish; Yoder, Derek; Becker, Michael; Ogata, Craig; Sanishvili, Ruslan; Venugopalan, Nagarajan; Smith, Janet L.; Fischetti, Robert F.

    2011-01-01

    The trio of macromolecular crystallography beamlines constructed by the General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT) in Sector 23 of the Advanced Photon Source (APS) have been in growing demand owing to their outstanding beam quality and capacity to measure data from crystals of only a few micrometres in size. To take full advantage of the state-of-the-art mechanical and optical design of these beamlines, a significant effort has been devoted to designing fast, convenient, intuitive and robust beamline controls that could easily accommodate new beamline developments. The GM/CA-CAT beamline controls are based on the power of EPICS for distributed hardware control, the rich Java graphical user interface of Eclipse RCP and the task-oriented philosophy as well as the look and feel of the successful SSRL BluIce graphical user interface for crystallography. These beamline controls feature a minimum number of software layers, the wide use of plug-ins that can be written in any language and unified motion controls that allow on-the-fly scanning and optimization of any beamline com­ponent. This paper describes the ways in which BluIce was combined with EPICS and converted into the Java-based JBluIce, discusses the solutions aimed at streamlining and speeding up operations and gives an overview of the tools that are provided by this new open-source control system for facilitating crystallo­graphic experiments, especially in the field of microcrystallo­graphy. PMID:21358048

  6. A simple quantitative model of macromolecular crowding effects on protein folding: Application to the murine prion protein(121-231)

    NASA Astrophysics Data System (ADS)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2013-06-01

    A model of protein folding kinetics is applied to study the effects of macromolecular crowding on protein folding rate and stability. Macromolecular crowding is found to promote a decrease of the entropic cost of folding of proteins that produces an increase of both the stability and the folding rate. The acceleration of the folding rate due to macromolecular crowding is shown to be a topology-dependent effect. The model is applied to the folding dynamics of the murine prion protein (121-231). The differential effect of macromolecular crowding as a function of protein topology suffices to make non-native configurations relatively more accessible.

  7. Leaching of organic acids from macromolecular organic matter by non-supercritical CO2

    NASA Astrophysics Data System (ADS)

    Sauer, P.; Glombitza, C.; Kallmeyer, J.

    2012-04-01

    The storage of CO2 in underground reservoirs is discussed controversly in the scientific literature. The worldwide search for suitable storage formations also considers coal-bearing strata. CO2 is already injected into seams for enhanced recovery of coal bed methane. However, the effects of increased CO2 concentration, especially on organic matter rich formations, are rarely investigated. The injected CO2 will dissolve in the pore water, causing a decrease in pH and resulting in acidic formation waters. Huge amounts of low molecular weight organic acids (LMWOAs) are chemically bound to the macromolecular matrix of sedimentary organic matter and may be liberated by hydrolysis, which is enhanced by the acidic porewater. Recent investigations outlined the importance of LMWOAs as a feedstock for microbial life in the subsurface [1]. Therefore, injection of CO2 into coal formations may result in enhanced nutrient supply for subsurface microbes. To investigate the effect of high concentrations of dissolved CO2 on the release of LMWOAs from coal we developed an inexpensive high-pressure high temperature system that allows manipulating the partial pressure of dissolved gases at pressures and temperatures up to 60 MPa and 120° C, respectively. In a reservoir vessel, gases are added to saturate the extraction medium to the desired level. Inside the extraction vessel hangs a flexible and inert PVDF sleeve (polyvinylidene fluoride, almost impermeable for gases), holding the sample and separating it from the pressure fluid. The flexibility of the sleeve allows for subsampling without loss of pressure. Coal samples from the DEBITS-1 well, Waikato Basin, NZ (R0 = 0.29, TOC = 30%). were extracted at 90° C and 5 MPa, either with pure or CO2-saturated water. Subsamples were taken at different time points during the extraction. The extracted LMWOAs such as formate, acetate and oxalate were analysed by ion chromatography. Yields of LMWOAs were higher with pure water than with CO2

  8. Connexin26 regulates assembly and maintenance of cochlear gap junction macromolecular complex for normal hearing

    NASA Astrophysics Data System (ADS)

    Kamiya, Kazusaku; Fukunaga, Ichiro; Hatakeyama, Kaori; Ikeda, Katsuhisa

    2015-12-01

    Hereditary deafness affects about 1 in 2000 children and GJB2 gene mutation is most frequent cause for this disease in the world. GJB2 encodes connexin26 (Cx26), a component in cochlear gap junction. Recently, we found macromolecular change of gap junction plaques with two different types of Cx26 mutation as major classification of clinical case, one is a model of dominant negative type, Cx26R75W+ and the other is conditional gene deficient mouse, Cx26f/fP0Cre as a model for insufficiency of gap junction protein [6]. Gap junction composed mainly of Cx26 and Cx30 in wild type mice formed large planar gap junction plaques (GJP). In contrast, Cx26R75W+ and Cx26f/fP0Cre showed fragmented small round GJPs around the cell border. In Cx26f/fP0Cre, some of the cells with Cx26 expression due to their cellular mosaicism showed normal large GJP with Cx26 and Cx30 only at the cell junction site between two Cx26 positive cells. These indicate that bilateral Cx26 expressions from both adjacent cells are essential for the formation of the cochlear linear GJP, and it is not compensated by other cochlear Connexins such as Connexin30. In the present study, we demonstrated a new molecular pathology in most common hereditary deafness with different types of Connexin26 mutations, and this machinery can be a new target for drag design of hereditary deafness.

  9. Contrast-induced transient cortical blindness.

    PubMed

    Shah, Parth R; Yohendran, Jayshan; Parker, Geoffrey D; McCluskey, Peter J

    2013-05-01

    We present a case of transient cortical blindness secondary to contrast medium toxicity. A 58-year-old man had successful endovascular coiling of a right posterior inferior cerebellar artery aneurysm but became confused and unable to see after the procedure. His visual acuity was no light perception bilaterally. Clinically, there was no new intra-ocular pathology. An urgent non-contrast computed tomography scan of the brain showed cortical hyperdensity in both parieto-occipital cortices, consistent with contrast medium leakage through the blood-brain barrier from the coiling procedure. The man remained completely blind for 72 hours, after which his visual acuity improved gradually back to his baseline level.

  10. Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography

    PubMed Central

    Chen, Julian C.-H.

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo­graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s. PMID:28250943

  11. Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

    DOE PAGES

    Chen, Julian C.-H.; Unkefer, Clifford Jay

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technicalmore » outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.« less

  12. Fifteen years of the Protein Crystallography Station: the coming of age of macromolecular neutron crystallography.

    PubMed

    Chen, Julian C-H; Unkefer, Clifford J

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002-2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo-graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.

  13. PDBe: improved accessibility of macromolecular structure data from PDB and EMDB

    PubMed Central

    Velankar, Sameer; van Ginkel, Glen; Alhroub, Younes; Battle, Gary M.; Berrisford, John M.; Conroy, Matthew J.; Dana, Jose M.; Gore, Swanand P.; Gutmanas, Aleksandras; Haslam, Pauline; Hendrickx, Pieter M. S.; Lagerstedt, Ingvar; Mir, Saqib; Fernandez Montecelo, Manuel A.; Mukhopadhyay, Abhik; Oldfield, Thomas J.; Patwardhan, Ardan; Sanz-García, Eduardo; Sen, Sanchayita; Slowley, Robert A.; Wainwright, Michael E.; Deshpande, Mandar S.; Iudin, Andrii; Sahni, Gaurav; Salavert Torres, Jose; Hirshberg, Miriam; Mak, Lora; Nadzirin, Nurul; Armstrong, David R.; Clark, Alice R.; Smart, Oliver S.; Korir, Paul K.; Kleywegt, Gerard J.

    2016-01-01

    The Protein Data Bank in Europe (http://pdbe.org) accepts and annotates depositions of macromolecular structure data in the PDB and EMDB archives and enriches, integrates and disseminates structural information in a variety of ways. The PDBe website has been redesigned based on an analysis of user requirements, and now offers intuitive access to improved and value-added macromolecular structure information. Unique value-added information includes lists of reviews and research articles that cite or mention PDB entries as well as access to figures and legends from full-text open-access publications that describe PDB entries. A powerful new query system not only shows all the PDB entries that match a given query, but also shows the ‘best structures’ for a given macromolecule, ligand complex or sequence family using data-quality information from the wwPDB validation reports. A PDBe RESTful API has been developed to provide unified access to macromolecular structure data available in the PDB and EMDB archives as well as value-added annotations, e.g. regarding structure quality and up-to-date cross-reference information from the SIFTS resource. Taken together, these new developments facilitate unified access to macromolecular structure data in an intuitive way for non-expert users and support expert users in analysing macromolecular structure data. PMID:26476444

  14. Contribution of Macromolecular Antioxidants to Dietary Antioxidant Capacity: A Study in the Spanish Mediterranean Diet.

    PubMed

    Pérez-Jiménez, Jara; Díaz-Rubio, M Elena; Saura-Calixto, Fulgencio

    2015-12-01

    Epidemiological and clinical studies show that diets with a high antioxidant capacity, such us those rich in plant food and beverages, are associated with significant decreases in the overall risk of cardiovascular disease or colorectal cancer. Current studies on dietary antioxidants and dietary antioxidant capacity focus exclusively on low molecular weight or soluble antioxidants (vitamins C and E, phenolic compounds and carotenoids), ignoring macromolecular antioxidants. These are polymeric phenolic compounds or polyphenols and carotenoids linked to plant food macromolecules that yield bioavailable metabolites by the action of the microbiota with significant effects either local and/or systemic after absorption. This study determined the antioxidant capacity of the Spanish Mediterranean diet including for the first time both soluble and macromolecular antioxidants. Antioxidant capacity and consumption data of the 54 most consumed plant foods and beverages were used. Results showed that macromolecular antioxidants are the major dietary antioxidants, contributing a 61% to the diet antioxidant capacity (8000 μmol Trolox, determined by ABTS method). The antioxidant capacity data for foods and beverages provided here may be used to estimate the dietary antioxidant capacity in different populations, where similar contributions of macromolecular antioxidants may be expected, and also to design antioxidant-rich diets. Including macromolecular antioxidants in mechanistic, intervention and observational studies on dietary antioxidants may contribute to a better understanding of the role of antioxidants in nutrition and health.

  15. Detection of Macromolecular Fractions in HCN Polymers Using Electrophoretic and Ultrafiltration Techniques.

    PubMed

    Marín-Yaseli, Margarita R; Cid, Cristina; Yagüe, Ana I; Ruiz-Bermejo, Marta

    2017-02-01

    Elucidating the origin of life involves synthetic as well as analytical challenges. Herein, for the first time, we describe the use of gel electrophoresis and ultrafiltration to fractionate HCN polymers. Since the first prebiotic synthesis of adenine by Oró, HCN polymers have gained much interest in studies on the origins of life due to the identification of biomonomers and related compounds within them. Here, we demonstrate that macromolecular fractions with electrophoretic mobility can also be detected within HCN polymers. The migration of polymers under the influence of an electric field depends not only on their sizes (one-dimensional electrophoresis) but also their different isoelectric points (two-dimensional electrophoresis, 2-DE). The same behaviour was observed for several macromolecular fractions detected in HCN polymers. Macromolecular fractions with apparent molecular weights as high as 250 kDa were detected by tricine-SDS gel electrophoresis. Cationic macromolecular fractions with apparent molecular weights as high as 140 kDa were also detected by 2-DE. The HCN polymers synthesized were fractionated by ultrafiltration. As a result, the molecular weight distributions of the macromolecular fractions detected in the HCN polymers directly depended on the synthetic conditions used to produce these polymers. The implications of these results for prebiotic chemistry will be discussed.

  16. PDBe: improved accessibility of macromolecular structure data from PDB and EMDB.

    PubMed

    Velankar, Sameer; van Ginkel, Glen; Alhroub, Younes; Battle, Gary M; Berrisford, John M; Conroy, Matthew J; Dana, Jose M; Gore, Swanand P; Gutmanas, Aleksandras; Haslam, Pauline; Hendrickx, Pieter M S; Lagerstedt, Ingvar; Mir, Saqib; Fernandez Montecelo, Manuel A; Mukhopadhyay, Abhik; Oldfield, Thomas J; Patwardhan, Ardan; Sanz-García, Eduardo; Sen, Sanchayita; Slowley, Robert A; Wainwright, Michael E; Deshpande, Mandar S; Iudin, Andrii; Sahni, Gaurav; Salavert Torres, Jose; Hirshberg, Miriam; Mak, Lora; Nadzirin, Nurul; Armstrong, David R; Clark, Alice R; Smart, Oliver S; Korir, Paul K; Kleywegt, Gerard J

    2016-01-04

    The Protein Data Bank in Europe (http://pdbe.org) accepts and annotates depositions of macromolecular structure data in the PDB and EMDB archives and enriches, integrates and disseminates structural information in a variety of ways. The PDBe website has been redesigned based on an analysis of user requirements, and now offers intuitive access to improved and value-added macromolecular structure information. Unique value-added information includes lists of reviews and research articles that cite or mention PDB entries as well as access to figures and legends from full-text open-access publications that describe PDB entries. A powerful new query system not only shows all the PDB entries that match a given query, but also shows the 'best structures' for a given macromolecule, ligand complex or sequence family using data-quality information from the wwPDB validation reports. A PDBe RESTful API has been developed to provide unified access to macromolecular structure data available in the PDB and EMDB archives as well as value-added annotations, e.g. regarding structure quality and up-to-date cross-reference information from the SIFTS resource. Taken together, these new developments facilitate unified access to macromolecular structure data in an intuitive way for non-expert users and support expert users in analysing macromolecular structure data.

  17. Fifteen years of the Protein Crystallography Station: The coming of age of macromolecular neutron crystallography

    SciTech Connect

    Chen, Julian C.-H.; Unkefer, Clifford Jay

    2017-01-01

    The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002–2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallography in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. As a result, this review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.

  18. Macromolecular properties and polymeric structure of canine tracheal mucins.

    PubMed Central

    Shankar, V; Virmani, A K; Naziruddin, B; Sachdev, G P

    1991-01-01

    Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an

  19. Synthesis and characterization of macromolecular layers grafted to polymer surfaces

    NASA Astrophysics Data System (ADS)

    Burtovyy, Oleksandr

    The composition and behavior of surfaces and interfaces play a pivotal role in dictating the overall efficiency of the majority of polymeric materials and devices. Surface properties of the materials can be altered using surface modification techniques. It is necessary to highlight that successful methods of surface modification should affect only the upper layer of the polymer material without changing bulk properties. The processes must introduce new functionalities to the surface, optimize surface roughness, lubrication, hydrophobicity, hydrophilicity, adhesion, conductivity, and/or biocompatibility. Research presented in this dissertation is dedicated to the synthesis, characterization, and application of thin macromolecular layers anchored to polymer substrates. Specifically, attachment of functional polymers via a "grafting to" approach has been extensively studied using PET and nylon model substrates. First, poly(glycidyl methacrylate) was used to introduce permanent functionalities to the model substrates by anchoring it to model films. Then, three different functional polymers were grafted on top of the previous layer. As one part of this study, the temperature and time dependence of grafting functional layers were studied. The surface coverage by hydrophobic polymer was determined from experimental data and predicted by a model. In general, the model has a high degree of predictive capability. Next, surface modification of polymeric fibers and membranes is presented as an important application of the polymer thin layers targeted in the study. Specifically, the procedures developed for surface modification of model substrates was employed for modification of PET, nylon, and cotton fabrics as well as PET track-etched membranes. Since epoxy groups are highly reactive in various chemical reactions, the approach becomes virtually universal, allowing both various surfaces and end-functionalized macromolecules to be used in the grafted layer synthesis. PET

  20. Path Similarity Analysis: A Method for Quantifying Macromolecular Pathways

    PubMed Central

    Seyler, Sean L.; Kumar, Avishek; Thorpe, M. F.; Beckstein, Oliver

    2015-01-01

    Diverse classes of proteins function through large-scale conformational changes and various sophisticated computational algorithms have been proposed to enhance sampling of these macromolecular transition paths. Because such paths are curves in a high-dimensional space, it has been difficult to quantitatively compare multiple paths, a necessary prerequisite to, for instance, assess the quality of different algorithms. We introduce a method named Path Similarity Analysis (PSA) that enables us to quantify the similarity between two arbitrary paths and extract the atomic-scale determinants responsible for their differences. PSA utilizes the full information available in 3N-dimensional configuration space trajectories by employing the Hausdorff or Fréchet metrics (adopted from computational geometry) to quantify the degree of similarity between piecewise-linear curves. It thus completely avoids relying on projections into low dimensional spaces, as used in traditional approaches. To elucidate the principles of PSA, we quantified the effect of path roughness induced by thermal fluctuations using a toy model system. Using, as an example, the closed-to-open transitions of the enzyme adenylate kinase (AdK) in its substrate-free form, we compared a range of protein transition path-generating algorithms. Molecular dynamics-based dynamic importance sampling (DIMS) MD and targeted MD (TMD) and the purely geometric FRODA (Framework Rigidity Optimized Dynamics Algorithm) were tested along with seven other methods publicly available on servers, including several based on the popular elastic network model (ENM). PSA with clustering revealed that paths produced by a given method are more similar to each other than to those from another method and, for instance, that the ENM-based methods produced relatively similar paths. PSA applied to ensembles of DIMS MD and FRODA trajectories of the conformational transition of diphtheria toxin, a particularly challenging example, showed that

  1. New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection

    PubMed Central

    Soltis, S. Michael; Cohen, Aina E.; Deacon, Ashley; Eriksson, Thomas; González, Ana; McPhillips, Scott; Chui, Hsui; Dunten, Pete; Hollenbeck, Michael; Mathews, Irimpan; Miller, Mitch; Moorhead, Penjit; Phizackerley, R. Paul; Smith, Clyde; Song, Jinhu; van dem Bedem, Henry; Ellis, Paul; Kuhn, Peter; McPhillips, Timothy; Sauter, Nicholas; Sharp, Kenneth; Tsyba, Irina; Wolf, Guenter

    2008-01-01

    Complete automation of the macromolecular crystallography experiment has been achieved at SSRL through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface. These highly reliable systems have enabled crystallography experiments to be carried out from the researchers’ home institutions and other remote locations while retaining complete control over even the most challenging systems. A breakthrough component of the system, the Stanford Auto-Mounter (SAM), has enabled the efficient mounting of cryocooled samples without human intervention. Taking advantage of this automation, researchers have successfully screened more than 200 000 samples to select the crystals with the best diffraction quality for data collection as well as to determine optimal crystallization and cryocooling conditions. These systems, which have been deployed on all SSRL macromolecular crystallography beamlines and several beamlines worldwide, are used by more than 80 research groups in remote locations, establishing a new paradigm for macromolecular crystallo­graphy experimentation. PMID:19018097

  2. Self-organization in macromolecular systems: the notion of adaptive value.

    PubMed

    Demetrius, L

    1984-10-01

    Self-organization in macromolecular systems refers to the transition from a random assembly of interacting oligomers to a system of stable heteropolymers. The concept of adaptive value describes the correlation between environmental variability and the variability in replication and mutation rates of the interacting oligomers. This paper describes a model of self-organization in macromolecular systems based on the concept of adaptive value. The equilibrium states of a set of interacting polymers are described by states that maximize the adaptive value. The analytic basis for this notion of equilibrium, which is called the adaptive value principle, is given and this principle is invoked to explain two examples of macromolecular self-assembly.

  3. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.

    PubMed

    Langer, Gerrit; Cohen, Serge X; Lamzin, Victor S; Perrakis, Anastassis

    2008-01-01

    ARP/wARP is a software suite to build macromolecular models in X-ray crystallography electron density maps. Structural genomics initiatives and the study of complex macromolecular assemblies and membrane proteins all rely on advanced methods for 3D structure determination. ARP/wARP meets these needs by providing the tools to obtain a macromolecular model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best-fitting ligand from a 'cocktail'; and finding ordered water molecules. All protocols are easy to handle by a nonexpert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours.

  4. Host responses and metabolic profiles of wood components in Dutch elm hybrids with a contrasting tolerance to Dutch elm disease

    PubMed Central

    Ďurkovič, Jaroslav; Kačík, František; Olčák, Dušan; Kučerová, Veronika; Krajňáková, Jana

    2014-01-01

    Background and Aims Changes occurring in the macromolecular traits of cell wall components in elm wood following attack by Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), are poorly understood. The purpose of this study was to compare host responses and the metabolic profiles of wood components for two Dutch elm (Ulmus) hybrids, ‘Groeneveld’ (a susceptible clone) and ‘Dodoens’ (a tolerant clone), that have contrasting survival strategies upon infection with the current prevalent strain of DED. Methods Ten-year-old plants of the hybrid elms were inoculated with O. novo-ulmi ssp. americana × novo-ulmi. Measurements were made of the content of main cell wall components and extractives, lignin monomer composition, macromolecular traits of cellulose and neutral saccharide composition. Key Results Upon infection, medium molecular weight macromolecules of cellulose were degraded in both the susceptible and tolerant elm hybrids, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels, but rarely in the fibres. The 13C nuclear magnetic resonance spectra revealed that loss of crystalline and non-crystalline cellulose regions occurred in parallel. The rate of cellulose degradation was influenced by the syringyl:guaiacyl ratio in lignin. Both hybrids commonly responded to the medium molecular weight cellulose degradation with the biosynthesis of high molecular weight macromolecules of cellulose, resulting in a significant increase in values for the degree of polymerization and polydispersity. Other responses of the hybrids included an increase in lignin content, a decrease in relative proportions of d-glucose, and an increase in proportions of d-xylose. Differential responses between the hybrids were found in the syringyl:guaiacyl ratio in lignin. Conclusions In susceptible ‘Groeneveld’ plants, syringyl-rich lignin provided a far greater degree of protection from cellulose degradation than in ‘Dodoens’, but

  5. Macromolecular Hydrogen Sulfide Donors Trigger Spatiotemporally Confined Changes in Cell Signaling.

    PubMed

    Ercole, Francesca; Mansfeld, Friederike M; Kavallaris, Maria; Whittaker, Michael R; Quinn, John F; Halls, Michelle L; Davis, Thomas P

    2016-01-11

    Hydrogen sulfide (H2S) is involved in a myriad of cell signaling processes that trigger physiological events ranging from vasodilation to cell proliferation. Moreover, disturbances to H2S signaling have been associated with numerous pathologies. As such, the ability to release H2S in a cellular environment and stimulate signaling events is of considerable interest. Herein we report the synthesis of macromolecular H2S donors capable of stimulating cell signaling pathways in both the cytosol and at the cell membrane. Specifically, copolymers having pendent oligo(ethylene glycol) and benzonitrile groups were synthesized, and the benzonitrile groups were subsequently transformed into primary aryl thioamide groups via thionation using sodium hydrosulfide. These thioamide moieties could be incorporated into a hydrophilic copolymer or a block copolymer (i.e., into either the hydrophilic or hydrophobic domain). An electrochemical sensor was used to demonstrate release of H2S under simulated physiological conditions. Subsequent treatment of HEK293 cells with a macromolecular H2S donor elicited a slow and sustained increase in cytosolic ERK signaling, as monitored using a FRET-based biosensor. The macromolecular donor was also shown to induce a small, fast and sustained increase in plasma membrane-localized PKC activity immediately following addition to cells. Studies using an H2S-selective fluorescent probe in live cells confirmed release of H2S from the macromolecular donor over physiologically relevant time scales consistent with the signaling observations. Taken together, these results demonstrate that by using macromolecular H2S donors it is possible to trigger spatiotemporally confined cell signaling events. Moreover, the localized nature of the observed signaling suggests that macromolecular donor design may provide an approach for selectively stimulating certain cellular biochemical pathways.

  6. Influence of protein crowder size on hydration structure and dynamics in macromolecular crowding

    NASA Astrophysics Data System (ADS)

    Wang, Po-hung; Yu, Isseki; Feig, Michael; Sugita, Yuji

    2017-03-01

    We investigate the effects of protein crowder sizes on hydration structure and dynamics in macromolecular crowded systems by all-atom MD simulations. The crowded systems consisting of only small proteins showed larger total surface areas than those of large proteins at the same volume fractions. As a result, more water molecules were trapped within the hydration shells, slowing down water diffusion. The simulation results suggest that the protein crowder size is another factor to determine the effect of macromolecular crowding and to explain the experimental kinetic data of proteins and DNAs in the presence of crowding agents.

  7. Visualizing Macromolecular Complexes with In Situ Liquid Scanning Transmission Electron Microscopy

    SciTech Connect

    Evans, James E.; Jungjohann, K. L.; Wong, Peony C. K.; Chiu, Po-Lin; Dutrow, Gavin H.; Arslan, Ilke; Browning, Nigel D.

    2012-11-01

    A central focus of biological research is understanding the structure/function relationship of macromolecular protein complexes. Yet conventional transmission electron microscopy techniques are limited to static observations. Here we present the first direct images of purified macromolecular protein complexes using in situ liquid scanning transmission electron microscopy. Our results establish the capability of this technique for visualizing the interface between biology and nanotechnology with high fidelity while also probing the interactions of biomolecules within solution. This method represents an important advancement towards allowing future high-resolution observations of biological processes and conformational dynamics in real-time.

  8. Visualizing macromolecular complexes with in situ liquid scanning transmission electron microscopy.

    PubMed

    Evans, James E; Jungjohann, Katherine L; Wong, Peony C K; Chiu, Po-Lin; Dutrow, Gavin H; Arslan, Ilke; Browning, Nigel D

    2012-11-01

    A central focus of biological research is understanding the structure/function relationship of macromolecular protein complexes. Yet conventional transmission electron microscopy techniques are limited to static observations. Here we present the first direct images of purified macromolecular protein complexes using in situ liquid scanning transmission electron microscopy. Our results establish the capability of this technique for visualizing the interface between biology and nanotechnology with high fidelity while also probing the interactions of biomolecules within solution. This method represents an important advancement towards allowing future high-resolution observations of biological processes and conformational dynamics in real-time.

  9. Accounting for large amplitude protein deformation during in silico macromolecular docking.

    PubMed

    Bastard, Karine; Saladin, Adrien; Prévost, Chantal

    2011-02-22

    Rapid progress of theoretical methods and computer calculation resources has turned in silico methods into a conceivable tool to predict the 3D structure of macromolecular assemblages, starting from the structure of their separate elements. Still, some classes of complexes represent a real challenge for macromolecular docking methods. In these complexes, protein parts like loops or domains undergo large amplitude deformations upon association, thus remodeling the surface accessible to the partner protein or DNA. We discuss the problems linked with managing such rearrangements in docking methods and we review strategies that are presently being explored, as well as their limitations and success.

  10. Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization

    NASA Astrophysics Data System (ADS)

    Rashid, Rafi; Chee, Stella Min Ling; Raghunath, Michael; Wohland, Thorsten

    2015-05-01

    Macromolecular crowding (MMC) has been used in various in vitro experimental systems to mimic in vivo physiology. This is because the crowded cytoplasm of cells contains many different types of solutes dissolved in an aqueous medium. MMC in the extracellular microenvironment is involved in maintaining stem cells in their undifferentiated state (niche) as well as in aiding their differentiation after they have travelled to new locations outside the niche. MMC at physiologically relevant fractional volume occupancies (FVOs) significantly enhances the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells during chemically induced adipogenesis. The mechanism by which MMC produces this enhancement is not entirely known. In the context of extracellular collagen deposition, we have recently reported the importance of optimizing the FVO while minimizing the bulk viscosity. Two opposing properties will determine the net rate of a biochemical reaction: the negative effect of bulk viscosity and the positive effect of the excluded volume, the latter being expressed by the FVO. In this study we have looked more closely at the effect of viscosity on reaction rates. We have used fluorimetry to measure the rate of actin polymerization and fluorescence correlation spectroscopy (FCS) to measure diffusion of various probes in solutions containing the crowder Ficoll at physiological concentrations. Similar to its effect on collagen, Ficoll enhanced the actin polymerization rate despite increasing the bulk viscosity. Our FCS measurements reveal a relatively minor component of anomalous diffusion. In addition, our measurements do suggest that microviscosity becomes relevant in a crowded environment. We ruled out bulk viscosity as a cause of the rate enhancement by performing the actin polymerization assay in glycerol. These opposite effects of Ficoll and glycerol led us to conclude that microviscosity becomes relevant at the length scale of the reacting

  11. Macromolecular crowding gives rise to microviscosity, anomalous diffusion and accelerated actin polymerization.

    PubMed

    Rashid, Rafi; Chee, Stella Min Ling; Raghunath, Michael; Wohland, Thorsten

    2015-04-30

    Macromolecular crowding (MMC) has been used in various in vitro experimental systems to mimic in vivo physiology. This is because the crowded cytoplasm of cells contains many different types of solutes dissolved in an aqueous medium. MMC in the extracellular microenvironment is involved in maintaining stem cells in their undifferentiated state (niche) as well as in aiding their differentiation after they have travelled to new locations outside the niche. MMC at physiologically relevant fractional volume occupancies (FVOs) significantly enhances the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells during chemically induced adipogenesis. The mechanism by which MMC produces this enhancement is not entirely known. In the context of extracellular collagen deposition, we have recently reported the importance of optimizing the FVO while minimizing the bulk viscosity. Two opposing properties will determine the net rate of a biochemical reaction: the negative effect of bulk viscosity and the positive effect of the excluded volume, the latter being expressed by the FVO. In this study we have looked more closely at the effect of viscosity on reaction rates. We have used fluorimetry to measure the rate of actin polymerization and fluorescence correlation spectroscopy (FCS) to measure diffusion of various probes in solutions containing the crowder Ficoll at physiological concentrations. Similar to its effect on collagen, Ficoll enhanced the actin polymerization rate despite increasing the bulk viscosity. Our FCS measurements reveal a relatively minor component of anomalous diffusion. In addition, our measurements do suggest that microviscosity becomes relevant in a crowded environment. We ruled out bulk viscosity as a cause of the rate enhancement by performing the actin polymerization assay in glycerol. These opposite effects of Ficoll and glycerol led us to conclude that microviscosity becomes relevant at the length scale of the reacting

  12. Hydropyrolysis: A new technique for the analysis of macromolecular material in meteorites

    NASA Astrophysics Data System (ADS)

    Sephton, Mark A.; Love, Gordon D.; Meredith, Will; Snape, Colin E.; Sun, Cheng-Gong; Watson, Jonathan S.

    2005-10-01

    The carbonaceous chondrite meteorites are fragments of asteroids that have remained relatively unprocessed since the formation of the Solar System 4.56 billion years ago. The major organic component in these meteorites is a macromolecular phase that is resistant to solvent extraction. The information contained within macromolecular material can be accessed by degradative techniques such as pyrolysis. Hydropyrolysis refers to pyrolysis assisted by high hydrogen gas pressures and a dispersed sulphided molybdenum catalyst. Hydropyrolysis of the Murchison macromolecular material successfully releases much greater quantities of hydrocarbons than traditional pyrolysis techniques (twofold greater than hydrous pyrolysis) including significant amounts of high molecular weight polyaromatic hydrocarbons (PAH) such as phenanthrene, carbazole, fluoranthene, pyrene, chrysene, perylene, benzoperylene and coronene units with varying degrees of alkylation. When hydropyrolysis products are collected using a silica trap immersed in liquid nitrogen, the technique enables the solubilisation and retention of compounds with a wide range of volatilities (i.e. benzene to coronene). This report describes the hydropyrolysis method and the information it can provide about meteorite macromolecular material constitution.

  13. Effects of macromolecular crowding on the inhibition of virus assembly and virus-cell receptor recognition.

    PubMed

    Rincón, Verónica; Bocanegra, Rebeca; Rodríguez-Huete, Alicia; Rivas, Germán; Mateu, Mauricio G

    2011-02-02

    Biological fluids contain a very high total concentration of macromolecules that leads to volume exclusion by one molecule to another. Theory and experiment have shown that this condition, termed macromolecular crowding, can have significant effects on molecular recognition. However, the influence of molecular crowding on recognition events involving virus particles, and their inhibition by antiviral compounds, is virtually unexplored. Among these processes, capsid self-assembly during viral morphogenesis and capsid-cell receptor recognition during virus entry into cells are receiving increasing attention as targets for the development of new antiviral drugs. In this study, we have analyzed the effect of macromolecular crowding on the inhibition of these two processes by peptides. Macromolecular crowding led to a significant reduction in the inhibitory activity of: 1), a capsid-binding peptide and a small capsid protein domain that interfere with assembly of the human immunodeficiency virus capsid, and 2), a RGD-containing peptide able to block the interaction between foot-and-mouth disease virus and receptor molecules on the host cell membrane (in this case, the effect was dependent on the conditions used). The results, discussed in the light of macromolecular crowding theory, are relevant for a quantitative understanding of molecular recognition processes during virus infection and its inhibition.

  14. Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase.

    PubMed

    Nadar, Shamraja S; Muley, Abhijeet B; Ladole, Mayur R; Joshi, Pranoti U

    2016-03-01

    Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase were prepared by precipitation and subsequent cross-linking. The non-toxic, biodegradable, biocompatible, renewable polysaccharide based macromolecular cross-linkers viz. agar, chitosan, dextran, and gum arabic were used as a substitute for traditional glutaraldehyde to augment activity recovery toward macromolecular substrate. Macromolecular cross-linkers were prepared by periodate mediated controlled oxidation of polysaccharides. The effects of precipitating agent, concentration and different cross-linkers on activity recovery of α-amylase CLEAs were investigated. α-Amylase aggregated with ammonium sulphate and cross-linked by dextran showed 91% activity recovery, whereas glutaraldehyde CLEAs (G-CLEAs) exhibited 42% activity recovery. M-CLEAs exhibited higher thermal stability in correlation with α-amylase and G-CLEAs. Moreover, dextran and chitosan M-CLEAs showed same affinity for starch hydrolysis as of free α-amylase. The changes in secondary structures revealed the enhancements in structural and conformational rigidity attributed by cross-linkers. Finally, after five consecutive cycles dextran M-CLEAs retained 1.25 times higher initial activity than G-CLEAs.

  15. The interplay of intrinsic disorder and macromolecular crowding on α-synuclein fibril formation

    NASA Astrophysics Data System (ADS)

    Shirai, Nobu C.; Kikuchi, Macoto

    2016-02-01

    α-synuclein (α-syn) is an intrinsically disordered protein which is considered to be one of the causes of Parkinson's disease. This protein forms amyloid fibrils when in a highly concentrated solution. The fibril formation of α-syn is induced not only by increases in α-syn concentration but also by macromolecular crowding. In order to investigate the coupled effect of the intrinsic disorder of α-syn and macromolecular crowding, we construct a lattice gas model of α-syn in contact with a crowding agent reservoir based on statistical mechanics. The main assumption is that α-syn can be expressed as coarse-grained particles with internal states coupled with effective volume; and disordered states are modeled by larger particles with larger internal entropy than other states. Thanks to the simplicity of the model, we can exactly calculate the number of conformations of crowding agents, and this enables us to prove that the original grand canonical ensemble with a crowding agent reservoir is mathematically equivalent to a canonical ensemble without crowding agents. In this expression, the effect of macromolecular crowding is absorbed in the internal entropy of disordered states; it is clearly shown that the crowding effect reduces the internal entropy. Based on Monte Carlo simulation, we provide scenarios of crowding-induced fibril formation. We also discuss the recent controversy over the existence of helically folded tetramers of α-syn, and suggest that macromolecular crowding is the key to resolving the controversy.

  16. Hypersensitivity to contrast media and dyes.

    PubMed

    Brockow, Knut; Sánchez-Borges, Mario

    2014-08-01

    This article updates current knowledge on hypersensitivity reactions to diagnostic contrast media and dyes. After application of a single iodinated radiocontrast medium (RCM), gadolinium-based contrast medium, fluorescein, or a blue dye, a hypersensitivity reaction is not a common finding; however, because of the high and still increasing frequency of those procedures, patients who have experienced severe reactions are nevertheless frequently encountered in allergy departments. Evidence on allergologic testing and management is best for iodinated RCM, limited for blue dyes, and insufficient for fluorescein. Skin tests can be helpful in the diagnosis of patients with hypersensitivity reactions to these compounds.

  17. Apparatus for separating particles utilizing engineered acoustic contrast capture particles

    DOEpatents

    Kaduchak, Gregory; Ward, Michael D

    2016-05-17

    An apparatus for separating particles from a medium includes a capillary defining a flow path therein that is in fluid communication with a medium source. The medium source includes engineered acoustic contrast capture particle having a predetermined acoustic contrast. The apparatus includes a vibration generator that is operable to produce at least one acoustic field within the flow path. The acoustic field produces a force potential minima for positive acoustic contrast particles and a force potential minima for negative acoustic contrast particles in the flow path and drives the engineered acoustic contrast capture particles to either the force potential minima for positive acoustic contrast particles or the force potential minima for negative acoustic contrast particles.

  18. Apparatus for separating particles utilizing engineered acoustic contrast capture particles

    DOEpatents

    Kaduchak, Gregory; Ward, Michael D.

    2011-12-27

    An apparatus for separating particles from a medium includes a capillary defining a flow path therein that is in fluid communication with a medium source. The medium source includes engineered acoustic contrast capture particle having a predetermined acoustic contrast. The apparatus includes a vibration generator that is operable to produce at least one acoustic field within the flow path. The acoustic field produces a force potential minima for positive acoustic contrast particles and a force potential minima for negative acoustic contrast particles in the flow path and drives the engineered acoustic contrast capture particles to either the force potential minima for positive acoustic contrast particles or the force potential minima for negative acoustic contrast particles.

  19. Apparatus for separating particles utilizing engineered acoustic contrast capture particles

    SciTech Connect

    Kaduchak, Gregory; Ward, Michael D

    2014-10-21

    An apparatus for separating particles from a medium includes a capillary defining a flow path therein that is in fluid communication with a medium source. The medium source includes engineered acoustic contrast capture particle having a predetermined acoustic contrast. The apparatus includes a vibration generator that is operable to produce at least one acoustic field within the flow path. The acoustic field produces a force potential minima for positive acoustic contrast particles and a force potential minima for negative acoustic contrast particles in the flow path and drives the engineered acoustic contrast capture particles to either the force potential minima for positive acoustic contrast particles or the force potential minima for negative acoustic contrast particles.

  20. Flexibility damps macromolecular crowding effects on protein folding dynamics: Application to the murine prion protein (121-231)

    NASA Astrophysics Data System (ADS)

    Bergasa-Caceres, Fernando; Rabitz, Herschel A.

    2014-01-01

    A model of protein folding kinetics is applied to study the combined effects of protein flexibility and macromolecular crowding on protein folding rate and stability. It is found that the increase in stability and folding rate promoted by macromolecular crowding is damped for proteins with highly flexible native structures. The model is applied to the folding dynamics of the murine prion protein (121-231). It is found that the high flexibility of the native isoform of the murine prion protein (121-231) reduces the effects of macromolecular crowding on its folding dynamics. The relevance of these findings for the pathogenic mechanism are discussed.

  1. The Contrastive Analysis Hypothesis

    ERIC Educational Resources Information Center

    Wardhaugh, Ronald

    1970-01-01

    Discusses the strong contrastive analysis hypothesis, which claims predictive powers for contrastive analysis, and the weak hypothesis, which claims only that contrastive analysis can help account for observed difficulties in second language learning. The strong hypothesis is found untenable, and difficulties with the weak hypothesis are discussed…

  2. The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes

    PubMed Central

    Boeri Erba, Elisabetta; Petosa, Carlo

    2015-01-01

    Mass spectrometry (MS) is a powerful tool for determining the mass of biomolecules with high accuracy and sensitivity. MS performed under so-called “native conditions” (native MS) can be used to determine the mass of biomolecules that associate noncovalently. Here we review the application of native MS to the study of protein−ligand interactions and its emerging role in elucidating the structure of macromolecular assemblies, including soluble and membrane protein complexes. Moreover, we discuss strategies aimed at determining the stoichiometry and topology of subunits by inducing partial dissociation of the holo-complex. We also survey recent developments in "native top-down MS", an approach based on Fourier Transform MS, whereby covalent bonds are broken without disrupting non-covalent interactions. Given recent progress, native MS is anticipated to play an increasingly important role for researchers interested in the structure of macromolecular complexes. PMID:25676284

  3. From "simple" DNA-protein interactions to the macromolecular machines of gene expression.

    PubMed

    von Hippel, Peter H

    2007-01-01

    The physicochemical concepts that underlie our present ideas on the structure and assembly of the "macromolecular machines of gene expression" are developed, starting with the structure and folding of the individual protein and DNA components, the thermodynamics and kinetics of their conformational rearrangements during complex assembly, and the molecular basis of the sequence specificity and recognition interactions of the final assemblies that include the DNA genome. The role of diffusion in reduced dimensions in the kinetics of the assembly of macromolecular machines from their components is also considered, and diffusion-driven reactions are compared with those fueled by ATP binding and hydrolysis, as well as by the specific covalent chemical modifications involved in rearranging chromatin and modifying signal transduction networks in higher organisms.

  4. PIMADb: A Database of Protein–Protein Interactions in Huge Macromolecular Assemblies

    PubMed Central

    Mathew, Oommen K.; Sowdhamini, Ramanathan

    2016-01-01

    Protein–protein interactions play a very important role in the process of cellular functionality. Intricate details about the interactions between the proteins in a macromolecular assembly are important to understand the function and significance of protein complexes. We are reporting about a database of protein–protein interactions in huge macromolecular assemblies (PIMADb) that records the intrinsic details of 189,532 interchain interactions in 40,049 complexes from the Protein Data Bank. These details include the results of the quantification and analysis of all the interactions in the complex. The availability of interprotomer interaction networks can enable the design of point mutation experiments. PIMADb can be accessed from the URL: http://caps.ncbs.res.in/pimadb PMID:27478368

  5. Size evolution of highly amphiphilic macromolecular solution assemblies via a distinct bimodal pathway

    PubMed Central

    Kelley, Elizabeth G.; Murphy, Ryan P.; Seppala, Jonathan E.; Smart, Thomas P.; Hann, Sarah D.

    2014-01-01

    The solution self-assembly of macromolecular amphiphiles offers an efficient, bottom-up strategy for producing well--defined nanocarriers, with applications ranging from drug delivery to nanoreactors. Typically, the generation of uniform nanocarrier architecturesis controlled by processing methods that rely upon cosolvent mixtures. These preparation strategies hinge on the assumption that macromolecular solution nanostructures are kinetically stable following transfer from an organic/aqueous cosolvent into aqueous solution. Herein we demonstrate that unequivocal step-change shifts in micelle populations occur over several weeks following transfer into a highly selective solvent. The unexpected micelle growth evolves through a distinct bimodal distribution separated by multiple fusion events and critically depends on solution agitation. Notably, these results underscore fundamental similarities between assembly processes in amphiphilic polymer, small molecule, and protein systems. Moreover, the non-equilibrium micelle size increase can have a major impact on the assumed stability of solution assemblies, for which performance is dictated by nanocarrier size and structure. PMID:24710204

  6. Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies

    PubMed Central

    Kumar, Pramod; Satyam, Abhigyan; Fan, Xingliang; Collin, Estelle; Rochev, Yury; Rodriguez, Brian J.; Gorelov, Alexander; Dillon, Simon; Joshi, Lokesh; Raghunath, Michael; Pandit, Abhay; Zeugolis, Dimitrios I.

    2015-01-01

    Therapeutic strategies based on the principles of tissue engineering by self-assembly put forward the notion that functional regeneration can be achieved by utilising the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, in dilute ex vivo microenvironments, prolonged culture time is required to develop an extracellular matrix-rich implantable device. Herein, we assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 h in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro. PMID:25736020

  7. PHENIX: a comprehensive Python-based system for macromolecular structure solution

    PubMed Central

    Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li-Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.

    2010-01-01

    Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. How­ever, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallo­graphic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms. PMID:20124702

  8. Macromolecular X-ray structure determination using weak, single-wavelength anomalous data

    SciTech Connect

    Bunkóczi, Gábor; McCoy, Airlie J.; Echols, Nathaniel; Grosse-Kunstleve, Ralf W.; Adams, Paul D.; Holton, James M.; Read, Randy J.; Terwilliger, Thomas C.

    2014-12-22

    We describe a likelihood-based method for determining the substructure of anomalously scattering atoms in macromolecular crystals that allows successful structure determination by single-wavelength anomalous diffraction (SAD) X-ray analysis with weak anomalous signal. With the use of partial models and electron density maps in searches for anomalously scattering atoms, testing of alternative values of parameters and parallelized automated model-building, this method has the potential to extend the applicability of the SAD method in challenging cases.

  9. Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers.

    PubMed

    Hattne, Johan; Echols, Nathaniel; Tran, Rosalie; Kern, Jan; Gildea, Richard J; Brewster, Aaron S; Alonso-Mori, Roberto; Glöckner, Carina; Hellmich, Julia; Laksmono, Hartawan; Sierra, Raymond G; Lassalle-Kaiser, Benedikt; Lampe, Alyssa; Han, Guangye; Gul, Sheraz; DiFiore, Dörte; Milathianaki, Despina; Fry, Alan R; Miahnahri, Alan; White, William E; Schafer, Donald W; Seibert, M Marvin; Koglin, Jason E; Sokaras, Dimosthenis; Weng, Tsu-Chien; Sellberg, Jonas; Latimer, Matthew J; Glatzel, Pieter; Zwart, Petrus H; Grosse-Kunstleve, Ralf W; Bogan, Michael J; Messerschmidt, Marc; Williams, Garth J; Boutet, Sébastien; Messinger, Johannes; Zouni, Athina; Yano, Junko; Bergmann, Uwe; Yachandra, Vittal K; Adams, Paul D; Sauter, Nicholas K

    2014-05-01

    X-ray free-electron laser (XFEL) sources enable the use of crystallography to solve three-dimensional macromolecular structures under native conditions and without radiation damage. Results to date, however, have been limited by the challenge of deriving accurate Bragg intensities from a heterogeneous population of microcrystals, while at the same time modeling the X-ray spectrum and detector geometry. Here we present a computational approach designed to extract meaningful high-resolution signals from fewer diffraction measurements.

  10. Porphyrin-Cored Polymer Nanoparticles: Macromolecular Models for Heme Iron Coordination.

    PubMed

    Rodriguez, Kyle J; Hanlon, Ashley M; Lyon, Christopher K; Cole, Justin P; Tuten, Bryan T; Tooley, Christian A; Berda, Erik B; Pazicni, Samuel

    2016-10-03

    Porphyrin-cored polymer nanoparticles (PCPNs) were synthesized and characterized to investigate their utility as heme protein models. Created using collapsible heme-centered star polymers containing photodimerizable anthracene units, these systems afford model heme cofactors buried within hydrophobic, macromolecular environments. Spectroscopic interrogations demonstrate that PCPNs display redox and ligand-binding reactivity similar to that of native systems and thus are potential candidates for modeling biological heme iron coordination.

  11. The R-factor gap in macromolecular crystallography: an untapped potential for insights on accurate structures

    PubMed Central

    Holton, James M; Classen, Scott; Frankel, Kenneth A; Tainer, John A

    2014-01-01

    In macromolecular crystallography, the agreement between observed and predicted structure factors (Rcryst and Rfree) is seldom better than 20%. This is much larger than the estimate of experimental error (Rmerge). The difference between Rcryst and Rmerge is the R-factor gap. There is no such gap in small-molecule crystallography, for which calculated structure factors are generally considered more accurate than the experimental measurements. Perhaps the true noise level of macromolecular data is higher than expected? Or is the gap caused by inaccurate phases that trap refined models in local minima? By generating simulated diffraction patterns using the program MLFSOM, and including every conceivable source of experimental error, we show that neither is the case. Processing our simulated data yielded values that were indistinguishable from those of real data for all crystallographic statistics except the final Rcryst and Rfree. These values decreased to 3.8% and 5.5% for simulated data, suggesting that the reason for high R-factors in macromolecular crystallography is neither experimental error nor phase bias, but rather an underlying inadequacy in the models used to explain our observations. The present inability to accurately represent the entire macromolecule with both its flexibility and its protein-solvent interface may be improved by synergies between small-angle X-ray scattering, computational chemistry and crystallography. The exciting implication of our finding is that macromolecular data contain substantial hidden and untapped potential to resolve ambiguities in the true nature of the nanoscale, a task that the second century of crystallography promises to fulfill. Database Coordinates and structure factors for the real data have been submitted to the Protein Data Bank under accession 4tws. PMID:25040949

  12. Developing genetic tools to exploit Chaetomium thermophilum for biochemical analyses of eukaryotic macromolecular assemblies

    PubMed Central

    Kellner, Nikola; Schwarz, Johannes; Sturm, Miriam; Fernandez-Martinez, Javier; Griesel, Sabine; Zhang, Wenzhu; Chait, Brian T.; Rout, Michael P.; Kück, Ulrich; Hurt, Ed

    2016-01-01

    We describe a method to genetically manipulate Chaetomium thermophilum, a eukaryotic thermophile, along with various biochemical applications. The transformation method depends on a thermostable endogenous selection marker operating at high temperatures combined with chromosomal integration of target genes. Our technique allows exploiting eukaryotic thermophiles as source for purifying thermostable native macromolecular complexes with an emphasis on the nuclear pore complex, holding great potential for applications in basic science and biotechnology. PMID:26864114

  13. A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.

    PubMed

    Wu, Yanning; Wang, Shuo; Farooq, Shukkur M; Castelvetere, Marcello P; Hou, Yuning; Gao, Ji-Liang; Navarro, Javier V; Oupicky, David; Sun, Fei; Li, Chunying

    2012-02-17

    Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.

  14. Folding dynamics of Trp-cage in the presence of chemical interference and macromolecular crowding. I

    NASA Astrophysics Data System (ADS)

    Samiotakis, Antonios; Cheung, Margaret S.

    2011-11-01

    Proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been well established that the so-called "macromolecular crowding" effect enhances the folding stability of proteins by destabilizing their unfolded states for selected proteins. On the other hand, chemical and thermal denaturation is often used in experiments as a tool to destabilize a protein by populating the unfolded states when probing its folding landscape and thermodynamic properties. However, little is known about the complicated effects of these synergistic perturbations acting on the kinetic properties of proteins, particularly when large structural fluctuations, such as protein folding, have been involved. In this study, we have first investigated the folding mechanism of Trp-cage dependent on urea concentration by coarse-grained molecular simulations where the impact of urea is implemented into an energy function of the side chain and/or backbone interactions derived from the all-atomistic molecular dynamics simulations with urea through a Boltzmann inversion method. In urea solution, the folding rates of a model miniprotein Trp-cage decrease and the folded state slightly swells due to a lack of contact formation between side chains at the terminal regions. In addition, the equilibrium m-values of Trp-cage from the computer simulations are in agreement with experimental measurements. We have further investigated the combined effects of urea denaturation and macromolecular crowding on Trp-cage's folding mechanism where crowding agents are modeled as hard-spheres. The enhancement of folding rates of Trp-cage is most pronounced by macromolecular crowding effect when the extended conformations of Trp-cast dominate at high urea concentration. Our study makes quantitatively testable predictions on protein folding dynamics in a complex environment involving both chemical denaturation and macromolecular crowding effects.

  15. Protein Self-Association Induced by Macromolecular Crowding: A Quantitative Analysis by Magnetic Relaxation Dispersion

    PubMed Central

    Snoussi, Karim; Halle, Bertil

    2005-01-01

    In the presence of high concentrations of inert macromolecules, the self-association of proteins is strongly enhanced through an entropic, excluded-volume effect variously called macromolecular crowding or depletion attraction. Despite the predicted large magnitude of this universal effect and its far-reaching biological implications, few experimental studies of macromolecular crowding have been reported. Here, we introduce a powerful new technique, fast field-cycling magnetic relaxation dispersion, for investigating crowding effects on protein self-association equilibria. By recording the solvent proton spin relaxation rate over a wide range of magnetic field strengths, we determine the populations of coexisting monomers and decamers of bovine pancreatic trypsin inhibitor in the presence of dextran up to a macromolecular volume fraction of 27%. Already at a dextran volume fraction of 14%, we find a 30-fold increase of the decamer population and 5105-fold increase of the association constant. The analysis of these results, in terms of a statistical-mechanical model that incorporates polymer flexibility as well as the excluded volume of the protein, shows that the dramatic enhancement of bovine pancreatic trypsin inhibitor self-association can be quantitatively rationalized in terms of hard repulsive interactions. PMID:15665132

  16. Design and application of a C{sup ++} macromolecular class library

    SciTech Connect

    Chang, W.; Shindyalov, I.N.; Bourne, P.E.

    1994-12-31

    PDBlib is an extensible object oriented class library written in C{sup ++} for representing the 3-dimensional structure of biological macromolecules. PDBlib forms the kernel of a larger software framework being developed for assisting in knowledge discovery from macromolecular structure data. The software design strategy used by PDBlib, how the library may be used and several prototype applications that use the library are summarized. PDBlib represents the structural features of proteins, DNA, RNA, and complexes thereof, at a level of detail on a par with that which can be parsed from a Protein Data Bank (PDB) entry. However, the memory resident representation of the macromolecule is independent of the PDB entry and can be obtained from other back-end data sources, for example, existing relational databases and our own object oriented database (OOPDB) built on top of the commercial object oriented database, ObjectStore. At the front-end are several prototype applications that use the library: Macromolecular Query Language (MMQL) is based on a separate class library (MMQLlib) for building complex queries pertaining to macromolecular structure; PDBtool is an interactive structure verification tool; and PDBview, is a structure rendering tool used either as a stand alone tool or as part of another application. Each of these software components are described.

  17. Hog barn dust extract increases macromolecular efflux from the hamster cheek pouch.

    PubMed

    Rubinstein, Israel; Von Essen, Susanna G

    2006-07-01

    The purpose of this study was to determine whether short-term exposure to an aqueous extract of hog barn dust increases macromolecular efflux from the intact hamster cheek pouch and, if so, to begin to determine the mechanism(s) underlying this response. By using intravital microscopy, we found that suffusion of hog barn dust extract onto the intact hamster cheek pouch for 60 min elicited a significant, concentration-dependent leaky site formation and increase in clearance of FITC-labeled dextran (molecular mass, 70 kDa). This response was significantly attenuated by suffusion of catalase (60 U/ml), but not by heat-inactivated catalase, and by pretreatment with dexamethasone (10 mg/kg iv) (P < 0.05). Catalase had no significant effects on adenosine-induced increase in macromolecular efflux from the cheek pouch. Suffusion of hog barn dust extract had no significant effects on arteriolar diameter in the cheek pouch. Taken together, these data indicate that hog barn dust extract increases macromolecular efflux from the in situ hamster cheek pouch, in part, through local elaboration of reactive oxygen species that are inactivated by catalase. This response is specific and attenuated by corticosteroids. We suggest that plasma exudation plays an important role in the genesis of upper airway dysfunction evoked by short-term exposure to hog barn dust.

  18. Homogenization Theory for the Prediction of Obstructed Solute Diffusivity in Macromolecular Solutions

    PubMed Central

    Donovan, Preston; Chehreghanianzabi, Yasaman; Rathinam, Muruhan; Zustiak, Silviya Petrova

    2016-01-01

    The study of diffusion in macromolecular solutions is important in many biomedical applications such as separations, drug delivery, and cell encapsulation, and key for many biological processes such as protein assembly and interstitial transport. Not surprisingly, multiple models for the a-priori prediction of diffusion in macromolecular environments have been proposed. However, most models include parameters that are not readily measurable, are specific to the polymer-solute-solvent system, or are fitted and do not have a physical meaning. Here, for the first time, we develop a homogenization theory framework for the prediction of effective solute diffusivity in macromolecular environments based on physical parameters that are easily measurable and not specific to the macromolecule-solute-solvent system. Homogenization theory is useful for situations where knowledge of fine-scale parameters is used to predict bulk system behavior. As a first approximation, we focus on a model where the solute is subjected to obstructed diffusion via stationary spherical obstacles. We find that the homogenization theory results agree well with computationally more expensive Monte Carlo simulations. Moreover, the homogenization theory agrees with effective diffusivities of a solute in dilute and semi-dilute polymer solutions measured using fluorescence correlation spectroscopy. Lastly, we provide a mathematical formula for the effective diffusivity in terms of a non-dimensional and easily measurable geometric system parameter. PMID:26731550

  19. Gorgon and Pathwalking: Macromolecular Modeling Tools for Subnanometer Resolution Density Maps

    PubMed Central

    Baker, Matthew L.; Baker, Mariah R.; Hryc, Corey F.; Ju, Tao; Chiu, Wah

    2013-01-01

    The complex interplay of proteins and other molecules, often in the form of large transitory assemblies, are critical to cellular function. Today, X-ray crystallography and electron cryo-microscopy (cryo-EM) are routinely used to image these macromolecular complexes, though often at limited resolutions. Despite the rapidly growing number of macromolecular structures, few tools exist for modeling and annotating structures in the range of 3-10Å resolution. To address this need, we have developed a number of utilities specifically targeting subnanometer resolution density maps. As part of the 2010 Cryo-EM Modeling Challenge, we demonstrated two of our latest de novo modeling tools, Pathwalking and Gorgon, as well as a tool for secondary structure identification (SSEHunter) and a new rigid-body/flexible fitting tool in Gorgon. In total, we submitted 30 structural models from ten different subnanometer resolution data sets in four of the six challenge categories. Each of our utlities produced accurate structural models and annotations across the various density maps. In the end, the utilities that we present here offer users a robust toolkit for analyzing and modeling protein structure in macromolecular assemblies at non-atomic resolutions. PMID:22696403

  20. A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines.

    PubMed

    Joglekar, Ajit; Chen, Renjie; Lawrimore, Joshua

    2013-01-01

    Macromolecular machines participate in almost every cell biological function. These machines can take the form of well-defined protein structures such as the kinetochore, or more loosely organized protein assemblies like the endocytic coat. The protein architecture of these machines-the arrangement of multiple copies of protein subunits at the nanoscale, is necessary for understanding their cell biological function and biophysical mechanism. Defining this architecture in vivo presents a major challenge. High density of protein molecules within macromolecular machines severely limits the effectiveness of super-resolution microscopy. However, this density is ideal for Forster Resonance Energy Transfer (FRET), which can determine the proximity between neighboring molecules. Here, we present a simple FRET quantitation scheme that calibrates a standard epifluorescence microscope for measuring donor-acceptor separations. This calibration can be used to deduce FRET efficiency fluorescence intensity measurements. This method will allow accurate determination of FRET efficiency over a wide range of values and FRET pair number. It will also allow dynamic FRET measurements with high spatiotemporal resolution under cell biological conditions. Although the poor maturation efficiency of genetically encoded fluorescent proteins presents a challenge, we show that its effects can be alleviated. To demonstrate this methodology, we probe the in vivo architecture of the γ-Tubulin Ring. Our technique can be applied to study the architecture and dynamics of a wide range of macromolecular machines.

  1. Space warping order parameters and symmetry: application to multiscale simulation of macromolecular assemblies.

    PubMed

    Singharoy, Abhishek; Joshi, Harshad; Miao, Yinglong; Ortoleva, Peter J

    2012-07-26

    Coarse-grained features of macromolecular assemblies are understood via a set of order parameters (OPs) constructed in terms of their all-atom configuration. OPs are shown to be slowly changing in time and capture the large-scale spatial features of macromolecular assemblies. The relationship of these variables to the classic notion of OPs based on symmetry breaking phase transitions is discussed. OPs based on space warping transformations are analyzed in detail as they naturally provide a connection between overall structure of an assembly and all-atom configuration. These OPs serve as the basis of a multiscale analysis that yields Langevin equations for OP dynamics. In this context, the characteristics of OPs and PCA modes are compared. The OPs enable efficient all-atom multiscale simulations of the dynamics of macromolecular assemblies in response to changes in microenvironmental conditions, as demonstrated on the structural transitions of cowpea chlorotic mottle virus capsid (CCMV) and RNA of the satellite tobacco mosaic virus (STMV).

  2. Macromolecular crowding can account for RNase-sensitive constraint of bacterial nucleoid structure

    SciTech Connect

    Foley, Patricia L.; Wilson, David B.; Shuler, Michael L.

    2010-04-23

    The shape and compaction of the bacterial nucleoid may affect the accessibility of genetic material to the transcriptional machinery in natural and synthetic systems. To investigate this phenomenon, the nature and contribution of RNA and protein to the compaction of nucleoids that had been gently released from Escherichia coli cells were investigated using fluorescent and transmission electron microscopy. We propose that the removal of RNA from the bacterial nucleoid affects nucleoid compaction by altering the branching density and molecular weight of the nucleoid. We show that a common detergent in nucleoid preparations, Brij 58, plays a previously unrecognized role as a macromolecular crowding agent. RNA-free nucleoids adopt a compact structure similar in size to exponential-phase nucleoids when the concentration of Brij 58 is increased, consistent with our hypothesis. We present evidence that control and protein-free nucleoids behave similarly in solutions containing a macromolecular crowding agent. These results show that the contribution to DNA compaction by nucleoid-associated proteins is small when compared to macromolecular crowding effects.

  3. Principles and Overview of Sampling Methods for Modeling Macromolecular Structure and Dynamics

    PubMed Central

    Moffatt, Ryan; Ma, Buyong; Nussinov, Ruth

    2016-01-01

    Investigation of macromolecular structure and dynamics is fundamental to understanding how macromolecules carry out their functions in the cell. Significant advances have been made toward this end in silico, with a growing number of computational methods proposed yearly to study and simulate various aspects of macromolecular structure and dynamics. This review aims to provide an overview of recent advances, focusing primarily on methods proposed for exploring the structure space of macromolecules in isolation and in assemblies for the purpose of characterizing equilibrium structure and dynamics. In addition to surveying recent applications that showcase current capabilities of computational methods, this review highlights state-of-the-art algorithmic techniques proposed to overcome challenges posed in silico by the disparate spatial and time scales accessed by dynamic macromolecules. This review is not meant to be exhaustive, as such an endeavor is impossible, but rather aims to balance breadth and depth of strategies for modeling macromolecular structure and dynamics for a broad audience of novices and experts. PMID:27124275

  4. Homogenization Theory for the Prediction of Obstructed Solute Diffusivity in Macromolecular Solutions.

    PubMed

    Donovan, Preston; Chehreghanianzabi, Yasaman; Rathinam, Muruhan; Zustiak, Silviya Petrova

    2016-01-01

    The study of diffusion in macromolecular solutions is important in many biomedical applications such as separations, drug delivery, and cell encapsulation, and key for many biological processes such as protein assembly and interstitial transport. Not surprisingly, multiple models for the a-priori prediction of diffusion in macromolecular environments have been proposed. However, most models include parameters that are not readily measurable, are specific to the polymer-solute-solvent system, or are fitted and do not have a physical meaning. Here, for the first time, we develop a homogenization theory framework for the prediction of effective solute diffusivity in macromolecular environments based on physical parameters that are easily measurable and not specific to the macromolecule-solute-solvent system. Homogenization theory is useful for situations where knowledge of fine-scale parameters is used to predict bulk system behavior. As a first approximation, we focus on a model where the solute is subjected to obstructed diffusion via stationary spherical obstacles. We find that the homogenization theory results agree well with computationally more expensive Monte Carlo simulations. Moreover, the homogenization theory agrees with effective diffusivities of a solute in dilute and semi-dilute polymer solutions measured using fluorescence correlation spectroscopy. Lastly, we provide a mathematical formula for the effective diffusivity in terms of a non-dimensional and easily measurable geometric system parameter.

  5. Competition between chemical denaturation and macromolecular crowding effects on the folding dynamics of proteins

    NASA Astrophysics Data System (ADS)

    Samiotakis, Antonios; Cheung, Margaret

    2011-03-01

    It is well known that proteins fold and function in the crowded environment of the cell's interior. In the recent years it has been established that the so-called ``macromolecular crowding'' effect can enhance the folding stability of proteins by destabilizing their unfolded states. On the other hand, chemical and thermal denaturation are often used in experiments as tools to destabilize protein structures when probing a protein's folding landscape. However, little is known about the combined effects of these competing phenomena on proteins. In this work, we use coarse-grained molecular simulations to study the thermodynamic and kinetic properties of the small peptide Trp-cage, in the combined presence of macromolecular crowders and chemical denaturant. With the use of an energy function derived by all-atomistic simulations in the presence of urea, we investigate the thermodynamics and kinetics of Trp-cage's folding mechanism at several concentrations of urea. The effects of the competition between stabilization by macromolecular crowding and destabilization by chemical denaturation will also be discussed. This work was supported by the National Science Foundation, Molecular & Cellular Biosciences (MCB0919974).

  6. Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination

    PubMed Central

    Wlodawer, Alexander; Minor, Wladek; Dauter, Zbigniew; Jaskolski, Mariusz

    2014-01-01

    The number of macromolecular structures deposited in the Protein Data Bank now approaches 100 000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation. PMID:24034303

  7. Liberation of microbial substrates from macromolecular organic matter by non-supercritical CO2

    NASA Astrophysics Data System (ADS)

    Sauer, P.; Glombitza, C.; Kallmeyer, J.

    2012-12-01

    The worldwide search for suitable underground storage formations for CO2 also considers coal-bearing strata. CO2 is already injected into coal seams for enhanced recovery of coal bed methane. However, the geochemical and microbiological effects of increased CO2 concentrations on organic matter rich formations are rarely investigated. The injected CO2 will dissolve in the pore water, causing a decrease in pH and resulting in acidic formation waters. Low molecular weight organic acids (LMWOAs) are chemically bound to the macromolecular matrix of sedimentary organic matter and may be liberated by hydrolysis, which is enhanced under acidic conditions. Recent investigations outlined the importance of LMWOAs as a feedstock for subsurface microbial life [1]. Therefore, injection of CO2 into coal formations may result in enhanced nutrient supply for subsurface microbes. To investigate the effects of highly CO2-saturated waters on the release of LMWOAs from coal, we developed an inexpensive high-pressure-high-temperature system that allows manipulating the concentration of dissolved gases up to 60 MPa and 120°C, respectively. The sample is placed in a flexible, gas-tight and inert PVDF sleeve, separating it from the pressure fluid and allowing for subsampling without loss of pressure. Lignite samples from the DEBITS-1 well, Waikato Basin, NZ and the Welzow-Süd open-cast mine, Niederlausitz, Germany, were extracted at 90° C and 5 MPa, with either pure water, CO2-saturated water, CO2/NO2 or CO2/SO2-saturated water. Subsamples were taken at different time points during the 72 hrs. long extraction. Extraction of LMWOAs from coal samples with our pressurised system resulted in yields that were up to four times higher than those reported for Soxhlet extraction [2]. These higher yields may be explained by the fact that during Soxhlet extraction the sample only gets into contact with freshly distilled water, whereas in our system the extraction fluid is circulated, resulting in

  8. High-resolution three-dimensional macromolecular proton fraction mapping for quantitative neuroanatomical imaging of the rodent brain in ultra-high magnetic fields.

    PubMed

    Naumova, Anna V; Akulov, Andrey E; Khodanovich, Marina Yu; Yarnykh, Vasily L

    2017-02-15

    A well-known problem in ultra-high-field MRI is generation of high-resolution three-dimensional images for detailed characterization of white and gray matter anatomical structures. T1-weighted imaging traditionally used for this purpose suffers from the loss of contrast between white and gray matter with an increase of magnetic field strength. Macromolecular proton fraction (MPF) mapping is a new method potentially capable to mitigate this problem due to strong myelin-based contrast and independence of this parameter of field strength. MPF is a key parameter determining the magnetization transfer effect in tissues and defined within the two-pool model as a relative amount of macromolecular protons involved into magnetization exchange with water protons. The objectives of this study were to characterize the two-pool model parameters in brain tissues in ultra-high magnetic fields and introduce fast high-field 3D MPF mapping as both anatomical and quantitative neuroimaging modality for small animal applications. In vivo imaging data were obtained from four adult male rats using an 11.7T animal MRI scanner. Comprehensive comparison of brain tissue contrast was performed for standard R1 and T2 maps and reconstructed from Z-spectroscopic images two-pool model parameter maps including MPF, cross-relaxation rate constant, and T2 of pools. Additionally, high-resolution whole-brain 3D MPF maps were obtained with isotropic 170µm voxel size using the single-point synthetic-reference method. MPF maps showed 3-6-fold increase in contrast between white and gray matter compared to other parameters. MPF measurements by the single-point synthetic reference method were in excellent agreement with the Z-spectroscopic method. MPF values in rat brain structures at 11.7T were similar to those at lower field strengths, thus confirming field independence of MPF. 3D MPF mapping provides a useful tool for neuroimaging in ultra-high magnetic fields enabling both quantitative tissue

  9. Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron

    PubMed Central

    Juanhuix, Jordi; Gil-Ortiz, Fernando; Cuní, Guifré; Colldelram, Carles; Nicolás, Josep; Lidón, Julio; Boter, Eva; Ruget, Claude; Ferrer, Salvador; Benach, Jordi

    2014-01-01

    BL13-XALOC is currently the only macromolecular crystallography beamline at the 3 GeV ALBA synchrotron near Barcelona, Spain. The optics design is based on an in-vacuum undulator, a Si(111) channel-cut crystal monochromator and a pair of KB mirrors. It allows three main operation modes: a focused configuration, where both mirrors can focus the beam at the sample position to 52 µm × 5.5 µm FWHM (H × V); a defocused configuration that can match the size of the beam to the dimensions of the crystals or to focus the beam at the detector; and an unfocused configuration, where one or both mirrors are removed from the photon beam path. To achieve a uniform defocused beam, the slope errors of the mirrors were reduced down to 55 nrad RMS by employing a novel method that has been developed at the ALBA high-accuracy metrology laboratory. Thorough commissioning with X-ray beam and user operation has demonstrated an excellent energy and spatial stability of the beamline. The end-station includes a high-accuracy single-axis diffractometer, a removable mini-kappa stage, an automated sample-mounting robot and a photon-counting detector that allows shutterless operation. The positioning tables of the diffractometer and the detector are based on a novel and highly stable design. This equipment, together with the operation flexibility of the beamline, allows a large variety of types of crystals to be tackled, from medium-sized crystals with large unit-cell parameters to microcrystals. Several examples of data collections measured during beamline commissioning are described. The beamline started user operation on 18 July 2012. PMID:24971961

  10. The effect of macromolecular crowding on the structure of the protein complex superoxide dismutase

    NASA Astrophysics Data System (ADS)

    Rajapaksha Mudalige, Ajith Rathnaweera

    Biological environments contain between 7 - 40% macromolecules by volume. This reduces the available volume for macromolecules and elevates the osmotic pressure relative to pure water. Consequently, biological macromolecules in their native environments tend to adopt more compact and dehydrated conformations than those in vitro. This effect is referred to as macromolecular crowding and constitutes an important physical difference between native biological environments and the simple solutions in which biomolecules are usually studied. We used small angle scattering (SAS) to measure the effects of macromolecular crowding on the size of a protein complex, superoxide dismutase (SOD). Crowding was induced using 400 MW polyethylene glycol (PEG), triethylene glycol (TEG), methyl-alpha-glucoside (alpha-MG) and trimethylamine N-oxide (TMAO). Parallel small angle neutron scattering (SANS) and small angle X-ray scattering (SAXS) allowed us to unambiguously attribute apparent changes in radius of gyration to changes in the structure of SOD. For a 40% PEG solution, we find that the volume of SOD was reduced by 9%. SAS coupled with osmotic pressure measurements allowed us to estimate a compressibility modulus for SOD. We believe this to be the first time the osmotic compressibility of a protein complex was measured. Molecular Dynamics (MD) simulations are widely used to obtain insights on biomolecular processes. However, it is not clear whether MD is capable of predicting subtle effects of macromolecular crowding. We used our experimentally observed compressibility of SOD to evaluate the ability of MD to predict macromolecular crowding. Effects of macromolecular crowding due to PEG on SOD were modeled using an all atom MD simulation with the CHARMM forcefield and the crystallographically resolved structures of SOD and PEG. Two parallel MD simulations were performed for SOD in water and SOD in 40% PEG for over 150~ns. Over the period of the simulation the SOD structure in 40

  11. Synthetic laser medium

    DOEpatents

    Stokowski, S.E.

    1987-10-20

    A laser medium is particularly useful in high average power solid state lasers. The laser medium includes a chromium dopant and preferably neodymium ions as codopant, and is primarily a gadolinium scandium gallium garnet, or an analog thereof. Divalent cations inhibit spiral morphology as large boules from which the laser medium is derived are grown, and a source of ions convertible between a trivalent state and a tetravalent state at a low ionization energy are in the laser medium to reduce an absorption coefficient at about one micron wavelength otherwise caused by the divalent cations. These divalent cations and convertible ions are dispersed in the laser medium. Preferred convertible ions are provided from titanium or cerium sources.

  12. Synthetic laser medium

    DOEpatents

    Stokowski, Stanley E.

    1989-01-01

    A laser medium is particularly useful in high average power solid state lasers. The laser medium includes a chormium dopant and preferably neodymium ions as codopant, and is primarily a gadolinium scandium gallium garnet, or an analog thereof. Divalent cations inhibit spiral morphology as large boules from which the laser medium is derived are grown, and a source of ions convertible between a trivalent state and a tetravalent state at a low ionization energy are in the laser medium to reduce an absorption coefficient at about one micron wavelength otherwise caused by the divalent cations. These divalent cations and convertible ions are dispersed in the laser medium. Preferred convertible ions are provided from titanium or cerium sources.

  13. Ultrasound contrast agents

    PubMed Central

    Ignee, Andre; Atkinson, Nathan S. S.; Schuessler, Gudrun; Dietrich, Christoph F.

    2016-01-01

    Endoscopic ultrasound (EUS) plays an important role in imaging of the mediastinum and abdominal organs. Since the introduction of US contrast agents (UCA) for transabdominal US, attempts have been made to apply contrast-enhanced US techniques also to EUS. Since 2003, specific contrast-enhanced imaging was possible using EUS. Important studies have been published regarding contrast-enhanced EUS and the characterization of focal pancreatic lesions, lymph nodes, and subepithelial tumors. In this manuscript, we describe the relevant UCA, their application, and specific image acquisition as well as the principles of image tissue characterization using contrast-enhanced EUS. Safety issues, potential future developments, and EUS-specific issues are reviewed. PMID:27824024

  14. Minimal effects of macromolecular crowding on an intrinsically disordered protein: a small-angle neutron scattering study.

    PubMed

    Goldenberg, David P; Argyle, Brian

    2014-02-18

    Small-angle neutron scattering was used to study the effects of macromolecular crowding by two globular proteins, i.e., bovine pancreatic trypsin inhibitor and equine metmyoglobin, on the conformational ensemble of an intrinsically disordered protein, the N protein of bacteriophage λ. The λ N protein was uniformly labeled with (2)H, and the concentrations of D2O in the samples were adjusted to match the neutron scattering contrast of the unlabeled crowding proteins, thereby masking their contribution to the scattering profiles. Scattering from the deuterated λ N was recorded for samples containing up to 0.12 g/mL bovine pancreatic trypsin inhibitor or 0.2 g/mL metmyoglobin. The radius of gyration of the uncrowded protein was estimated to be 30 Å and was found to be remarkably insensitive to the presence of crowders, varying by <2 Å for the highest crowder concentrations. The scattering profiles were also used to estimate the fractal dimension of λ N, which was found to be ∼1.8 in the absence or presence of crowders, indicative of a well-solvated and expanded random coil under all of the conditions examined. These results are contrary to the predictions of theoretical treatments and previous experimental studies demonstrating compaction of unfolded proteins by crowding with polymers such as dextran and Ficoll. A computational simulation suggests that some previous treatments may have overestimated the effective volumes of disordered proteins and the variation of these volumes within an ensemble. The apparent insensitivity of λ N to crowding may also be due in part to weak attractive interactions with the crowding proteins, which may compensate for the effects of steric exclusion.

  15. Biodegradable polydisulfide dendrimer nanoclusters as MRI contrast agents.

    PubMed

    Huang, Ching-Hui; Nwe, Kido; Al Zaki, Ajlan; Brechbiel, Martin W; Tsourkas, Andrew

    2012-11-27

    Gadolinium-conjugated dendrimer nanoclusters (DNCs) are a promising platform for the early detection of disease; however, their clinical utility is potentially limited due to safety concerns related to nephrogenic systemic fibrosis (NSF). In this paper, biodegradable DNCs were prepared with polydisulfide linkages between the individual dendrimers to facilitate excretion. Further, DNCs were labeled with premetalated Gd chelates to eliminate the risk of free Gd becoming entrapped in dendrimer cavities. The biodegradable polydisulfide DNCs possessed a circulation half-life of >1.6 h in mice and produced significant contrast enhancement in the abdominal aorta and kidneys for as long as 4 h. The DNCs were reduced in circulation as a result of thiol-disulfide exchange, and the degradation products were rapidly excreted via renal filtration. These agents demonstrated effective and prolonged in vivo contrast enhancement and yet minimized Gd tissue retention. Biodegradable polydisulfide DNCs represent a promising biodegradable macromolecular MRI contrast agent for magnetic resonance angiography and can potentially be further developed into target-specific MRI contrast agents.

  16. Compressive Phase Contrast Tomography

    SciTech Connect

    Maia, Filipe; MacDowell, Alastair; Marchesini, Stefano; Padmore, Howard A.; Parkinson, Dula Y.; Pien, Jack; Schirotzek, Andre; Yang, Chao

    2010-09-01

    When x-rays penetrate soft matter, their phase changes more rapidly than their amplitude. Interference effects visible with high brightness sources creates higher contrast, edge enhanced images. When the object is piecewise smooth (made of big blocks of a few components), such higher contrast datasets have a sparse solution. We apply basis pursuit solvers to improve SNR, remove ring artifacts, reduce the number of views and radiation dose from phase contrast datasets collected at the Hard X-Ray Micro Tomography Beamline at the Advanced Light Source. We report a GPU code for the most computationally intensive task, the gridding and inverse gridding algorithm (non uniform sampled Fourier transform).

  17. [Contrast-induced nephropathy: An update].

    PubMed

    Spagnoli, V; Azzalini, L; Tadros, V X; Picard, F; Ly, H Q

    2016-04-01

    Contrast-induced nephropathy (CIN) is common in hospitalized patients. Its occurrence is associated with an increased hospitalization stay and cost, morbidity and mortality. Thus, preventives strategies remain a major issue. Patients that are referred for cardiac catheterization are among the most vulnerable to develop CIN due to their comorbidities. Moreover, in some cases, such preventives measures cannot be introduced due to emergent clinical settings. After a summary regarding the properties of iodinated contrast medium, the aim of this work was to review the definition, pathophysiology, diagnosis and preventive strategies related to CIN.

  18. [Macromolecular aromatic network characteristics of Chinese power coal analyzed by synchronous fluorescence and X-ray diffraction].

    PubMed

    Ye, Cui-Ping; Feng, Jie; Li, Wen-Ying

    2012-07-01

    Coal structure, especially the macromolecular aromatic skeleton structure, has a strong influence on coke reactivity and coal gasification, so it is the key to grasp the macromolecular aromatic skeleton coal structure for getting the reasonable high efficiency utilization of coal. However, it is difficult to acquire their information due to the complex compositions and structure of coal. It has been found that the macromolecular aromatic network coal structure would be most isolated if small molecular of coal was first extracted. Then the macromolecular aromatic skeleton coal structure would be clearly analyzed by instruments, such as X-ray diffraction (XRD), fluorescence spectroscopy with synchronous mode (Syn-F), Gel permeation chromatography (GPC) etc. Based on the previous results, according to the stepwise fractional liquid extraction, two Chinese typical power coals, PS and HDG, were extracted by silica gel as stationary phase and acetonitrile, tetrahydrofuran (THF), pyridine and 1-methyl-2-pyrollidinone (NMP) as a solvent group for sequential elution. GPC, Syn-F and XRD were applied to investigate molecular mass distribution, condensed aromatic structure and crystal characteristics. The results showed that the size of aromatic layers (La) is small (3-3.95 nm) and the stacking heights (Lc) are 0.8-1.2 nm. The molecular mass distribution of the macromolecular aromatic network structure is between 400 and 1 130 amu, with condensed aromatic numbers of 3-7 in the structure units.

  19. The influence of interchain coupling on intramolecular oscillation mobility in coupled macromolecular chains: The case of coplanar parallel chains

    NASA Astrophysics Data System (ADS)

    Čevizović, D.; Petković, S.; Galović, S.; Chizhov, A.; Reshetnyak, A.

    2015-10-01

    We enlarge our results from the study of the hopping mechanism of the oscillation excitation transport in 1D model of one biologica-likel macromolecular chain to the case of a system composed from two 1D parallel macromolecular chains with consideration of the properties of intramolecular oscillation excitations. We suppose, that due to the exciton interaction with thermal oscillation (generated by mechanical phonon subsystem) of structural elements (consisting of the peptide group) of the chains, the exciton becomes by self trapped and forms the polaron state. We suggest a model which generalizes the modified Holstein polaron model to the case of two macromolecular chains and find that because of the interchain coupling, the exciton energy band is splitted into two subbands. The hopping process of exciton migration along the macromolecular chains is studied in dependence of system parameters and temperature. We pay an special attention to the temperature range (near T = 300 K) in which living cells operate. It is found that for the certain values of the system parameters there exists the abrupt change of the exciton migration nature from practically free (light) exciton motion to an immobile (heavy, dressed by phonon cloud) quasiparticle We discuss an application of the obtained results to the exciton transport both within deoxyribonucleic acid molecule and in the 2D polymer films organized from such macromolecular chains.

  20. Enhanced delivery of the RAPTA-C macromolecular chemotherapeutic by conjugation to degradable polymeric micelles.

    PubMed

    Blunden, Bianca M; Lu, Hongxu; Stenzel, Martina H

    2013-12-09

    Macromolecular ruthenium complexes are a promising avenue to better and more selective chemotherapeutics. We have previously shown that RAPTA-C [RuCl2(p-cymene)(PTA)], with the water-soluble 1,3,5-phosphaadamantane (PTA) ligand, could be attached to a polymer moiety via nucleophilic substitution of an available iodide with an amide in the PTA ligand. To increase the cell uptake of this macromolecule, we designed an amphiphilic block copolymer capable of self-assembling into polymeric micelles. The block copolymer was prepared by ring-opening polymerization of d,l-lactide (3,6-dimethyl-1,4-dioxane-2,5-dione) using a RAFT agent with an additional hydroxyl functionality, followed by the RAFT copolymerization of 2-hydroxyethyl acrylate (HEA) and 2-chloroethyl methacrylate (CEMA). The Finkelstein reaction and reaction with PTA led to polymers that can readily react with the dimer of RuCl2(p-cymene) to create a macromolecular RAPTA-C drug. RAPTA-C conjugation, micellization, and subsequent cytotoxicity and cell uptake of these polymeric moieties was tested on ovarian cancer A2780, A2780cis, and Ovcar-3 cell lines. Confocal microscopy images confirmed cell uptake of the micelles into the lysosome of the cells, indicative of an endocytic pathway. On average, a 10-fold increase in toxicity was found for the macromolecular drugs when compared to the RAPTA-C molecule. Furthermore, the cell uptake of ruthenium was analyzed and a significant increase was found for the micelles compared to RAPTA-C. Notably, micelles prepared from the polymer containing fewer HEA units had the highest cytotoxicity, the best cell uptake of ruthenium and were highly effective in suppressing the colony-forming ability of cells.

  1. Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect.

    PubMed

    Coquel, Anne-Sophie; Jacob, Jean-Pascal; Primet, Mael; Demarez, Alice; Dimiccoli, Mariella; Julou, Thomas; Moisan, Lionel; Lindner, Ariel B; Berry, Hugues

    2013-04-01

    Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of "soft" intracellular structuring (based on macromolecular

  2. Optimized beamline design for macromolecular crystallography at the Cornell High Energy Synchrotron Source (CHESS) (abstract)

    NASA Astrophysics Data System (ADS)

    Schildkamp, Wilfried; Bilderback, Donald; Moffat, Keith

    1989-07-01

    The A1 station on the CHESS wiggler beamline has been the workhorse for most macromolecular crystallographic experiments. This station is equipped with a fixed energy focusing germanium (111) monochromator and a focusing total reflection mirror. Our macromolecular crystallographers made full use of the high flux of more than 1012 photons/s/mm2 and the stable beam conditions, both in position and energy resolution. As a result, the A1 station was heavily oversubscribed. CHESS is presently expanding its capabilities and a new diffraction station for macromolecular crystallography is under construction. This beamline will be powered by a 24-pole hybrid permanent magnet wiggler with a critical energy of 25 keV. A focusing monochromator, which handles a specific heat load of 10 W/mm2, will have a range of tunability which covers all relevant absorption edges from 7 to 15 keV using a Ge(111) crystal. The energy resolution and the focusing properties remain constant within a factor of 2 over the entire tunability range. We expect a brilliance of about 1013 photons/s/mm2/mrad2/0.1% bandpass. The diffraction station will be equipped with an oscillation camera which can be used with x-ray film of 5×5 or 8×10 in. size or alternatively with Kodak storage phosphors. A wide variety of clamp-on accessories, like crystal coolers, fast shutters, helium pathways, polarimeter, etc. are available. The station will contain a beampipe system, which can also be used for small angle scattering experiments with sample-to-detector distances of up to 3000 mm. The entire diffraction station, its control area, a biological preparation area, and a darkroom are to be embedded in a biological safety containment of the level BL3. This will allow diffraction studies of virulent strains of viruses and other biohazards, which could not previously be studied at synchrotron radiation sources before without causing major disruption to the normal laboratory procedure.

  3. Macromolecular crowding effects on protein-protein binding affinity and specificity

    NASA Astrophysics Data System (ADS)

    Kim, Young C.; Best, Robert B.; Mittal, Jeetain

    2010-11-01

    Macromolecular crowding in cells is recognized to have a significant impact on biological function, yet quantitative models for its effects are relatively undeveloped. The influence of crowding on protein-protein interactions is of particular interest, since these mediate many processes in the cell, including the self-assembly of larger complexes, recognition, and signaling. We use a residue-level coarse-grained model to investigate the effects of macromolecular crowding on the assembly of protein-protein complexes. Interactions between the proteins are treated using a fully transferable energy function, and interactions of protein residues with the spherical crowders are repulsive. We show that the binding free energy for two protein complexes, ubiquitin/UIM1 and cytochrome c/cytochrome c peroxidase, decreases modestly as the concentration of crowding agents increases. To obtain a quantitative description of the stabilizing effect, we map the aspherical individual proteins and protein complexes onto spheres whose radii are calculated from the crowder-excluded protein volumes. With this correspondence, we find that the change in the binding free energy due to crowding can be quantitatively described by the scaled particle theory model without any fitting parameters. The effects of a mixture of different-size crowders—as would be found in a real cell—are predicted by the same model with an additivity ansatz. We also obtain the remarkable result that crowding increases the fraction of specific complexes at the expense of nonspecific transient encounter complexes in a crowded environment. This result, due to the greater excluded volume of the nonspecific complexes, demonstrates that macromolecular crowding can have subtle functional effects beyond the relative stability of bound and unbound complexes.

  4. Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect

    PubMed Central

    Coquel, Anne-Sophie; Jacob, Jean-Pascal; Primet, Mael; Demarez, Alice; Dimiccoli, Mariella; Julou, Thomas; Moisan, Lionel

    2013-01-01

    Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular

  5. A Web Resource for Standardized Benchmark Datasets, Metrics, and Rosetta Protocols for Macromolecular Modeling and Design.

    PubMed

    Ó Conchúir, Shane; Barlow, Kyle A; Pache, Roland A; Ollikainen, Noah; Kundert, Kale; O'Meara, Matthew J; Smith, Colin A; Kortemme, Tanja

    2015-01-01

    The development and validation of computational macromolecular modeling and design methods depend on suitable benchmark datasets and informative metrics for comparing protocols. In addition, if a method is intended to be adopted broadly in diverse biological applications, there needs to be information on appropriate parameters for each protocol, as well as metrics describing the expected accuracy compared to experimental data. In certain disciplines, there exist established benchmarks and public resources where experts in a particular methodology are encouraged to supply their most efficient implementation of each particular benchmark. We aim to provide such a resource for protocols in macromolecular modeling and design. We present a freely accessible web resource (https://kortemmelab.ucsf.edu/benchmarks) to guide the development of protocols for protein modeling and design. The site provides benchmark datasets and metrics to compare the performance of a variety of modeling protocols using different computational sampling methods and energy functions, providing a "best practice" set of parameters for each method. Each benchmark has an associated downloadable benchmark capture archive containing the input files, analysis scripts, and tutorials for running the benchmark. The captures may be run with any suitable modeling method; we supply command lines for running the benchmarks using the Rosetta software suite. We have compiled initial benchmarks for the resource spanning three key areas: prediction of energetic effects of mutations, protein design, and protein structure prediction, each with associated state-of-the-art modeling protocols. With the help of the wider macromolecular modeling community, we hope to expand the variety of benchmarks included on the website and continue to evaluate new iterations of current methods as they become available.

  6. Contrasting guest binding interaction of cucurbit[7-8]urils with neutral red dye: controlled exchange of multiple guests.

    PubMed

    Shaikh, Mhejabeen; Choudhury, Sharmistha Dutta; Mohanty, Jyotirmayee; Bhasikuttan, Achikanath C; Pal, Haridas

    2010-07-14

    Interactions among macrocyclic hosts and dyes/drugs have been explored extensively for their direct usage in controlled uptake and release of large number of potential drug molecules. In this paper we report the non-covalent interaction of cucurbit[8]uril macrocycle (CB8) with a biologically important dye, neutral red, by absorption and fluorescence spectroscopy. A comparative analysis with the complexation behaviour of the dye with CB7, the lower homologue of CB8, indicates contrasting guest binding behaviour with significant changes in the photophysical characteristics of the dye. While CB7 interaction leads to a 1 ratio 1 stoichiometry resulting in approximately 6 fold enhancement in the fluorescence emission of the dye, CB8 displays signatures for a 1 ratio 2 host-guest stoichiometry with drastic reduction in the fluorescence emission. Apart from the evaluation of approximately 2 unit shift in the protolytic equilibrium on complexation (pK(a) shift), the measurements with tryptophan established a selective guest exchange to favour a co-localized dimer inside the CB8 cavity. In a protein medium (BSA), the 1 ratio 2 complex was converted to a 1 ratio 1 ratio 1 CB8-NRH(+)-BSA complex. The finding that NRH(+) can be transferred from CB8 to BSA, even though the binding constant for NRH(+)-CB8 is much higher than NRH(+)-BSA, is projected for a controlled slow release of NRH(+) towards BSA. Since the release and activity of drugs can be controlled by regulating the protolytic equilibrium, the macromolecular encapsulation and release of NRH(+) demonstrated here provide information relevant to host-guest based drug delivery systems and its applications.

  7. The Stanford Automated Mounter: Pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    SciTech Connect

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; Cohen, Aina E.

    2016-02-24

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. As a result, sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.

  8. Structural analysis of macromolecular levan produced by Bacillus megaterium GJT321 based on enzymatic method.

    PubMed

    Yu, Xiaomeng; Li, Liyan; Zhang, Jingliang; Shen, Zhaopeng; Zhu, Changliang; Wang, Peng; Jiang, Xiaolu

    2016-12-01

    Extracellular polysaccharide (EPS) produced by Bacillus megaterium GJT321 was isolated from fermentation broth and further purified by gel filtration chromatography. The molecular weight of EPS was estimated as 1946kDa by gel permeation chromatography (GPC), so this EPS was determined as macromolecular polysaccharide. Fourier transform infrared spectrometry (FT-IR) showed that the kind of heterocyclic compound composing EPS was furanose. The structural characteristics of EPS were investigated by means of enzymatic method, electrospray ionization mass spectrometry (ESI-MS) and NMR spectra analysis. The structure elucidation of the EPS was accomplished, and it was β - (2, 6) -D- fructofuranose, namely levan.

  9. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    Soares, A.S.; Schneider, D. K.; Skinner, J. M.; Cowan, M.; Buono, R.; Robinson, H. H.; Heroux, A.; Carlucci-Dayton, M.; Saxena, A.; Sweet, R. M.

    2008-09-01

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  10. Remote Access to the PXRR Macromolecular Crystallography Facilities at the NSLS

    SciTech Connect

    A Soares; D Schneider; J Skinner; M Cowan; R Buono; H Robinson; A Heroux; M Carlucci-Dayton; A Saxena; R Sweet

    2011-12-31

    The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments.

  11. Simulation of macromolecular liquids with the adaptive resolution molecular dynamics technique

    NASA Astrophysics Data System (ADS)

    Peters, J. H.; Klein, R.; Delle Site, L.

    2016-08-01

    We extend the application of the adaptive resolution technique (AdResS) to liquid systems composed of alkane chains of different lengths. The aim of the study is to develop and test the modifications of AdResS required in order to handle the change of representation of large molecules. The robustness of the approach is shown by calculating several relevant structural properties and comparing them with the results of full atomistic simulations. The extended scheme represents a robust prototype for the simulation of macromolecular systems of interest in several fields, from material science to biophysics.

  12. The Stanford Automated Mounter: Pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    DOE PAGES

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; ...

    2016-02-24

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. As a result, sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade.

  13. Scale invariance of the density fluctuations in films and macromolecular aggregates in poly(styrene) solutions

    NASA Astrophysics Data System (ADS)

    Novikov, D. V.; Krasovskiĭ, A. N.; Osmolovskaya, N. A.; Efremov, V. I.

    2007-02-01

    The specific features of the transformation of a polymer solution into a solid state (film) of an amorphous polymer are investigated using electron microscopy. The correspondence between the characteristics of fractal macromolecular aggregates in a solution and the parameters of the spatial distribution of density fluctuations at the surface of the film is established using a linear atactic poly(styrene) as an example. The correspondence exists under the condition that the packing density of coils does not exceed a critical value at the liquid-solid phase transition point and the polymer concentration in the solution provides the formation of a continuous network of entangled macromolecules.

  14. Biologically active macromolecular forms of oxytocin. [8-Lysine]oxytocin as a suitable ligand.

    PubMed Central

    Snell, C R; Smyth, D G

    1977-01-01

    [8-Lysine]oxytocin was synthesized on a solid support and possessed an oxytocic activity of 100 +/- 6 units mumol on the isolated rat uterus. The epsilon-carbamoyl, epsilon-3-carboxypropionyl and epsilon-3-carboxybutryl derivatives were prepared and had uterotonic activities of 400, 55 and 50 units/mumol respectively. [8-Lysine]oxytocin was coupled unambiguously through the epsilon-amino group to the carboxyl groups of carboxymethylated dextrans or epsilon-3-carboxypropionly-gelatin. The macromolecular oxytocins were water-soluble and retained signigicant oxytocic activity. [8-Lysine]oxytocin should prove a useful ligand for affinity chromatography of oxytocin-binding proteins. PMID:889573

  15. The "macromolecular tourist": universal temperature dependence of thermal diffusion in aqueous colloidal suspensions.

    PubMed

    Iacopini, S; Rusconi, R; Piazza, R

    2006-01-01

    By performing measurements on a large class of macromolecular and colloidal systems, we show that thermophoresis (particle drift induced by thermal gradients) in aqueous solvents displays a distinctive universal dependence on temperature. For systems of particles interacting via temperature-independent forces, this behavior is strictly related to the solvent thermal expansivity, while an additional, T-independent term is needed to account for the behavior of "thermophilic" (migrating to the warmth) particles. The former relation between thermophoresis and thermal expansion may be exploited to envisage other fruitful studies of colloidal diffusion in inhomogeneous fluids.

  16. The Stanford Automated Mounter: pushing the limits of sample exchange at the SSRL macromolecular crystallography beamlines

    PubMed Central

    Russi, Silvia; Song, Jinhu; McPhillips, Scott E.; Cohen, Aina E.

    2016-01-01

    The Stanford Automated Mounter System, a system for mounting and dismounting cryo-cooled crystals, has been upgraded to increase the throughput of samples on the macromolecular crystallography beamlines at the Stanford Synchrotron Radiation Lightsource. This upgrade speeds up robot maneuvers, reduces the heating/drying cycles, pre-fetches samples and adds an air-knife to remove frost from the gripper arms. Sample pin exchange during automated crystal quality screening now takes about 25 s, five times faster than before this upgrade. PMID:27047309

  17. Macromolecular and nanotechnological modification of camptothecin and its analogs to improve the efficacy.

    PubMed

    Onishi, Hiraku; Machida, Yoshiharu

    2005-09-01

    Camptothecin (CPT) and its analogs are some of the most potent antitumor agents known. However, their poor water-solubility and high toxicity require changes of their physicochemical and biological characteristics. Active lactone forms have provoked interest in the utility of CPT and its analogs again. Macromolecular chemical modifications and nanotechnological formulations have been used to obtain improved systems of CPT-related compounds. In these systems, one of the most important concepts is the enhanced permeability and retention (EPR) effect. The outcomes obtained by these approaches are displayed by introducing concrete examples.

  18. Chirality as a physical aspect of structure formation in biological macromolecular systems

    NASA Astrophysics Data System (ADS)

    Malyshko, E. V.; Tverdislov, V. A.

    2016-08-01

    A novel regularity of hierarchical structures is found in the formation of chiral biological macromolecular systems. The formation of structures with alternating chirality (helical structures) serves as an instrument of stratification. The ability of a carbon atom to form chiral compounds is an important factor that determined the carbon basis of living systems on the Earth as well as their development through a series of chiral bifurcations. In the course of biological evolution, the helical structures became basic elements of the molecular machines in the cell. The discreteness of structural levels allowed the mechanical degrees of freedom formation in the molecular machines in the cell.

  19. Ground Based Program for the Physical Analysis of Macromolecular Crystal Growth

    NASA Technical Reports Server (NTRS)

    Malkin, Alexander J.

    1998-01-01

    During the past year we have focused on application of in situ Atomic Force Microscopy (AFM) for studies of the growth mechanisms and kinetics of crystallization for different macromolecular systems. Mechanisms of macrostep formation and their decay, which are important in understanding of defect formation, were studied on the surfaces of thaumatin, catalase, canavalin and lysozyme crystals. Experiments revealed that step bunching on crystalline surfaces occurred either due to two- or three-dimensional nucleation on the terraces of vicinal slopes or as a result of uneven step generation by complex dislocation sources. No step bunching arising from interaction of individual steps in the course of the experiment was observed. The molecular structure of the growth steps for thaumatin and lipase crystals were deduced. It was further shown that growth step advance occurs by incorporation of single protein molecules. In singular directions growth steps move by one-dimensional nucleation on step edges followed by lateral growth. One-dimensional nuclei have different sizes, less then a single unit cell, varying for different directions of step movement. There is no roughness due to thermal fluctuations, and each protein molecule which incorporated into the step remained. Growth kinetics for catalase crystals was investigated over wide supersaturation ranges. Strong directional kinetic anisotropy in the tangential step growth rates in different directions was seen. The influence of impurities on growth kinetics and cessation of macromolecular crystals was studied. Thus, for catalase, in addition to pronounced impurity effects on the kinetics of crystallization, we were also able to directly observe adsorption of some impurities. At low supersaturation we repeatedly observed filaments which formed from impurity molecules sedimenting on the surfaces. Similar filaments were observed on the surfaces of thaumatin, canavalin and STMV crystals as well, but the frequency was low

  20. Phase-contrast radiography.

    PubMed

    Gao, D; Pogany, A; Stevenson, A W; Wilkins, S W

    1998-01-01

    For the past 100 years, the paradigm for radiography has been premised on absorption as the sole means of contrast formation and on ray optics as the basis for image interpretation. A new conceptual approach to radiography has been developed that includes phase (ie, refractive) contrast and requires wave optics for proper treatment. This new approach greatly increases the amount of information that can be obtained with radiographic techniques and is particularly well suited to the imaging of soft tissue and of very small features in biologic samples. A key feature of the present technique of phase-contrast radiography is the use of a microfocus x-ray source about an order of magnitude (< or = 20 microm) smaller than that used in conventional radiography. Phase-contrast radiography offers a number of improvements over conventional radiography in a clinical setting, especially in soft-tissue imaging. These improvements include increased contrast resulting in improved visualization of anatomic detail, reduced absorbed dose to the patient, inherent image magnification and high spatial resolution, use of harder x rays, and relative ease of implementation. More technologically advanced detectors are currently being developed and commercialized, which will help fully realize the considerable potential of phase-contrast imaging.

  1. Automated particle picking for low-contrast macromolecules in cryo-electron microscopy

    PubMed Central

    Langlois, Robert; Pallesen, Jesper; Ash, Jordan T.; Ho, Danny Nam; Rubinstein, John L.; Frank, Joachim

    2014-01-01

    Cryo-electron microscopy is an increasingly popular tool for studying the structure and dynamics of biological macromolecules at high resolution. A crucial step in automating single-particle reconstruction of a biological sample is the selection of particle images from a micrograph. We present a novel algorithm for selecting particle images in low-contrast conditions; it proves more effective than the human eye on close-to-focus micrographs, yielding improved or comparable resolution in reconstructions of two macromolecular complexes. PMID:24607413

  2. Macromolecular liquids

    SciTech Connect

    Safinya, C.R.; Safran, S.A. ); Pincus, P.A. )

    1990-01-01

    Liquids include a broad range of material systems which are of high scientific and technological interest. Generally speaking, these are partially ordered or disordered phases where the individual molecular species have organized themselves on length scales which are larger than simple fluids, typically between 10 Angstroms and several microns. The specific systems reported on in this book include membranes, microemulsions, micelles, liquid crystals, colloidal suspensions, and polymers. They have a major impact on a broad spectrum of technological industries such as displays, plastics, soap and detergents, chemicals and petroleum, and pharmaceuticals.

  3. Psychophysical contrast calibration

    PubMed Central

    To, Long; Woods, Russell L; Goldstein, Robert B; Peli, Eli

    2013-01-01

    Electronic displays and computer systems offer numerous advantages for clinical vision testing. Laboratory and clinical measurements of various functions and in particular of (letter) contrast sensitivity require accurately calibrated display contrast. In the laboratory this is achieved using expensive light meters. We developed and evaluated a novel method that uses only psychophysical responses of a person with normal vision to calibrate the luminance contrast of displays for experimental and clinical applications. Our method combines psychophysical techniques (1) for detection (and thus elimination or reduction) of display saturating nonlinearities; (2) for luminance (gamma function) estimation and linearization without use of a photometer; and (3) to measure without a photometer the luminance ratios of the display’s three color channels that are used in a bit-stealing procedure to expand the luminance resolution of the display. Using a photometer we verified that the calibration achieved with this procedure is accurate for both LCD and CRT displays enabling testing of letter contrast sensitivity to 0.5%. Our visual calibration procedure enables clinical, internet and home implementation and calibration verification of electronic contrast testing. PMID:23643843

  4. Radiation damage within nucleoprotein complexes studied by macromolecular X-ray crystallography

    NASA Astrophysics Data System (ADS)

    Bury, Charles S.; Carmichael, Ian; McGeehan, John E.; Garman, Elspeth F.

    2016-11-01

    In X-ray crystallography, for the determination of the 3-D structure of macromolecules, radiation damage is still an inherent problem at modern third generation synchrotron sources, even when utilising cryo-crystallographic techniques (sample held at 100 K). At doses of just several MGy, at which a typical diffraction dataset is collected, site-specific radiation-induced chemical changes are known to manifest within protein crystals, and a wide body of literature is now devoted to understanding the mechanisms behind such damage. Far less is known regarding radiation-induced damage to crystalline nucleic acids and the wider class of nucleoprotein complexes during macromolecular X-ray crystallography (MX) data collection. As the MX structural biology community now strives to solve structures for increasingly larger and complex macromolecular assemblies, it essential to understand how such structures are affected by the X-ray radiation used to solve them. The purpose of this review is to summarise advances in the field of specific damage to nucleoprotein complexes and to present case studies of MX damage investigations on both protein-DNA (C.Esp1396I) and protein-RNA (TRAP) complexes. To motivate further investigations into MX damage mechanisms within nucleoprotein complexes, current and emerging protocols for investigating specific damage within Fobs(n)-Fobs(1) electron density difference maps are discussed.

  5. Progressive macromolecular self-assembly: from biomimetic chemistry to bio-inspired materials.

    PubMed

    Zhao, Yu; Sakai, Fuji; Su, Lu; Liu, Yijiang; Wei, Kongchang; Chen, Guosong; Jiang, Ming

    2013-10-04

    Macromolecular self-assembly (MSA) has been an active and fruitful research field since the 1980s, especially in this new century, which is promoted by the remarkable developments in controlled radical polymerization in polymer chemistry, etc. and driven by the demands in bio-related investigations and applications. In this review, we try to summarize the trends and recent progress in MSA in relation to biomimetic chemistry and bio-inspired materials. Our paper covers representative achievements in the fabrication of artificial building blocks for life, cell-inspired biomimetic materials, and macromolecular assemblies mimicking the functions of natural materials and their applications. It is true that the current status of the deliberately designed and obtained nano-objects based on MSA including a variety of micelles, multicompartment vesicles, and some hybrid and complex nano-objects is at their very first stage to mimic nature, but significant and encouraging progress has been made in achieving a certain similarity in morphologies or properties to that of natural ones. Such achievements also demonstrate that MSA has played an important and irreplaceable role in the grand and long-standing research of biomimetic and bio-inspired materials, the future success of which depends on mutual and persistent efforts in polymer science, material science, supramolecular chemistry, and biology.

  6. Influence of macromolecular architecture on necking in polymer extrusion film casting process

    SciTech Connect

    Pol, Harshawardhan; Banik, Sourya; Azad, Lal Busher; Doshi, Pankaj; Lele, Ashish; Thete, Sumeet

    2015-05-22

    Extrusion film casting (EFC) is an important polymer processing technique that is used to produce several thousand tons of polymer films/coatings on an industrial scale. In this research, we are interested in understanding quantitatively how macromolecular chain architecture (for example long chain branching (LCB) or molecular weight distribution (MWD or PDI)) influences the necking and thickness distribution of extrusion cast films. We have used different polymer resins of linear and branched molecular architecture to produce extrusion cast films under controlled experimental conditions. The necking profiles of the films were imaged and the velocity profiles during EFC were monitored using particle tracking velocimetry (PTV) technique. Additionally, the temperature profiles were captured using an IR thermography and thickness profiles were calculated. The experimental results are compared with predictions of one-dimensional flow model of Silagy et al{sup 1} wherein the polymer resin rheology is modeled using molecular constitutive equations such as the Rolie-Poly (RP) and extended Pom Pom (XPP). We demonstrate that the 1-D flow model containing the molecular constitutive equations provides new insights into the role of macromolecular chain architecture on film necking.{sup 1}D. Silagy, Y. Demay, and J-F. Agassant, Polym. Eng. Sci., 36, 2614 (1996)

  7. Force interacts with macromolecular structure in activation of TGF-β.

    PubMed

    Dong, Xianchi; Zhao, Bo; Iacob, Roxana E; Zhu, Jianghai; Koksal, Adem C; Lu, Chafen; Engen, John R; Springer, Timothy A

    2017-02-02

    Integrins are adhesion receptors that transmit force across the plasma membrane between extracellular ligands and the actin cytoskeleton. In activation of the transforming growth factor-β1 precursor (pro-TGF-β1), integrins bind to the prodomain, apply force, and release the TGF-β growth factor. However, we know little about how integrins bind macromolecular ligands in the extracellular matrix or transmit force to them. Here we show how integrin αVβ6 binds pro-TGF-β1 in an orientation biologically relevant for force-dependent release of TGF-β from latency. The conformation of the prodomain integrin-binding motif differs in the presence and absence of integrin binding; differences extend well outside the interface and illustrate how integrins can remodel extracellular matrix. Remodelled residues outside the interface stabilize the integrin-bound conformation, adopt a conformation similar to earlier-evolving family members, and show how macromolecular components outside the binding motif contribute to integrin recognition. Regions in and outside the highly interdigitated interface stabilize a specific integrin/pro-TGF-β orientation that defines the pathway through these macromolecules which actin-cytoskeleton-generated tensile force takes when applied through the integrin β-subunit. Simulations of force-dependent activation of TGF-β demonstrate evolutionary specializations for force application through the TGF-β prodomain and through the β- and not α-subunit of the integrin.

  8. Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta

    PubMed Central

    Wang, Ray Yu-Ruei; Song, Yifan; Barad, Benjamin A; Cheng, Yifan; Fraser, James S; DiMaio, Frank

    2016-01-01

    Cryo-EM has revealed the structures of many challenging yet exciting macromolecular assemblies at near-atomic resolution (3–4.5Å), providing biological phenomena with molecular descriptions. However, at these resolutions, accurately positioning individual atoms remains challenging and error-prone. Manually refining thousands of amino acids – typical in a macromolecular assembly – is tedious and time-consuming. We present an automated method that can improve the atomic details in models that are manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate that the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps. DOI: http://dx.doi.org/10.7554/eLife.17219.001 PMID:27669148

  9. Assessing physio-macromolecular effects of lactic acid on Zygosaccharomyces bailii cells during microaerobic fermentation.

    PubMed

    Kuanyshev, Nurzhan; Ami, Diletta; Signori, Lorenzo; Porro, Danilo; Morrissey, John P; Branduardi, Paola

    2016-08-01

    The ability of Zygosaccharomyces bailii to grow at low pH and in the presence of considerable amounts of weak organic acids, at lethal condition for Saccharomyces cerevisiae, increased the interest in the biotechnological potential of the yeast. To understand the mechanism of tolerance and growth effect of weak acids on Z. bailii, we evaluated the physiological and macromolecular changes of the yeast exposed to sub lethal concentrations of lactic acid. Lactic acid represents one of the important commodity chemical which can be produced by microbial fermentation. We assessed physiological effect of lactic acid by bioreactor fermentation using synthetic media at low pH in the presence of lactic acid. Samples collected from bioreactors were stained with propidium iodide (PI) which revealed that, despite lactic acid negatively influence the growth rate, the number of PI positive cells is similar to that of the control. Moreover, we have performed Fourier Transform Infra-Red (FTIR) microspectroscopy analysis on intact cells of the same samples. This technique has been never applied before to study Z. bailii under this condition. The analyses revealed lactic acid induced macromolecular changes in the overall cellular protein secondary structures, and alterations of cell wall and membrane physico-chemical properties.

  10. Macromolecular Crowding Studies of Amino Acids Using NMR Diffusion Measurements and Molecular Dynamics Simulations

    NASA Astrophysics Data System (ADS)

    Virk, Amninder; Stait-Gardner, Timothy; Willis, Scott; Torres, Allan; Price, William

    2015-02-01

    Molecular crowding occurs when the total concentration of macromolecular species in a solution is so high that a considerable proportion of the volume is physically occupied and therefore not accessible to other molecules. This results in significant changes in the solution properties of the molecules in such systems. Macromolecular crowding is ubiquitous in biological systems due to the generally high intracellular protein concentrations. The major hindrance to understanding crowding is the lack of direct comparison of experimental data with theoretical or simulated data. Self-diffusion is sensitive to changes in the molecular weight and shape of the diffusing species, and the available diffusion space (i.e., diffusive obstruction). Consequently, diffusion measurements are a direct means for probing crowded systems including the self-association of molecules. In this work, nuclear magnetic resonance measurements of the self-diffusion of four amino acids (glycine, alanine, valine and phenylalanine) up to their solubility limit in water were compared directly with molecular dynamics simulations. The experimental data were then analyzed using various models of aggregation and obstruction. Both experimental and simulated data revealed that the diffusion of both water and the amino acids were sensitive to the amino acid concentration. The direct comparison of the simulated and experimental data afforded greater insights into the aggregation and obstruction properties of each amino acid.

  11. Effects of macromolecular crowding on alkaline phosphatase unfolding, conformation and stability.

    PubMed

    Jia, Jiajia; Peng, Xin; Qi, Wei; Su, Rongxin; He, Zhimin

    2017-03-23

    The interior of the cell is tightly packed with various biological macromolecules, which affects physiological processes, especially protein folding process. To explore how macromolecular crowding may influence protein folding process, alkaline phosphatase (ALP) was chosen as a model protein, and the unfolding process of ALP induced by GdnHCl was studied in the presence of crowding agents such as PEG 4000, Dextran 70 and Ficoll 70. The effect of macromolecular crowding on the denatured state of ALP was directly probed by measuring enzyme activities, fluorescence spectroscopy and circular dichroism. From the results of circular dichroism, GdnHCl induced a biphasic change, suggesting that a three-state unfolding mechanism was involved in the denaturation process irrespective of the absence or presence of crowding agents. It was also found that crowding agents had a little impact on the unfolding process of ALP. The results of phase diagrams also demonstrated that the unfolding process of ALP induced by GdnHCl was three-state mechanism. Moreover, the results of fluorescence spectra demonstrated that with the increase of GdnHCl concentration, the structure of protein had changed, but existence of crowding agents can make protein structure more stable. Our results can provide valuable information for understanding the protein folding in vivo.

  12. Influence of macromolecular architecture on necking in polymer extrusion film casting process

    NASA Astrophysics Data System (ADS)

    Pol, Harshawardhan; Banik, Sourya; Azad, Lal Busher; Thete, Sumeet; Doshi, Pankaj; Lele, Ashish

    2015-05-01

    Extrusion film casting (EFC) is an important polymer processing technique that is used to produce several thousand tons of polymer films/coatings on an industrial scale. In this research, we are interested in understanding quantitatively how macromolecular chain architecture (for example long chain branching (LCB) or molecular weight distribution (MWD or PDI)) influences the necking and thickness distribution of extrusion cast films. We have used different polymer resins of linear and branched molecular architecture to produce extrusion cast films under controlled experimental conditions. The necking profiles of the films were imaged and the velocity profiles during EFC were monitored using particle tracking velocimetry (PTV) technique. Additionally, the temperature profiles were captured using an IR thermography and thickness profiles were calculated. The experimental results are compared with predictions of one-dimensional flow model of Silagy et al1 wherein the polymer resin rheology is modeled using molecular constitutive equations such as the Rolie-Poly (RP) and extended Pom Pom (XPP). We demonstrate that the 1-D flow model containing the molecular constitutive equations provides new insights into the role of macromolecular chain architecture on film necking.1D. Silagy, Y. Demay, and J-F. Agassant, Polym. Eng. Sci., 36, 2614 (1996).

  13. Fast Method for Computing Chemical Potentials and Liquid-Liquid Phase Equilibria of Macromolecular Solutions.

    PubMed

    Qin, Sanbo; Zhou, Huan-Xiang

    2016-08-25

    Chemical potential is a fundamental property for determining thermodynamic equilibria involving exchange of molecules, such as between two phases of molecular systems. Previously, we developed the fast Fourier transform (FFT)-based method for Modeling Atomistic Protein-crowder interactions (FMAP) to calculate excess chemical potentials according to the Widom insertion. Intermolecular interaction energies were expressed as correlation functions and evaluated via FFT. Here, we extend this method to calculate liquid-liquid phase equilibria of macromolecular solutions. Chemical potentials are calculated by FMAP over a wide range of molecular densities, and the condition for coexistence of low- and high-density phases is determined by the Maxwell equal-area rule. When benchmarked on Lennard-Jones fluids, our method produces an accurate phase diagram at 18% of the computational cost of the current best method. Importantly, the gain in computational speed increases dramatically as the molecules become more complex, leading to many orders of magnitude in speed up for atomistically represented proteins. We demonstrate the power of FMAP by reporting the first results for the liquid-liquid coexistence curve of γII-crystallin represented at the all-atom level. Our method may thus open the door to accurate determination of phase equilibria for macromolecular mixtures such as protein-protein mixtures and protein-RNA mixtures, that are known to undergo liquid-liquid phase separation, both in vitro and in vivo.

  14. Ion channel macromolecular complexes in cardiomyocytes: roles in sudden cardiac death.

    PubMed

    Abriel, Hugues; Rougier, Jean-Sébastien; Jalife, José

    2015-06-05

    The movement of ions across specific channels embedded on the membrane of individual cardiomyocytes is crucial for the generation and propagation of the cardiac electric impulse. Emerging evidence over the past 20 years strongly suggests that the normal electric function of the heart is the result of dynamic interactions of membrane ion channels working in an orchestrated fashion as part of complex molecular networks. Such networks work together with exquisite temporal precision to generate each action potential and contraction. Macromolecular complexes play crucial roles in transcription, translation, oligomerization, trafficking, membrane retention, glycosylation, post-translational modification, turnover, function, and degradation of all cardiac ion channels known to date. In addition, the accurate timing of each cardiac beat and contraction demands, a comparable precision on the assembly and organizations of sodium, calcium, and potassium channel complexes within specific subcellular microdomains, where physical proximity allows for prompt and efficient interaction. This review article, part of the Compendium on Sudden Cardiac Death, discusses the major issues related to the role of ion channel macromolecular assemblies in normal cardiac electric function and the mechanisms of arrhythmias leading to sudden cardiac death. It provides an idea of how these issues are being addressed in the laboratory and in the clinic, which important questions remain unanswered, and what future research will be needed to improve knowledge and advance therapy.

  15. Ion Channel Macromolecular Complexes in Cardiomyocytes: Roles in Sudden Cardiac Death

    PubMed Central

    Abriel, Hugues; Rougier, Jean-Sébastien; Jalife, José

    2015-01-01

    The movement of ions across specific channels embedded on the membrane of individual cardiomyocytes is crucial for the generation and propagation of the cardiac electrical impulse. Emerging evidence over the last 20 years strongly suggests that the normal electrical function of the heart is the result of dynamic interactions of membrane ion channels working in an orchestrated fashion as part of complex molecular networks. Such networks work together with exquisite temporal precision to generate each action potential and contraction. Macromolecular complexes play crucial roles in transcription, translation, oligomerization, trafficking, membrane retention, glycosylation, posttranslational modification, turnover, function and degradation of all cardiac ion channels known to date. In addition, the accurate timing of each cardiac beat and contraction demands, a comparable precision on the assembly and organizations of sodium, calcium and potassium channel complexes within specific subcellular microdomains, where physical proximity allows for prompt and efficient interaction. This review article, part of the Compendium on Sudden Cardiac Death, discusses the major issues related to the role of ion channel macromolecular assemblies in normal cardiac electrical function and the mechanisms of arrhythmias leading to sudden cardiac death. It provides an idea of how these issues are being addressed in the laboratory and in the clinic, which important questions remain unanswered, and what future research will be needed to improve knowledge and advance therapy. PMID:26044251

  16. The Neurobiologist's Guide to Structural Biology: A Primer on Why Macromolecular Structure Matters and How to Evaluate Structural Data

    PubMed Central

    Minor, Daniel L.

    2010-01-01

    Structural biology now plays a prominent role in addressing questions central to understanding how excitable cells function. Although interest in the insights gained from the definition and dissection of macromolecular anatomy is high, many neurobiologists remain unfamiliar with the methods employed. This primer aims to help neurobiologists understand approaches for probing macromolecular structure and where the limits and challenges remain. Using examples of macromolecules with neurobiological importance, the review covers X-ray crystallography, electron microscopy (EM), small-angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) and biophysical methods with which these approaches are often paired: isothermal titration calorimetry (ITC), equilibrium analytical ultracentifugation, and molecular dynamics (MD). PMID:17521566

  17. The neurobiologist's guide to structural biology: a primer on why macromolecular structure matters and how to evaluate structural data.

    PubMed

    Minor, Daniel L

    2007-05-24

    Structural biology now plays a prominent role in addressing questions central to understanding how excitable cells function. Although interest in the insights gained from the definition and dissection of macromolecular anatomy is high, many neurobiologists remain unfamiliar with the methods employed. This primer aims to help neurobiologists understand approaches for probing macromolecular structure and where the limits and challenges remain. Using examples of macromolecules with neurobiological importance, the review covers X-ray crystallography, electron microscopy (EM), small-angle X-ray scattering (SAXS), and nuclear magnetic resonance (NMR) and biophysical methods with which these approaches are often paired: isothermal titration calorimetry (ITC), equilibrium analytical ultracentifugation, and molecular dynamics (MD).

  18. Survey of contrast media used in coronary angiograph

    SciTech Connect

    Weikl, A.; Hubmann, M.

    1982-01-01

    In recent years, various contrast media have been developed for use in coronary angiography. These contrast media may be divided into ionic contrast media of high osmolality, those of low osmolality, and nonionic contrast materials. We conducted our own clinical studies with 40 patients. In random succession a standard contrast medium (ionic, of high osmolality) and a new-generation contrast medium (either nonionic or ionic with low osmolality) were injected into the right and left coronary arteries. After each injection we measured the systolic and diastolic blood pressure using a liquid-filled coronary catheter. In addition, the change in the length of the cardiac cycle was registered in terms of the R-R interval (in ms) and at the same time, leads I, II, and III of the ECG were recorded. We studied the influence of the various contrast media on the activity of ATPase in in vitro experiments, using Lasser and Lang's. When ionic contrast media of low osmolality and nonionic contrast media were utilized the heart rate showed no change. Disturbances of rhythm such as ventricular tachycardia or fibrillation were not observed. All of the contrast media used produced the same ECG changes. These changes can be ascribed to the inhibition of ATPase. The arterial blood pressure was lowered significantly only by ionic contrast media of high osmolality only.

  19. Macromolecular crowding: chemistry and physics meet biology (Ascona, Switzerland, 10-14 June 2012)

    NASA Astrophysics Data System (ADS)

    Foffi, G.; Pastore, A.; Piazza, F.; Temussi, P. A.

    2013-08-01

    More than 60 years of biochemical and biophysical studies have accustomed us to think of proteins as highly purified entities that act in isolation, more or less freely diffusing until they find their cognate partner to bind to. While in vitro experiments that reproduce these conditions largely remain the only way to investigate the intrinsic properties of molecules, this approach ignores an important factor: in their natural milieu , proteins are surrounded by several other molecules of different chemical nature, and this crowded environment can considerably modify their behaviour. About 40% of the cellular volume on average is occupied by all sorts of molecules. Furthermore, biological macromolecules live and operate in an extremely structured and complex environment within the cell (endoplasmic reticulum, Golgi apparatus, cytoskeletal structures, etc). Hence, to further complicate the picture, the interior of the cell is by no means a simply crowded medium, rather, a most crowded and confining one. In recent times, several approaches have been developed in the attempt to take into account important factors such as the ones mentioned above, at both theoretical and experimental levels, so that this field of research is now emerging as one of the most thriving in molecular and cell biology (see figure 1). Figure 1. Figure 1. Left: number of articles containing the word 'crowding' as a keyword limited to the biological and chemical science domains (source: ISI Web of Science). The arrow flags the 2003 'EMBO Workshop on Biological Implications of Macromolecular Crowding' (Embo, 2012). Right: number of citations to articles containing the word 'crowding' limited to the same domains (bars) and an exponential regression curve (source: Elsevier Scopus). To promote the importance of molecular crowding and confinement and provide researchers active in this field an interdisciplinary forum for meeting and exchanging ideas, we recently organized an international conference

  20. Macromolecular crowding: chemistry and physics meet biology (Ascona, Switzerland, 10-14 June 2012).

    PubMed

    Foffi, G; Pastore, A; Piazza, F; Temussi, P A

    2013-08-02

    More than 60 years of biochemical and biophysical studies have accustomed us to think of proteins as highly purified entities that act in isolation, more or less freely diffusing until they find their cognate partner to bind to. While in vitro experiments that reproduce these conditions largely remain the only way to investigate the intrinsic properties of molecules, this approach ignores an important factor: in their natural milieu , proteins are surrounded by several other molecules of different chemical nature, and this crowded environment can considerably modify their behaviour. About 40% of the cellular volume on average is occupied by all sorts of molecules. Furthermore, biological macromolecules live and operate in an extremely structured and complex environment within the cell (endoplasmic reticulum, Golgi apparatus, cytoskeletal structures, etc). Hence, to further complicate the picture, the interior of the cell is by no means a simply crowded medium, rather, a most crowded and confining one. In recent times, several approaches have been developed in the attempt to take into account important factors such as the ones mentioned above, at both theoretical and experimental levels, so that this field of research is now emerging as one of the most thriving in molecular and cell biology (see figure 1). [Formula: see text] Figure 1. Left: number of articles containing the word 'crowding' as a keyword limited to the biological and chemical science domains (source: ISI Web of Science). The arrow flags the 2003 'EMBO Workshop on Biological Implications of Macromolecular Crowding' (Embo, 2012). Right: number of citations to articles containing the word 'crowding' limited to the same domains (bars) and an exponential regression curve (source: Elsevier Scopus). To promote the importance of molecular crowding and confinement and provide researchers active in this field an interdisciplinary forum for meeting and exchanging ideas, we recently organized an international

  1. Phonation in Tonal Contrasts

    ERIC Educational Resources Information Center

    Kuang, Jianjing

    2013-01-01

    Phonation is used in many tonal languages, but how it should be incorporated into tonal systems is not well understood. The purpose of this dissertation thus is to examine the role of phonation in tonal contrasts, and to investigate how phonation and pitch interact in the tonal space. This dissertation presents close studies of tonal contrasts…

  2. Simultaneous blur contrast

    NASA Astrophysics Data System (ADS)

    Webster, Shernaaz M.; Webster, Michael A.; Taylor, John; Jaikumar, Jaikishan; Verma, Richa

    2001-06-01

    How well-focused an image appears can be strongly influenced by the surroundings context. A blurred surround can cause a central image to appear too sharp, while sharped surrounds can induce blur. We examined some spatial properties and stimulus selectivities of this 'simultaneous blur contrast.' Observers adjusted the focus of a central test image by a 2AFC staircase procedure that varied the slope of the image amplitude spectrum. The test were surrounded by 8 identical images with biased spectra, that were presented concurrently with the test for 0.5 sec on a uniform gray background. Contrast effects were comparable in magnitude for image sizes ranging from 1-deg to 4-deg in visual angle, but were stronger for test that were viwe4 in the periphery rather than fixated directly. Consistent biases were found for different types of grayscale images, including natural images, filtered noise, and simple edges. However, effects were weaker when surrounds and tests were drawn from different images, or differed in contrast-polarity or color, and thus do not depend on blur or on average spatial- frequency content per se. These induction effects may in part reflect a manifestation of selective contrast gain control

  3. Contrasting Views on Censorship.

    ERIC Educational Resources Information Center

    Riel, Arthur R., Jr.

    This paper asserts that public demands for book censorship are but one aspect of a deep public dissatisfaction with the educational establishment, and develops the thesis that the cause of this dissatisfaction is the contrast in world views and religions of those in the educational establishment and the censors. The educational establishment…

  4. Modulation of 3-methylcholanthrene toxicity in cultured neoplastic keratinocytes by glucocorticoids and retinoids is not accounted for by macromolecular adduct formation.

    PubMed Central

    Rubin, A L; Rice, R H

    1989-01-01

    3-Methylcholanthrene (3-MC) greatly inhibits the growth of two lines of human squamous carcinoma cells, SCC-9 and SCC-12B2. Exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin alone was much less effective and, in the presence of 3-MC, did not alter the sensitivity (EC50 = 0.3 microM) or extent of growth inhibition by the latter. The degree of 3-MC-mediated inhibition, however, was markedly alleviated by inclusion of retinoic acid (EC50 greater than or equal to 0.7 microM) and hydrocortisone (EC50 = 40 nM) or dexamethasone (EC50 = 3 nM) in the culture medium. These physiological effectors, which are known to have opposing actions on keratinocyte character in SCC cells, did not significantly alter either aryl hydrocarbon hydroxylase activity or macromolecular adduct formation. Further analysis of the cellular responses indicated that hydrocortisone and, in some experiments, retinoids increased the growth rate in 3-MC-exposed cultures, while 3-MC increased the saturation density in retinoic acid-exposed cultures, an example of interference with a physiological response of the cells. These results indicate that alteration of the differentiated state, regardless of the direction of the change, can alter the sensitivity of the cells to toxic stimuli. Further investigation of the bases of such toxic responses and their modulation by the microenvironment may enhance our understanding of the target cell specificity of polycyclic aromatic hydrocarbons. Images PMID:2468166

  5. Hypermedia as medium

    NASA Technical Reports Server (NTRS)

    Dede, Christopher J.

    1990-01-01

    Claims and rebuttals that hypermedia (the associative, nonlinear interconnection of multimedia materials) is a fundamentally innovative means of thinking and communicating are described. This representational architecture has many advantages that make it a major advance over other media; however, it also has several intrinsic problems that severly limits its effectiveness as a medium. These advantages and limits in applications are discussed.

  6. Holographic recording medium

    NASA Technical Reports Server (NTRS)

    Gange, Robert Allen (Inventor)

    1977-01-01

    A holographic recording medium comprising a conductive substrate, a photoconductive layer and an electrically alterable layer of a linear, low molecular weight hydrocarbon polymer has improved fatigue resistance. An acrylic barrier layer can be interposed between the photoconductive and electrically alterable layers.

  7. MCNP simulation of absorbed energy and dose by iodinated contrast agent

    NASA Astrophysics Data System (ADS)

    He, Wenjun; Mah, Eugene; Huda, Walter; Yao, Hai

    2012-03-01

    The purpose of this study is to investigate the absorbed dose and energy by iodinated contrast medium in diagnostic radiology. A simulation geometry in which an inner sphere (d = 0.2cm, 1cm, 5cm) filled with iodinated contrast medium (or water) is located at the center of a 20cm diameter water sphere was used in simulations performed with MCNP5 codes. Monoenergetic x-rays with energies ranging from 40 to 80keV from a cone beam source were utilized and contrast medium concentration ranged from 100 to 1mg/ml. Absorbed dose ratio (RD) to inner sphere and total absorbed energies ratio (RE) to the whole phantom with and without iodinated contrast medium were investigated. The maximum RD was ~13 for the 0.2cm diameter sphere with 100mg/ml contrast medium. The maximum RE was ~1.05 for the 5cm diameter contrast sphere at 80keV with 100mg/ml contrast medium. Under the same incident photon energy, increasing the inner sphere size from 0.2cm to 5cm caused a ~63% increase in the RD on average. Decreasing the contrast medium concentration from 100 to 10 mg/ml caused a decrease of RD of ~ 76%. A conclusion was reached that although local absorbed dose increase caused by iodinated contrast agent could be high; the increase in total absorbed energy is negligible.

  8. Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

    PubMed Central

    Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O’Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

    2016-01-01

    Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes. PMID:27478033

  9. Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis

    NASA Astrophysics Data System (ADS)

    Cigognini, Daniela; Gaspar, Diana; Kumar, Pramod; Satyam, Abhigyan; Alagesan, Senthilkumar; Sanz-Nogués, Clara; Griffin, Matthew; O’Brien, Timothy; Pandit, Abhay; Zeugolis, Dimitrios I.

    2016-08-01

    Modular tissue engineering is based on the cells’ innate ability to create bottom-up supramolecular assemblies with efficiency and efficacy still unmatched by man-made devices. Although the regenerative potential of such tissue substitutes has been documented in preclinical and clinical setting, the prolonged culture time required to develop an implantable device is associated with phenotypic drift and/or cell senescence. Herein, we demonstrate that macromolecular crowding significantly enhances extracellular matrix deposition in human bone marrow mesenchymal stem cell culture at both 20% and 2% oxygen tension. Although hypoxia inducible factor - 1α was activated at 2% oxygen tension, increased extracellular matrix synthesis was not observed. The expression of surface markers and transcription factors was not affected as a function of oxygen tension and macromolecular crowding. The multilineage potential was also maintained, albeit adipogenic differentiation was significantly reduced in low oxygen tension cultures, chondrogenic differentiation was significantly increased in macromolecularly crowded cultures and osteogenic differentiation was not affected as a function of oxygen tension and macromolecular crowding. Collectively, these data pave the way for the development of bottom-up tissue equivalents based on physiologically relevant developmental processes.

  10. Probing the Interplay of Size, Shape, and Solution Environment in Macromolecular Diffusion Using a Simple Refraction Experiment

    ERIC Educational Resources Information Center

    Mankidy, Bijith D.; Coutinho, Cecil A.; Gupta, Vinay K.

    2010-01-01

    The diffusion coefficient of polymers is a critical parameter in biomedicine, catalysis, chemical separations, nanotechnology, and other industrial applications. Here, measurement of macromolecular diffusion in solutions is described using a visually instructive, undergraduate-level optical refraction experiment based on Weiner's method. To…

  11. Chromatography: concepts and contrasts

    SciTech Connect

    Miller, J.M.

    1988-01-01

    As the author states in the Preface, this text attempts to provide a unified approach to chromatography (hence the title) by way of contrasting similarities and differences between gas chromatography (GC), column liquid chromatography (LC), and thin-layer chromatography (TLC). This book is also said to be pitched at an elementary level, suitable for most newcomers to the field (e.g., advanced undergraduates and beginning graduate students in the academic world, as well as bench-level chemists in industry).

  12. Polychromatic diffraction contrast tomography

    SciTech Connect

    King, A.; Reischig, P.; Adrien, J.; Peetermans, S.; Ludwig, W.

    2014-11-15

    This tutorial review introduces the use of polychromatic radiation for 3D grain mapping using X-ray diffraction contrast tomography. The objective is to produce a 3D map of the grain shapes and orientations within a bulk, millimeter-sized polycrystalline sample. The use of polychromatic radiation enables the standard synchrotron X-ray technique to be applied in a wider range of contexts: 1) Using laboratory X-ray sources allows a much wider application of the diffraction contrast tomography technique. 2) Neutron sources allow large samples, or samples containing high Z elements to be studied. 3) Applied to synchrotron sources, smaller samples may be treated, or faster measurements may be possible. Challenges and particularities in the data acquisition and processing, and the limitations of the different variants, are discussed. - Highlights: • We present a tutorial review of polychromatic diffraction contrast tomography techniques. • The use of polychromatic radiation allows the standard synchrotron DCT technique to be extended to a range of other sources. • The characteristics and limitations of all variants of the techniques are derived, discussed and compared. • Examples using laboratory X-ray and cold neutron radiation are presented. • Suggestions for the future development of these techniques are presented.

  13. Anisotropic contrast optical microscope.

    PubMed

    Peev, D; Hofmann, T; Kananizadeh, N; Beeram, S; Rodriguez, E; Wimer, S; Rodenhausen, K B; Herzinger, C M; Kasputis, T; Pfaunmiller, E; Nguyen, A; Korlacki, R; Pannier, A; Li, Y; Schubert, E; Hage, D; Schubert, M

    2016-11-01

    An optical microscope is described that reveals contrast in the Mueller matrix images of a thin, transparent, or semi-transparent specimen located within an anisotropic object plane (anisotropic filter). The specimen changes the anisotropy of the filter and thereby produces contrast within the Mueller matrix images. Here we use an anisotropic filter composed of a semi-transparent, nanostructured thin film with sub-wavelength thickness placed within the object plane. The sample is illuminated as in common optical microscopy but the light is modulated in its polarization using combinations of linear polarizers and phase plate (compensator) to control and analyze the state of polarization. Direct generalized ellipsometry data analysis approaches permit extraction of fundamental Mueller matrix object plane images dispensing with the need of Fourier expansion methods. Generalized ellipsometry model approaches are used for quantitative image analyses. These images are obtained from sets of multiple images obtained under various polarizer, analyzer, and compensator settings. Up to 16 independent Mueller matrix images can be obtained, while our current setup is limited to 11 images normalized by the unpolarized intensity. We demonstrate the anisotropic contrast optical microscope by measuring lithographically defined micro-patterned anisotropic filters, and we quantify the adsorption of an organic self-assembled monolayer film onto the anisotropic filter. Comparison with an isotropic glass slide demonstrates the image enhancement obtained by our method over microscopy without the use of an anisotropic filter. In our current instrument, we estimate the limit of detection for organic volumetric mass within the object plane of ≈49 fg within ≈7 × 7 μm(2) object surface area. Compared to a quartz crystal microbalance with dissipation instrumentation, where contemporary limits require a total load of ≈500 pg for detection, the instrumentation demonstrated here improves

  14. Anisotropic contrast optical microscope

    NASA Astrophysics Data System (ADS)

    Peev, D.; Hofmann, T.; Kananizadeh, N.; Beeram, S.; Rodriguez, E.; Wimer, S.; Rodenhausen, K. B.; Herzinger, C. M.; Kasputis, T.; Pfaunmiller, E.; Nguyen, A.; Korlacki, R.; Pannier, A.; Li, Y.; Schubert, E.; Hage, D.; Schubert, M.

    2016-11-01

    An optical microscope is described that reveals contrast in the Mueller matrix images of a thin, transparent, or semi-transparent specimen located within an anisotropic object plane (anisotropic filter). The specimen changes the anisotropy of the filter and thereby produces contrast within the Mueller matrix images. Here we use an anisotropic filter composed of a semi-transparent, nanostructured thin film with sub-wavelength thickness placed within the object plane. The sample is illuminated as in common optical microscopy but the light is modulated in its polarization using combinations of linear polarizers and phase plate (compensator) to control and analyze the state of polarization. Direct generalized ellipsometry data analysis approaches permit extraction of fundamental Mueller matrix object plane images dispensing with the need of Fourier expansion methods. Generalized ellipsometry model approaches are used for quantitative image analyses. These images are obtained from sets of multiple images obtained under various polarizer, analyzer, and compensator settings. Up to 16 independent Mueller matrix images can be obtained, while our current setup is limited to 11 images normalized by the unpolarized intensity. We demonstrate the anisotropic contrast optical microscope by measuring lithographically defined micro-patterned anisotropic filters, and we quantify the adsorption of an organic self-assembled monolayer film onto the anisotropic filter. Comparison with an isotropic glass slide demonstrates the image enhancement obtained by our method over microscopy without the use of an anisotropic filter. In our current instrument, we estimate the limit of detection for organic volumetric mass within the object plane of ≈49 fg within ≈7 × 7 μm2 object surface area. Compared to a quartz crystal microbalance with dissipation instrumentation, where contemporary limits require a total load of ≈500 pg for detection, the instrumentation demonstrated here improves

  15. Phenix - a comprehensive python-based system for macromolecular structure solution

    SciTech Connect

    Terwilliger, Thomas C; Hung, Li - Wei; Adams, Paul D; Afonine, Pavel V; Bunkoczi, Gabor; Chen, Vincent B; Davis, Ian; Echols, Nathaniel; Headd, Jeffrey J; Grosse Kunstleve, Ralf W; Mccoy, Airlie J; Moriarty, Nigel W; Oeffner, Robert; Read, Randy J; Richardson, David C; Richardson, Jane S; Zwarta, Peter H

    2009-01-01

    Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages, and the repeated use of interactive three-dimensional graphics. Phenix has been developed to provide a comprehensive system for crystallographic structure solution with an emphasis on automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand, and finally the development of a framework that allows a tight integration between the algorithms.

  16. Nalidixic Acid and Macromolecular Metabolism in Tetrahymena pyriformis: Effects on Protein Synthesis

    PubMed Central

    de Castro, J. F.; Carvalho, J. F. O.; Moussatché, N.; de Castro, F. T.

    1975-01-01

    A study on the effect of nalidixic acid on macromolecular metabolism, particularly of protein, in Tetrahymena pyriformis was performed. It was shown that the compound is a potent inhibitor of deoxyribonucleic acid, ribonucleic acid, and protein synthesis for this organism. A conspicuous breakdown of polysomes, accompanied by the accumulation of 80S ribosomes, occurred in cells incubated for 10 min with the drug; polysome formation was prevented. The accumulating 80S particles were shown to be run-off ribosomal units. The incorporation of amino acids by a cell-free system is not affected by nalidixic acid. In nonproliferating cells the incorporation was also not prevented, unless the cells were previously incubated with the drug. These results are discussed in terms of the possible mechanism of action of nalidixic acid in T. pyriformis. PMID:807153

  17. Phase transitions of macromolecular microsphere composite hydrogels based on the stochastic Cahn–Hilliard equation

    SciTech Connect

    Li, Xiao Ji, Guanghua Zhang, Hui

    2015-02-15

    We use the stochastic Cahn–Hilliard equation to simulate the phase transitions of the macromolecular microsphere composite (MMC) hydrogels under a random disturbance. Based on the Flory–Huggins lattice model and the Boltzmann entropy theorem, we develop a reticular free energy suit for the network structure of MMC hydrogels. Taking the random factor into account, with the time-dependent Ginzburg-Landau (TDGL) mesoscopic simulation method, we set up a stochastic Cahn–Hilliard equation, designated herein as the MMC-TDGL equation. The stochastic term in the equation is constructed appropriately to satisfy the fluctuation-dissipation theorem and is discretized on a spatial grid for the simulation. A semi-implicit difference scheme is adopted to numerically solve the MMC-TDGL equation. Some numerical experiments are performed with different parameters. The results are consistent with the physical phenomenon, which verifies the good simulation of the stochastic term.

  18. RosettaScripts: a scripting language interface to the Rosetta macromolecular modeling suite.

    PubMed

    Fleishman, Sarel J; Leaver-Fay, Andrew; Corn, Jacob E; Strauch, Eva-Maria; Khare, Sagar D; Koga, Nobuyasu; Ashworth, Justin; Murphy, Paul; Richter, Florian; Lemmon, Gordon; Meiler, Jens; Baker, David

    2011-01-01

    Macromolecular modeling and design are increasingly useful in basic research, biotechnology, and teaching. However, the absence of a user-friendly modeling framework that provides access to a wide range of modeling capabilities is hampering the wider adoption of computational methods by non-experts. RosettaScripts is an XML-like language for specifying modeling tasks in the Rosetta framework. RosettaScripts provides access to protocol-level functionalities, such as rigid-body docking and sequence redesign, and allows fast testing and deployment of complex protocols without need for modifying or recompiling the underlying C++ code. We illustrate these capabilities with RosettaScripts protocols for the stabilization of proteins, the generation of computationally constrained libraries for experimental selection of higher-affinity binding proteins, loop remodeling, small-molecule ligand docking, design of ligand-binding proteins, and specificity redesign in DNA-binding proteins.

  19. Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes

    PubMed Central

    Ilca, Serban L.; Kotecha, Abhay; Sun, Xiaoyu; Poranen, Minna M.; Stuart, David I.; Huiskonen, Juha T.

    2015-01-01

    Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems. PMID:26534841

  20. Macromolecular crowding develops heterogeneous environments of gene expression in picoliter droplets

    PubMed Central

    Hansen, Maike M. K.; Meijer, Lenny H. H.; Spruijt, Evan; Maas, Roel J. M.; Roquelles, Marta Ventosa; Groen, Joost; Heus, Hans A.; Huck, Wilhelm T. S.

    2015-01-01

    Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalised biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in cell-free gene expression of two genes compartmentalised within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous micro-environments of mRNA as local production rates exceed diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery to stay in the same microenvironment, directly increasing the system’s stochasticity. PMID:26501750

  1. CheckMyMetal: a macromolecular metal-binding validation tool

    PubMed Central

    Porebski, Przemyslaw J.

    2017-01-01

    Metals are essential in many biological processes, and metal ions are modeled in roughly 40% of the macromolecular structures in the Protein Data Bank (PDB). However, a significant fraction of these structures contain poorly modeled metal-binding sites. CheckMyMetal (CMM) is an easy-to-use metal-binding site validation server for macromolecules that is freely available at http://csgid.org/csgid/metal_sites. The CMM server can detect incorrect metal assignments as well as geometrical and other irregularities in the metal-binding sites. Guidelines for metal-site modeling and validation in macromolecules are illustrated by several practical examples grouped by the type of metal. These examples show CMM users (and crystallographers in general) problems they may encounter during the modeling of a specific metal ion. PMID:28291757

  2. Ground Based Program for the Physical Analysis of Macromolecular Crystal Growth

    NASA Technical Reports Server (NTRS)

    Malkin, Alexander J.

    1999-01-01

    In a reported period in situ atomic force microscopy was utilized in our laboratory to study mechanisms of growth and kinetics of crystallization of ten protein and virus crystals. These included canavalin, thaumatin, apoferritin, lipase, catalase, t-RNA, lysozyme, xylanase, turnip yellow mosaic virus (TYMV) and satellite tobacco mosaic virus (STMV). We have also designed and constructed in our laboratory both in situ conventional two-beam Michelson and phase shift Mach-Zenhder interferometers. Computer software for the processing of the interferometric images was developed as well. Interferometric techniques were applied for studies of growth kinetics and transport phenomena in crystallization of several macromolecular crystals. As a result of this work we have published 21 papers and have given many presentations at international and national meetings. A list of these publications and conference presentations is attached.

  3. The New Macromolecular Crystallography Stations At MAX-lab: The MAD Station

    SciTech Connect

    Ursby, Thomas; Svensson, Christer; Sommarin, Bengt; Mammen, Christian B.; Als-Nielsen, Jens; Cerenius, Yngve; Fodje, Michel N.; Logan, Derek T.; Thunnissen, Marjolein M. G. M.; Liljas, Anders; Larsen, Sine

    2004-05-12

    A new beamline, Cassiopeia, at MAX II is about to come into operation. It consists of an energy-tunable station and four side stations intended for macromolecular crystallography. The X-ray source is a 3.5 T superconducting multipole wiggler installed in the 1.5 GeV MAX II storage ring. The energy-tunable station use grazing incidence Rh-coated silicon mirrors and an internally water-cooled Si(111) double-crystal monochromator while the four side stations use bent diamond and germanium monochromators and multilayer mirrors. This paper concentrates on the optics design of the energy-tunable station and also briefly describes other beamline components.

  4. Polymer segregation under confinement: Influences of macromolecular crowding and the interaction between the polymer and crowders

    NASA Astrophysics Data System (ADS)

    Chen, Yuhao; Yu, Wancheng; Wang, Jiajun; Luo, Kaifu

    2015-10-01

    Entropy driven polymer segregation in confinements as a model for chromosome separation in bacteria has attracted wide attention; however, the effects of macromolecular crowding and the interaction between the binding protein and the newly replicated DNA on the segregation dynamics are not clear. Using Langevin dynamics simulations, we investigate the influences of crowders and the attractive interaction between the polymer and a small number of crowders on segregation of two overlapping polymers under a cylindrical confinement. We find that the segregation time increases with increasing the volume fraction of crowders due to the slower chain diffusion in crowded environments. For a fixed volume fraction of crowders, the segregation time decreases with increasing the size of crowders. Moreover, the attractive interaction between the polymer and a small number of crowders can significantly facilitate the chain segregation. These results are important for understanding the chromosome segregation in living cells.

  5. Function and dynamics of macromolecular complexes explored by integrative structural and computational biology.

    PubMed

    Purdy, Michael D; Bennett, Brad C; McIntire, William E; Khan, Ali K; Kasson, Peter M; Yeager, Mark

    2014-08-01

    Three vignettes exemplify the potential of combining EM and X-ray crystallographic data with molecular dynamics (MD) simulation to explore the architecture, dynamics and functional properties of multicomponent, macromolecular complexes. The first two describe how EM and X-ray crystallography were used to solve structures of the ribosome and the Arp2/3-actin complex, which enabled MD simulations that elucidated functional dynamics. The third describes how EM, X-ray crystallography, and microsecond MD simulations of a GPCR:G protein complex were used to explore transmembrane signaling by the β-adrenergic receptor. Recent technical advancements in EM, X-ray crystallography and computational simulation create unprecedented synergies for integrative structural biology to reveal new insights into heretofore intractable biological systems.

  6. Parallel macromolecular delivery and biochemical/electrochemical interface to cells employing nanostructures

    SciTech Connect

    McKnight, Timothy E; Melechko, Anatoli V; Griffin, Guy D; Guillorn, Michael A; Merkulov, Vladimir L; Simpson, Michael L

    2015-03-31

    Systems and methods are described for parallel macromolecular delivery and biochemical/electrochemical interface to whole cells employing carbon nanostructures including nanofibers and nanotubes. A method includes providing a first material on at least a first portion of a first surface of a first tip of a first elongated carbon nanostructure; providing a second material on at least a second portion of a second surface of a second tip of a second elongated carbon nanostructure, the second elongated carbon nanostructure coupled to, and substantially parallel to, the first elongated carbon nanostructure; and penetrating a boundary of a biological sample with at least one member selected from the group consisting of the first tip and the second tip.

  7. Flexible torsion-angle noncrystallographic symmetry restraints for improved macromolecular structure refinement

    PubMed Central

    Headd, Jeffrey J.; Echols, Nathaniel; Afonine, Pavel V.; Moriarty, Nigel W.; Gildea, Richard J.; Adams, Paul D.

    2014-01-01

    One of the great challenges in refining macromolecular crystal structures is a low data-to-parameter ratio. Historically, knowledge from chemistry has been used to help to improve this ratio. When a macromolecule crystallizes with more than one copy in the asymmetric unit, the noncrystallographic symmetry relationships can be exploited to provide additional restraints when refining the working model. However, although globally similar, NCS-related chains often have local differences. To allow for local differences between NCS-related molecules, flexible torsion-based NCS restraints have been introduced, coupled with intelligent rotamer handling for protein chains, and are available in phenix.refine for refinement of models at all resolutions. PMID:24816103

  8. The kinetic dose limit in room-temperature time-resolved macromolecular crystallography

    PubMed Central

    Schmidt, M.; Šrajer, V.; Purwar, N.; Tripathi, S.

    2012-01-01

    Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored. PMID:22338689

  9. Macromolecular Approaches to Prevent Thrombosis and Intimal Hyperplasia Following Percutaneous Coronary Intervention

    PubMed Central

    2015-01-01

    Cardiovascular disease remains one of the largest contributors to death worldwide. Improvements in cardiovascular technology leading to the current generation of drug-eluting stents, bioresorbable stents, and drug-eluting balloons, coupled with advances in antirestenotic therapeutics developed by pharmaceutical community, have had a profound impact on quality of life and longevity. However, these procedures and devices contribute to both short- and long-term complications. Thus, room for improvement and development of new, alternative strategies exists. Two major approaches have been investigated to improve outcomes following percutaneous coronary intervention including perivascular delivery and luminal paving. For both approaches, polymers play a major role as controlled research vehicles, carriers for cells, and antithrombotic coatings. With improvements in catheter delivery devices and increases in our understanding of the biology of healthy and diseased vessels, the time is ripe for development of novel macromolecular coatings that can protect the vessel lumen following balloon angioplasty and promote healthy vascular healing. PMID:24964369

  10. Imaging of doxorubicin release from theranostic macromolecular prodrugs via fluorescence resonance energy transfer.

    PubMed

    Krüger, Harald R; Schütz, Irene; Justies, Aileen; Licha, Kai; Welker, Pia; Haucke, Volker; Calderón, Marcelo

    2014-11-28

    Herein we present a FRET-based theranostic macromolecular prodrug (TMP) composed of (a) dendritic polyglycerol (PG) as polymeric nanocarrier, (b) doxorubicin (Dox) linked via a pH-sensitive hydrazone to (c) a tri-functional linker, and (d) an indodicarbocyanine dye (IDCC) attached in close proximity to Dox. The drug fluorescence is quenched via intramolecular FRET until the pH-sensitive hydrazone bond between the TMP and Dox is cleaved at acidic pH. By measuring its fluorescence, we characterized the TMP cleavage kinetics at different pH values in vitro. The intracellular release of Dox from the carrier was monitored in real time in intact cancer cells, giving more insight into the mode of action of a polymer drug conjugate.

  11. Structure, function and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding.

    NASA Astrophysics Data System (ADS)

    Samiotakis, Antonios; Dhar, Apratim; Ebbinghaus, Simon; Nienhaus, Lea; Homouz, Dirar; Gruebele, Martin; Cheung, Margaret

    2010-10-01

    We combine experiment and computer simulation to show how macromolecular crowding dramatically affects the structure, function and folding landscape of phosphoglycerate kinase (PGK). Fluorescence labeling shows that compact states of yeast PGK are populated as the amount of crowding agents (Ficoll 70) increases. Coarse-grained molecular simulations reveal three compact ensembles: C (crystal structure), CC (collapsed crystal) and Sph (spherical compact). With an adjustment for viscosity, crowded wild type PGK and fluorescent PGK are about 15 times or more active in 200 mg/ml Ficoll than in aqueous solution. Our results suggest a new solution to the classic problem of how the ADP and diphosphoglycerate binding sites of PGK come together to make ATP: rather than undergoing a hinge motion, the ADP and substrate sites are already located in proximity under crowded conditions that mimic the in vivo conditions under which the enzyme actually operates.

  12. The macromolecular crystallography beamline I911-3 at the MAX IV laboratory

    PubMed Central

    Ursby, Thomas; Unge, Johan; Appio, Roberto; Logan, Derek T.; Fredslund, Folmer; Svensson, Christer; Larsson, Krister; Labrador, Ana; Thunnissen, Marjolein M. G. M.

    2013-01-01

    The macromolecular crystallography beamline I911-3, part of the Cassiopeia/I911 suite of beamlines, is based on a superconducting wiggler at the MAX II ring of the MAX IV Laboratory in Lund, Sweden. The beamline is energy-tunable within a range between 6 and 18 keV. I911-3 opened for users in 2005. In 2010–2011 the experimental station was completely rebuilt and refurbished such that it has become a state-of-the-art experimental station with better possibilities for rapid throughput, crystal screening and work with smaller samples. This paper describes the complete I911-3 beamline and how it is embedded in the Cassiopeia suite of beamlines. PMID:23765310

  13. Reliable and efficient solution of genome-scale models of Metabolism and macromolecular Expression

    NASA Astrophysics Data System (ADS)

    Ma, Ding; Yang, Laurence; Fleming, Ronan M. T.; Thiele, Ines; Palsson, Bernhard O.; Saunders, Michael A.

    2017-01-01

    Constraint-Based Reconstruction and Analysis (COBRA) is currently the only methodology that permits integrated modeling of Metabolism and macromolecular Expression (ME) at genome-scale. Linear optimization computes steady-state flux solutions to ME models, but flux values are spread over many orders of magnitude. Data values also have greatly varying magnitudes. Standard double-precision solvers may return inaccurate solutions or report that no solution exists. Exact simplex solvers based on rational arithmetic require a near-optimal warm start to be practical on large problems (current ME models have 70,000 constraints and variables and will grow larger). We have developed a quadruple-precision version of our linear and nonlinear optimizer MINOS, and a solution procedure (DQQ) involving Double and Quad MINOS that achieves reliability and efficiency for ME models and other challenging problems tested here. DQQ will enable extensive use of large linear and nonlinear models in systems biology and other applications involving multiscale data.

  14. Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography.

    PubMed

    Foadi, James; Aller, Pierre; Alguel, Yilmaz; Cameron, Alex; Axford, Danny; Owen, Robin L; Armour, Wes; Waterman, David G; Iwata, So; Evans, Gwyndaf

    2013-08-01

    The availability of intense microbeam macromolecular crystallography beamlines at third-generation synchrotron sources has enabled data collection and structure solution from microcrystals of <10 µm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large numbers of data sets from many crystals of the same protein structure. The associated analysis and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the solution of a novel membrane protein.

  15. Anion Recognition in Water: Recent Advances from a Supramolecular and Macromolecular Perspective

    PubMed Central

    Langton, Matthew J.

    2015-01-01

    Abstract The recognition of anions in water remains a key challenge in modern supramolecular chemistry, and is essential if proposed applications in biological, medical, and environmental arenas that typically require aqueous conditions are to be achieved. However, synthetic anion receptors that operate in water have, in general, been the exception rather than the norm to date. Nevertheless, a significant step change towards routinely conducting anion recognition in water has been achieved in the past few years, and this Review highlights these approaches, with particular focus on controlling and using the hydrophobic effect, as well as more exotic interactions such as C−H hydrogen bonding and halogen bonding. We also look beyond the field of small‐molecule recognition into the macromolecular domain, covering recent advances in anion recognition based on biomolecules, polymers, and nanoparticles. PMID:26612067

  16. CheckMyMetal: a macromolecular metal-binding validation tool.

    PubMed

    Zheng, Heping; Cooper, David R; Porebski, Przemyslaw J; Shabalin, Ivan G; Handing, Katarzyna B; Minor, Wladek

    2017-03-01

    Metals are essential in many biological processes, and metal ions are modeled in roughly 40% of the macromolecular structures in the Protein Data Bank (PDB). However, a significant fraction of these structures contain poorly modeled metal-binding sites. CheckMyMetal (CMM) is an easy-to-use metal-binding site validation server for macromolecules that is freely available at http://csgid.org/csgid/metal_sites. The CMM server can detect incorrect metal assignments as well as geometrical and other irregularities in the metal-binding sites. Guidelines for metal-site modeling and validation in macromolecules are illustrated by several practical examples grouped by the type of metal. These examples show CMM users (and crystallographers in general) problems they may encounter during the modeling of a specific metal ion.

  17. Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions.

    PubMed

    Brautigam, Chad A; Zhao, Huaying; Vargas, Carolyn; Keller, Sandro; Schuck, Peter

    2016-05-01

    Isothermal titration calorimetry (ITC) is a powerful and widely used method to measure the energetics of macromolecular interactions by recording a thermogram of differential heating power during a titration. However, traditional ITC analysis is limited by stochastic thermogram noise and by the limited information content of a single titration experiment. Here we present a protocol for bias-free thermogram integration based on automated shape analysis of the injection peaks, followed by combination of isotherms from different calorimetric titration experiments into a global analysis, statistical analysis of binding parameters and graphical presentation of the results. This is performed using the integrated public-domain software packages NITPIC, SEDPHAT and GUSSI. The recently developed low-noise thermogram integration approach and global analysis allow for more precise parameter estimates and more reliable quantification of multisite and multicomponent cooperative and competitive interactions. Titration experiments typically take 1-2.5 h each, and global analysis usually takes 10-20 min.

  18. A Test of Macromolecular Crystallization in Microgravity: Large, Well-Ordered Insulin Crystals

    NASA Technical Reports Server (NTRS)

    Borgstahl, Gloria E. O.; Vahedi-Faridi, Ardeschir; Lovelace, Jeff; Bellamy, Henry D.; Snell, Edward H.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    Crystals of insulin grown in microgravity on space shuttle mission STS-95 were extremely well-ordered and unusually large (many > 2 mm). The physical characteristics of six microgravity and six earth-grown crystals were examined by X-ray analysis employing superfine f slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of reflections to be precisely examined for each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had 7 times lower mosaicity, had 54 times higher reflection peak heights and diffracted to significantly higher resolution than their earth grown counterparts. A single mosaic domain model could account for reflections in microgravity crystals whereas reflections from earth crystals required a model with multiple mosaic domains. This statistically significant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

  19. A Nonlinear Elasticity Model of Macromolecular Conformational Change Induced by Electrostatic Forces

    PubMed Central

    Zhou, Y. C.; Holst, Michael; McCammon, J. Andrew

    2008-01-01

    In this paper we propose a nonlinear elasticity model of macromolecular conformational change (deformation) induced by electrostatic forces generated by an implicit solvation model. The Poisson-Boltzmann equation for the electrostatic potential is analyzed in a domain varying with the elastic deformation of molecules, and a new continuous model of the electrostatic forces is developed to ensure solvability of the nonlinear elasticity equations. We derive the estimates of electrostatic forces corresponding to four types of perturbations to an electrostatic potential field, and establish the existance of an equilibrium configuration using a fixed-point argument, under the assumption that the change in the ionic strength and charges due to the additional molecules causing the deformation are sufficiently small. The results are valid for elastic models with arbitrarily complex dielectric interfaces and cavities, and can be generalized to large elastic deformation caused by high ionic strength, large charges, and strong external fields by using continuation methods. PMID:19461946

  20. A Nonlinear Elasticity Model of Macromolecular Conformational Change Induced by Electrostatic Forces.

    PubMed

    Zhou, Y C; Holst, Michael; McCammon, J Andrew

    2008-04-01

    In this paper we propose a nonlinear elasticity model of macromolecular conformational change (deformation) induced by electrostatic forces generated by an implicit solvation model. The Poisson-Boltzmann equation for the electrostatic potential is analyzed in a domain varying with the elastic deformation of molecules, and a new continuous model of the electrostatic forces is developed to ensure solvability of the nonlinear elasticity equations. We derive the estimates of electrostatic forces corresponding to four types of perturbations to an electrostatic potential field, and establish the existance of an equilibrium configuration using a fixed-point argument, under the assumption that the change in the ionic strength and charges due to the additional molecules causing the deformation are sufficiently small. The results are valid for elastic models with arbitrarily complex dielectric interfaces and cavities, and can be generalized to large elastic deformation caused by high ionic strength, large charges, and strong external fields by using continuation methods.

  1. Recent Major Improvements to the ALS Sector 5 MacromolecularCrystallography Beamlines

    SciTech Connect

    Morton, Simon A.; Glossinger, James; Smith-Baumann, Alexis; McKean, John P.; Trame, Christine; Dickert, Jeff; Rozales, Anthony; Dauz,Azer; Taylor, John; Zwart, Petrus; Duarte, Robert; Padmore, Howard; McDermott, Gerry; Adams, Paul

    2007-07-01

    Although the Advanced Light Source (ALS) was initially conceived primarily as a low energy (1.9GeV) 3rd generation source of VUV and soft x-ray radiation it was realized very early in the development of the facility that a multipole wiggler source coupled with high quality, (brightness preserving), optics would result in a beamline whose performance across the optimal energy range (5-15keV) for macromolecular crystallography (MX) would be comparable to, or even exceed, that of many existing crystallography beamlines at higher energy facilities. Hence, starting in 1996, a suite of three beamlines, branching off a single wiggler source, was constructed, which together formed the ALS Macromolecular Crystallography Facility. From the outset this facility was designed to cater equally to the needs of both academic and industrial users with a heavy emphasis placed on the development and introduction of high throughput crystallographic tools, techniques, and facilities--such as large area CCD detectors, robotic sample handling and automounting facilities, a service crystallography program, and a tightly integrated, centralized, and highly automated beamline control environment for users. This facility was immediately successful, with the primary Multiwavelength Anomalous Diffraction beamline (5.0.2) in particular rapidly becoming one of the foremost crystallographic facilities in the US--responsible for structures such as the 70S ribosome. This success in-turn triggered enormous growth of the ALS macromolecular crystallography community and spurred the development of five additional ALS MX beamlines all utilizing the newly developed superconducting bending magnets ('superbends') as sources. However in the years since the original Sector 5.0 beamlines were built the performance demands of macromolecular crystallography users have become ever more exacting; with growing emphasis placed on studying larger complexes, more difficult structures, weakly diffracting or smaller

  2. Macromolecular semi-rigid nanocavities for cooperative recognition of specific large molecular shapes.

    PubMed

    Imaoka, Takane; Kawana, Yuki; Kurokawa, Takuto; Yamamoto, Kimihisa

    2013-01-01

    Molecular shape recognition for larger guest molecules (typically over 1 nm) is a difficult task because it requires cooperativity within a wide three-dimensional nanospace coincidentally probing every molecular aspect (size, outline shape, flexibility and specific groups). Although the intelligent functions of proteins have fascinated many researchers, the reproduction by artificial molecules remains a significant challenge. Here we report the construction of large, well-defined cavities in macromolecular hosts. Through the use of semi-rigid dendritic phenylazomethine backbones, even subtle differences in the shapes of large guest molecules (up to ~2 nm) may be discriminated by the cooperative mechanism. A conformationally fixed complex with the best-fitting guest is supported by a three-dimensional model based on a molecular simulation. Interestingly, the simulated cavity structure also predicts catalytic selectivity by a ruthenium porphyrin centre, demonstrating the high shape persistence and wide applicability of the cavity.

  3. Site-selective electroless nickel plating on patterned thin films of macromolecular metal complexes.

    PubMed

    Kimura, Mutsumi; Yamagiwa, Hiroki; Asakawa, Daisuke; Noguchi, Makoto; Kurashina, Tadashi; Fukawa, Tadashi; Shirai, Hirofusa

    2010-12-01

    We demonstrate a simple route to depositing nickel layer patterns using photocross-linked polymer thin films containing palladium catalysts, which can be used as adhesive interlayers for fabrication of nickel patterns on glass and plastic substrates. Electroless nickel patterns can be obtained in three steps: (i) the pattern formation of partially quaterized poly(vinyl pyridine) by UV irradiation, (ii) the formation of macromolecular metal complex with palladium, and (iii) the nickel metallization using electroless plating bath. Metallization is site-selective and allows for a high resolution. And the resulting nickel layered structure shows good adhesion with glass and plastic substrates. The direct patterning of metallic layers onto insulating substrates indicates a great potential for fabricating micro/nano devices.

  4. New computational tools for H/D determination in macromolecular structures from neutron data.

    PubMed

    Siliqi, Dritan; Caliandro, Rocco; Carrozzini, Benedetta; Cascarano, Giovanni Luca; Mazzone, Annamaria

    2010-11-01

    Two new computational methods dedicated to neutron crystallography, called n-FreeLunch and DNDM-NDM, have been developed and successfully tested. The aim in developing these methods is to determine hydrogen and deuterium positions in macromolecular structures by using information from neutron density maps. Of particular interest is resolving cases in which the geometrically predicted hydrogen or deuterium positions are ambiguous. The methods are an evolution of approaches that are already applied in X-ray crystallography: extrapolation beyond the observed resolution (known as the FreeLunch procedure) and a difference electron-density modification (DEDM) technique combined with the electron-density modification (EDM) tool (known as DEDM-EDM). It is shown that the two methods are complementary to each other and are effective in finding the positions of H and D atoms in neutron density maps.

  5. Destruction of Tissue, Cells and Organelles in Type 1 Diabetic Rats Presented at Macromolecular Resolution

    PubMed Central

    Ravelli, Raimond B. G.; Kalicharan, Ruby D.; Avramut, M. Cristina; Sjollema, Klaas A.; Pronk, Joachim W.; Dijk, Freark; Koster, Abraham J.; Visser, Jeroen T. J.; Faas, Frank G. A.; Giepmans, Ben N. G.

    2013-01-01

    Finding alternatives for insulin therapy and making advances in etiology of type 1 diabetes benefits from a full structural and functional insight into Islets of Langerhans. Electron microscopy (EM) can visualize Islet morphology at the highest possible resolution, however, conventional EM only provides biased snapshots and lacks context. We developed and employed large scale EM and compiled a resource of complete cross sections of rat Islets during immuno-destruction to provide unbiased structural insight of thousands of cells at macromolecular resolution. The resource includes six datasets, totalling 25.000 micrographs, annotated for cellular and ultrastructural changes during autoimmune diabetes. Granulocytes are attracted to the endocrine tissue, followed by extravasation of a pleiotrophy of leukocytes. Subcellullar changes in beta cells include endoplasmic reticulum stress, insulin degranulation and glycogen accumulation. Rare findings include erythrocyte extravasation and nuclear actin-like fibers. While we focus on a rat model of autoimmune diabetes, our approach is general applicable. PMID:23652855

  6. Harvesting and cryo-cooling crystals of membrane proteins grown in lipidic mesophases for structure determination by macromolecular crystallography.

    PubMed

    Li, Dianfan; Boland, Coilín; Aragao, David; Walsh, Kilian; Caffrey, Martin

    2012-09-02

    An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases(1-5), has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field(6-21) (www.mpdb.tcd.ie). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting(22,23). Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)(24,25) are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection. The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies(4,26). The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been

  7. Experimental determination of optimal root-mean-square deviations of macromolecular bond lengths and angles from their restrained ideal values.

    PubMed

    Tickle, Ian J

    2007-12-01

    A number of inconsistencies are apparent in the recent research paper by Jaskolski et al. [(2007), Acta Cryst. D63, 611-620] concerning their recommendations for the values of the magnitude and resolution-dependence of the root-mean-square deviations (RMSDs) of bond lengths and angles from their restrained ideal values in macromolecular refinement, as well as their suggestions for the use of variable standard uncertainties dependent on atomic displacement parameters (ADPs) and occupancies. Whilst many of the comments and suggestions in the paper regarding updates for the ideal geometry values proposed by Engh and Huber are entirely reasonable and supported by the experimental evidence, the recommendations concerning the optimal values of RMSDs appear to be in conflict with previous experimental and theoretical work in this area [Tickle et al. (1998), Acta Cryst. D54, 243-252] and indeed appear to be based on a misunderstanding of the distinction between RMSD and standard uncertainty (SU). In contrast, it is proposed here that the optimal values of all desired weighting parameters, in particular the weighting parameters for the ADP differences and for the diffraction terms, be estimated by the purely objective procedure of maximizing the experiment-based log(free likelihood). In principle, this allows all weighting parameters that are not known accurately a priori to be scaled globally, relative to those that are known accurately, for an optimal refinement. The RMS Z score (RMSZ) is recommended as a more satisfactory statistic than the RMSD to assess the extent to which the geometry deviates from the ideal values and a theoretical rationale for the results obtained is presented in which the optimal RMSZ is identified as the calculated versus true Z-score correlation coefficient, the latter being a monotonic function of the resolution cutoff of the data. Regarding the proposal to use variable standard uncertainties, it is suggested that any departure from the current

  8. D3, the new diffractometer for the macromolecular crystallography beamlines of the Swiss Light Source

    PubMed Central

    Fuchs, Martin R.; Pradervand, Claude; Thominet, Vincent; Schneider, Roman; Panepucci, Ezequiel; Grunder, Marcel; Gabadinho, Jose; Dworkowski, Florian S. N.; Tomizaki, Takashi; Schneider, Jörg; Mayer, Aline; Curtin, Adrian; Olieric, Vincent; Frommherz, Uli; Kotrle, Goran; Welte, Jörg; Wang, Xinyu; Maag, Stephan; Schulze-Briese, Clemens; Wang, Meitian

    2014-01-01

    A new diffractometer for microcrystallography has been developed for the three macromolecular crystallography beamlines of the Swiss Light Source. Building upon and critically extending previous developments realised for the high-resolution endstations of the two undulator beamlines X06SA and X10SA, as well as the super-bend dipole beamline X06DA, the new diffractometer was designed to the following core design goals. (i) Redesign of the goniometer to a sub-micrometer peak-to-peak cylinder of confusion for the horizontal single axis. Crystal sizes down to at least 5 µm and advanced sample-rastering and scanning modes are supported. In addition, it can accommodate the new multi-axis goniometer PRIGo (Parallel Robotics Inspired Goniometer). (ii) A rapid-change beam-shaping element system with aperture sizes down to a minimum of 10 µm for microcrystallography measurements. (iii) Integration of the on-axis microspectrophotometer MS3 for microscopic sample imaging with 1 µm image resolution. Its multi-mode optical spectroscopy module is always online and supports in situ UV/Vis absorption, fluorescence and Raman spectroscopy. (iv) High stability of the sample environment by a mineral cast support construction and by close containment of the cryo-stream. Further features are the support for in situ crystallization plate screening and a minimal achievable detector distance of 120 mm for the Pilatus 6M, 2M and the macromolecular crystallography group’s planned future area detector Eiger 16M. PMID:24562555

  9. Do Macromolecular Crowding Agents Exert Only an Excluded Volume Effect? A Protein Solvation Study.

    PubMed

    Mukherjee, Sanjib K; Gautam, Saurabh; Biswas, Saikat; Kundu, Jayanta; Chowdhury, Pramit K

    2015-11-05

    The effect of macromolecular crowding on protein structure and dynamics has mostly been explained on the basis of the excluded volume effect, its origin being entropic. In recent times a progressive shift in this view has been taking place with increasing emphasis on soft interactions that are enthalpic by nature. Using very low concentrations (1-10 g/L) of both synthetic (dextran- and poly(ethylene glycol) (PEG)-based) and protein (α-synuclein and myoglobin)-based crowders, we have shown that the solvation of probe molecule ANS (1-anilinonapthalene-8-sulfonate) bound to serum proteins bovine serum albumin (BSA) and human serum albumin (HSA) is significantly modulated in both a protein- and crowder-dependent fashion. Since under such conditions the effect of excluded volume is appreciably low, we propose that our observations are direct evidence of soft interactions between the macromolecular crowding agents used and the serum proteins. Moreover, our data reveal, that since at these low crowder concentrations major perturbations to the protein structure are unlikely to take place while minor perturbations might not be readily visible, protein solvation provides a unique spectral signature for capturing such local dynamics, thereby allowing one to decouple hard-sphere interactions from soft sphere ones. Furthermore, since fast fluctuations are known to play a major role in determining the functional characteristics of proteins and enzymes, our results suggest that such motions are prone to be modulated even when the cellular crowding conditions are quite relaxed. In other words, by the time the excluded volume effects come into the picture in the physiological milieu, modulations of functionally important protein motions that need a relatively lower activation energy have already taken place as a result of the aforementioned enthalpic (soft) interactions.

  10. Effects of Hydrophobic Macromolecular Crowders on Amyloid β (16–22) Aggregation

    PubMed Central

    Latshaw, David C.; Hall, Carol K.

    2015-01-01

    In Alzheimer’s disease (AD), the amyloid β (Aβ) peptide aggregates in the brain to form progressively larger oligomers, fibrils, and plaques. The aggregation process is strongly influenced by the presence of other macromolecular species, called crowders, that can exert forces on the proteins. One very common attribute of macromolecular crowders is their hydrophobicity. We examined the effect of hydrophobic crowders on protein aggregation by using discontinuous molecular dynamics (DMD) simulations in combination with an intermediate resolution protein model, PRIME20. The systems considered contained 48 Aβ (16–22) peptides and crowders with diameters of 5 Å, 20 Å, and 40 Å, represented by hard spheres or spheres with square-well/square-shoulder interactions, at a crowder volume fraction of ϕ = 0.10. Results show that low levels of crowder hydrophobicity are capable of increasing the fibrillation lag time and high levels of crowder hydrophobicity can fully prevent the formation of fibrils. The types of structures that remain during the final stages of the simulations are summarized in a global phase diagram that shows fibril, disordered oligomer, or β-sheet phases in the space spanned by crowder size and crowder hydrophobicity. In particular, at high levels of hydrophobicity, simulations with 5 Å crowders result in only disordered oligomers and simulations with 40 Å crowders result in only β-sheets. The presence of hydrophobic crowders reduces the antiparallel β-sheet content of fibrils, whereas hard sphere crowders increase it. Finally, strong hydrophobic crowders alter the secondary structure of the Aβ (16–22) monomers, bending them into a shape that is incapable of forming ordered β-sheets or fibrils. These results qualitatively agree with previous theoretical and experimental work. PMID:26153709

  11. Transformations of the macromolecular landscape at mitochondria during DNA-damage-induced apoptotic cell death.

    PubMed

    Yadav, N; Pliss, A; Kuzmin, A; Rapali, P; Sun, L; Prasad, P; Chandra, D

    2014-10-09

    Apoptosis is a dynamic process regulated by mitochondrion critical for cellular respiration and survival. Execution of apoptosis is mediated by multiple protein signaling events at mitochondria. Initiation and progression of apoptosis require numerous apoptogenic factors that are either released from or sequestered in mitochondria, which may transform the biomolecular makeup of the organelle. In this communication, using Raman microspectroscopy, we demonstrate that transformation in biomolecular composition of mitochondrion may be used as apoptosis marker in an individual cell. For the first time, we show that significant changes occur in the concentrations of RNA, DNA, protein, and lipid constituents of mitochondria during apoptosis. The structural analysis of proteins on mitochondria demonstrated a decrease in α-helix secondary structure content, and an increase in the levels of random coils and β-sheets on mitochondria. This may represent an additional hallmark of apoptosis. Strikingly, we observed nearly identical changes in macromolecular content of mitochondria both in the presence and absence of a key proapoptotic protein, Bax (Bcl-2-associated X protein). Increased DNA level in mitochondria corresponded with higher mitochondrial DNA (mtDNA), cellular reactive oxygen species (ROS), and mitochondrial ROS production. Upregulation of polymerase-γ (POLG), mitochondrial helicase Twinkle, and mitochondrial transcription factor A (Tfam) in response to DNA damage correlated with increased mtDNA and RNA synthesis. Elevated activity of oxidative phosphorylation complexes supports functional mitochondrial respiration during apoptosis. Thus, we define previously unknown dynamic correlation of macromolecular structure of mitochondria and apoptosis progression in the presence and absence of Bax protein. These findings open up a new approach for monitoring physiological status of cells by non invasive single-cell method.

  12. Building macromolecular assemblies by information-driven docking: introducing the HADDOCK multibody docking server.

    PubMed

    Karaca, Ezgi; Melquiond, Adrien S J; de Vries, Sjoerd J; Kastritis, Panagiotis L; Bonvin, Alexandre M J J

    2010-08-01

    Over the last years, large scale proteomics studies have generated a wealth of information of biomolecular complexes. Adding the structural dimension to the resulting interactomes represents a major challenge that classical structural experimental methods alone will have difficulties to confront. To meet this challenge, complementary modeling techniques such as docking are thus needed. Among the current docking methods, HADDOCK (High Ambiguity-Driven DOCKing) distinguishes itself from others by the use of experimental and/or bioinformatics data to drive the modeling process and has shown a strong performance in the critical assessment of prediction of interactions (CAPRI), a blind experiment for the prediction of interactions. Although most docking programs are limited to binary complexes, HADDOCK can deal with multiple molecules (up to six), a capability that will be required to build large macromolecular assemblies. We present here a novel web interface of HADDOCK that allows the user to dock up to six biomolecules simultaneously. This interface allows the inclusion of a large variety of both experimental and/or bioinformatics data and supports several types of cyclic and dihedral symmetries in the docking of multibody assemblies. The server was tested on a benchmark of six cases, containing five symmetric homo-oligomeric protein complexes and one symmetric protein-DNA complex. Our results reveal that, in the presence of either bioinformatics and/or experimental data, HADDOCK shows an excellent performance: in all cases, HADDOCK was able to generate good to high quality solutions and ranked them at the top, demonstrating its ability to model symmetric multicomponent assemblies. Docking methods can thus play an important role in adding the structural dimension to interactomes. However, although the current docking methodologies were successful for a vast range of cases, considering the variety and complexity of macromolecular assemblies, inclusion of some kind of

  13. Transition modes in Ising networks: an approximate theory for macromolecular recognition.

    PubMed Central

    Keating, S; Di Cera, E

    1993-01-01

    For a statistical lattice, or Ising network, composed of N identical units existing in two possible states, 0 and 1, and interacting according to a given geometry, a set of values can be found for the mean free energy of the 0-->1 transition of a single unit. Each value defines a transition mode in an ensemble of nu N = 3N - 2N possible values and reflects the role played by intermediate states in shaping the energetics of the system as a whole. The distribution of transition modes has a number of intriguing properties. Some of them apply quite generally to any Ising network, regardless of its dimension, while others are specific for each interaction geometry and dimensional embedding and bear on fundamental aspects of analytical number theory. The landscape of transition modes encapsulates all of the important thermodynamic properties of the network. The free energy terms defining the partition function of the system can be derived from the modes by simple transformations. Classical mean-field expressions can be obtained from consideration of the properties of transition modes in a rather straightforward way. The results obtained in the analysis of the transition mode distributions have been used to develop an approximate treatment of the problem of macromolecular recognition. This phenomenon is modeled as a cooperative process that involves a number of recognition subsites across an interface generated by the binding of two macromolecular components. The distribution of allowed binding free energies for the system is shown to be a superposition of Gaussian terms with mean and variance determined a priori by the theory. Application to the analysis of the biologically interaction of thrombin with hirudin has provided some useful information on basic aspects of the interaction, such as the number of recognition subsites involved and the energy balance for binding and cooperative coupling among them. Our results agree quite well with information derived independently

  14. Dynamic simulation of concentrated macromolecular solutions with screened long-range hydrodynamic interactions: Algorithm and limitations

    PubMed Central

    Ando, Tadashi; Chow, Edmond; Skolnick, Jeffrey

    2013-01-01

    Hydrodynamic interactions exert a critical effect on the dynamics of macromolecules. As the concentration of macromolecules increases, by analogy to the behavior of semidilute polymer solutions or the flow in porous media, one might expect hydrodynamic screening to occur. Hydrodynamic screening would have implications both for the understanding of macromolecular dynamics as well as practical implications for the simulation of concentrated macromolecular solutions, e.g., in cells. Stokesian dynamics (SD) is one of the most accurate methods for simulating the motions of N particles suspended in a viscous fluid at low Reynolds number, in that it considers both far-field and near-field hydrodynamic interactions. This algorithm traditionally involves an O(N3) operation to compute Brownian forces at each time step, although asymptotically faster but more complex SD methods are now available. Motivated by the idea of hydrodynamic screening, the far-field part of the hydrodynamic matrix in SD may be approximated by a diagonal matrix, which is equivalent to assuming that long range hydrodynamic interactions are completely screened. This approximation allows sparse matrix methods to be used, which can reduce the apparent computational scaling to O(N). Previously there were several simulation studies using this approximation for monodisperse suspensions. Here, we employ newly designed preconditioned iterative methods for both the computation of Brownian forces and the solution of linear systems, and consider the validity of this approximation in polydisperse suspensions. We evaluate the accuracy of the diagonal approximation method using an intracellular-like suspension. The diffusivities of particles obtained with this approximation are close to those with the original method. However, this approximation underestimates intermolecular correlated motions, which is a trade-off between accuracy and computing efficiency. The new method makes it possible to perform large-scale and

  15. Liquid chromatographic extraction medium

    DOEpatents

    Horwitz, E.P.; Dietz, M.L.

    1994-09-13

    A method and apparatus are disclosed for extracting strontium and technetium values from biological, industrial and environmental sample solutions using a chromatographic column. An extractant medium for the column is prepared by generating a solution of a diluent containing a Crown ether and dispersing the solution on a resin substrate material. The sample solution is highly acidic and is introduced directed to the chromatographic column and strontium or technetium is eluted using deionized water. 1 fig.

  16. Liquid chromatographic extraction medium

    DOEpatents

    Horwitz, E. Philip; Dietz, Mark L.

    1994-01-01

    A method and apparatus for extracting strontium and technetium values from biological, industrial and environmental sample solutions using a chromatographic column is described. An extractant medium for the column is prepared by generating a solution of a diluent containing a Crown ether and dispersing the solution on a resin substrate material. The sample solution is highly acidic and is introduced directed to the chromatographic column and strontium or technetium is eluted using deionized water.

  17. Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast

    PubMed Central

    Dong, Biqin; Almassalha, Luay M.; Stypula-Cyrus, Yolanda; Urban, Ben E.; Chandler, John E.; Nguyen, The-Quyen; Sun, Cheng; Zhang, Hao F.; Backman, Vadim

    2016-01-01

    Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging. PMID:27535934

  18. Determination of contrast media administration to achieve a targeted contrast enhancement in CT

    NASA Astrophysics Data System (ADS)

    Sahbaee, Pooyan; Li, Yuan; Segars, Paul; Marin, Daniele; Nelson, Rendon; Samei, Ehsan

    2015-03-01

    Contrast enhancement is a key component of CT imaging and offer opportunities for optimization. The design and optimization of new techniques however requires orchestration with the scan parameters and further a methodology to relate contrast enhancement and injection function. In this study, we used such a methodology to develop a method, analytical inverse method, to predict the required injection function to achieve a desired contrast enhancement in a given organ by incorporation of a physiologically based compartmental model. The method was evaluated across 32 different target contrast enhancement functions for aorta, kidney, stomach, small intestine, and liver. The results exhibited that the analytical inverse method offers accurate performance with error in the range of 10% deviation between the predicted and desired organ enhancement curves. However, this method is incapable of predicting the injection function based on the liver enhancement. The findings of this study can be useful in optimizing contrast medium injection function as well as the scan timing to provide more consistency in the way that the contrast enhanced CT examinations are performed. To our knowledge, this work is one of the first attempts to predict the contrast material injection function for a desired organ enhancement curve.

  19. High Contrast CRT.

    DTIC Science & Technology

    1980-02-01

    form brown sulfides or sulfates. By con- trast, No. 1720 glass does not acquire a brown coloration . How- ever, preliminary tests with 1723 glass show...TR-77-2639-F NL -mmo mhhmhul IIII,. BwII ---- i 11111--- IIIIIN III i 8’ II.I25 .11111 I .6 MCROCOPY RESOLUTION TEST CHART NATIONAL BUREAU Of...High Contrast Displays Two- Color CRT Laminar Flow Electron Gun Thin Film Phosphor Color Penetration Tube 2% AS~iTACT (ConIlm. a" Pove.. 0fdo if

  20. Contrast and depth perception: effects of texture contrast and area contrast.

    PubMed

    Ichihara, Shigeru; Kitagawa, Norimichi; Akutsu, Hiromi

    2007-01-01

    Many objects in natural scenes have textures on their surfaces. Contrast of the texture surfaces (the texture contrast) reduces when the viewing distance increases. Similarly, contrast between the surfaces of the objects and the background (the area contrast) reduces when the viewing distance increases. The texture contrast and the area contrast were defined by the contrast between random dots, and by the contrast between the average luminance of the dot pattern and the luminance of the background, respectively. To examine how these two types of contrast influence depth perception, we ran two experiments. In both experiments two areas of random-dot patterns were presented against a uniform background, and participants rated relative depth between the two areas. We found that the rated depth of the patterned areas increased with increases in texture contrast. Furthermore, the effect of the texture contrast on depth judgment increased when the area contrast became low.

  1. Attenuation contrast between biomolecular and inorganic materials at terahertz frequencies

    NASA Astrophysics Data System (ADS)

    Chan, T. L. J.; Bjarnason, J. E.; Lee, A. W. M.; Celis, M. A.; Brown, E. R.

    2004-09-01

    Wideband photomixing spectroscopy is used in the present work to contrast the transmission spectra of macromolecules commonly found in biomaterials such as potato starch, wheat flour and cornstarch, and proteins (Cytoplex™), and micromolecules such as sucrose, and inorganic materials such as sodium bicarbonate, and calcium sulfate. Powdered samples were measured at 0.1-0.5THz frequencies. A significant difference in attenuation is found between these samples. At 300GHz starch shows an absorption coefficient of ˜6cm-1 whereas Cytoplex shows 1-3cm-1, while inorganic micromolecules have ˜1cm-1. The absorption in starch increases rapidly with frequency tending to follow a power law α =fn with n typically between 1.5 and 2.0. In contrast, protein materials display a slower dependence on frequency with n between 1.0 and 1.5, and simple molecules show the least n among all three categories. The difference between these ubiquitous macromolecular and micromolecular materials is explained in terms of water content and molecular structure.

  2. EMatch: an efficient method for aligning atomic resolution subunits into intermediate-resolution cryo-EM maps of large macromolecular assemblies

    SciTech Connect

    Dror, Oranit Lasker, Keren; Nussinov, Ruth; Wolfson, Haim

    2007-01-01

    A method for detecting structural homologs of components in an intermediate resolution cryo-EM map and their spatial configuration is presented. Structural analysis of biological machines is essential for inferring their function and mechanism. Nevertheless, owing to their large size and instability, deciphering the atomic structure of macromolecular assemblies is still considered as a challenging task that cannot keep up with the rapid advances in the protein-identification process. In contrast, structural data at lower resolution is becoming more and more available owing to recent advances in cryo-electron microscopy (cryo-EM) techniques. Once a cryo-EM map is acquired, one of the basic questions asked is what are the folds of the components in the assembly and what is their configuration. Here, a novel knowledge-based computational method, named EMatch, towards tackling this task for cryo-EM maps at 6–10 Å resolution is presented. The method recognizes and locates possible atomic resolution structural homologues of protein domains in the assembly. The strengths of EMatch are demonstrated on a cryo-EM map of native GroEL at 6 Å resolution.

  3. Facile Fabrication of Water Dispersible Latex Particles with Homogeneous or Chain-Segregated Surface from RAFT Polymerization Using a Mixture of Two Macromolecular Chain Transfer Agents.

    PubMed

    Sun, Li; Hong, Liangzhi; Wang, Chaoyang

    2016-04-01

    Water dispersible latex particles with randomly mixed shells or chain segregated surface are synthesized from one-pot reversible addition-fragmentation chain transfer heterogeneous polymerization of benzyl methacrylate (BzMA) using a mixture of poly(glycerol monomethacrylate) (PGMA) and poly(2,3-bis(succinyloxy)propyl methacrylate) (PBSPMA) macromolecular chain transfer agents. In methanol, the two in situ synthesized PGMA-b-PBzMA and PBSPMA-b-PBzMA diblock copolymers coaggregate into spherical micelles, which contain PBzMA core and discrete PGMA and PBSPMA nanodomains on the shell. In contrast, in water-methanol mixture (V/V = 9/1), latex particles with homogeneous distribution of PGMA and PBSPMA polymer chains on the shell are obtained. The reasons leading to formation of latex particles with homogenous or chain-segregated surface are discussed, and polymerization kinetics and physical state of PBSPMA in methanol and water-methanol mixtures are ascribed. These polymeric micelles with patterned functional group on the surface are potentially important for application in supracolloidal hierarchical assemblies and catalysis.

  4. Saphenous Venous Ablation with Hot Contrast in a Canine Model

    SciTech Connect

    Prasad, Amit Qian Zhong; Kirsch, David; Eissa, Marna; Narra, Pavan; Lopera, Jorge; Espinoza, Carmen G.; Castaneda, Wifrido

    2008-01-15

    Purpose. To determine the feasibility, efficacy, and safety of thermal ablation of the saphenous vein with hot contrast medium. Methods. Twelve saphenous veins of 6 dogs were percutaneously ablated with hot contrast medium. In all animals, ablation was performed in the vein of one leg, followed by ablation in the contralateral side 1 month later. An occlusion balloon catheter was placed in the infragenicular segment of the saphenous vein via a jugular access to prevent unwanted thermal effects on the non-target segment of the saphenous vein. After inflation of the balloon, 10 ml of hot contrast medium was injected under fluoroscopic control through a sheath placed in the saphenous vein above the ankle. A second 10 ml injection of hot contrast medium was made after 5 min in each vessel. Venographic follow-up of the ablated veins was performed at 1 month (n = 12) and 2 months (n = 6). Results. Follow-up venograms showed that all ablated venous segments were occluded at 1 month. In 6 veins which were followed up to 2 months, 4 (66%) remained occluded, 1 (16%) was partially patent, and the remaining vein (16%) was completely patent. In these latter 2 cases, an inadequate amount of hot contrast was delivered to the lumen due to a closed balloon catheter downstream which did not allow contrast to displace blood within the vessel. Discussion. Hot contrast medium thermal ablation of the saphenous vein appears feasible, safe, and effective in the canine model, provided an adequate amount of embolization agent is used.

  5. On Establishing Underlying Tonal Contrast

    ERIC Educational Resources Information Center

    Snider, Keith

    2014-01-01

    Phonological field work is largely about establishing contrast in comparable environments. The notion of phonological contrast, however, can be confusing, particularly in its application to tone analysis. Does it mean phonemic contrast in the structuralist sense, or does it mean underlying contrast in the generative sense? Many linguists, in…

  6. Contrasting Martian Terrains

    NASA Technical Reports Server (NTRS)

    2004-01-01

    NASA's Mars Exploration Rover Spirit captured this interesting view of martian topography just below the 'West Spur' portion of the 'Columbia Hills' on sol 208 (Aug. 2, 2004). The view is looking southwest. The rover's wheel tracks show the contrast between soft martian soil and the harder 'Clovis' rock outcrop, which scientists are now studying.

    The angle of the horizon indicates the tilt of the rover to be about 20 degrees. On the horizon is a small peak informally named 'Grissom Hill,' about 15 kilometers (9.3 miles) away. To the right of the peak is the edge of a 2-kilometer-wide (1.2-mile-wide) crater. A few weeks ago, Spirit stopped to conduct scientific studies of rocks in 'Hank's Hollow,' located on the right side of the image approximately one-third of the way down from the top. This photo was taken with Spirit's right rear hazard-avoidance camera.

  7. On motion in a resisting medium: A historical perspective

    NASA Astrophysics Data System (ADS)

    Hackborn, William W.

    2016-02-01

    This paper examines, compares, and contrasts ideas about motion, especially the motion of a body in a resisting medium, proposed by Galileo, Newton, and Tartaglia, the author of the first text on exterior ballistics, within the context of the Aristotelian philosophy prevalent when these scholars developed their ideas. This historical perspective offers insights on the emergence of a scientific paradigm for motion, particularly with respect to the challenge of incorporating into this paradigm the role played by the medium.

  8. [An improved differential medium, CA medium, for differentiating Shigella].

    PubMed

    Tokoro, M; Nagano, I; Goto, K; Nakamura, A

    1990-07-01

    We devised a Citrate-Acetate (CA) medium for rapidly differentiating Shigella. The medium consisted of 3.0 g of sodium citrate, 2.0 g of sodium acetate, 0.2 g of glucose, 1.0 g of dipotassium phosphate, 1.0 g of mono ammonium phosphate, 0.2 g of magnesium sulfate, 5.0 g of sodium chloride, 0.08 g of brom thymol blue, 15.0 g of agar, and 1000 ml of distilled water. An evaluation was made of the CA medium, for the rapid differentiation of 23 Shigella strains, 129 Escherichia coli strains and 130 isolates, that formed colourless colonies suspected to be Shigella on SS agar plate, from feces of healthy people. The results obtained were as follows 1) On the CA medium, all Shigella strains did not grow and there was no change in colour. 2) Positive growth rates of E. coli strains after incubation for 24 hr at 37 degrees C on CA medium, sodium acetate medium (Acet) and Christensen citrate medium (C-Cit) were 96.0%, 95.2% and 28.0%, respectively. Therefore, the positive growth rate of E. coli strains after incubation for 24 hr on CA medium was significantly higher (p less than 0.01) than that on C-Cit medium. 3) Positive growth rates of isolates after incubation for 24 hr at 37 degrees C on CA medium, Acet medium and C-Cit medium were 95.4%, 83.1% and 71.5%, respectively. Therefore, the positive growth rates of isolates after incubation for 24 hr on CA medium was significantly higher (p less than 0.01) than that on Acet medium and C-Cit medium.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. DENSE MEDIUM CYCLONE OPTIMIZATON

    SciTech Connect

    Gerald H. Luttrell; Chris J. Barbee; Peter J. Bethell; Chris J. Wood

    2005-06-30

    Dense medium cyclones (DMCs) are known to be efficient, high-tonnage devices suitable for upgrading particles in the 50 to 0.5 mm size range. This versatile separator, which uses centrifugal forces to enhance the separation of fine particles that cannot be upgraded in static dense medium separators, can be found in most modern coal plants and in a variety of mineral plants treating iron ore, dolomite, diamonds, potash and lead-zinc ores. Due to the high tonnage, a small increase in DMC efficiency can have a large impact on plant profitability. Unfortunately, the knowledge base required to properly design and operate DMCs has been seriously eroded during the past several decades. In an attempt to correct this problem, a set of engineering tools have been developed to allow producers to improve the efficiency of their DMC circuits. These tools include (1) low-cost density tracers that can be used by plant operators to rapidly assess DMC performance, (2) mathematical process models that can be used to predict the influence of changes in operating and design variables on DMC performance, and (3) an expert advisor system that provides plant operators with a user-friendly interface for evaluating, optimizing and trouble-shooting DMC circuits. The field data required to develop these tools was collected by conducting detailed sampling and evaluation programs at several industrial plant sites. These data were used to demonstrate the technical, economic and environmental benefits that can be realized through the application of these engineering tools.

  10. AutoDrug: fully automated macromolecular crystallography workflows for fragment-based drug discovery

    SciTech Connect

    Tsai, Yingssu; McPhillips, Scott E.; González, Ana; McPhillips, Timothy M.; Zinn, Daniel; Cohen, Aina E.; Feese, Michael D.; Bushnell, David; Tiefenbrunn, Theresa; Stout, C. David; Ludaescher, Bertram; Hedman, Britt; Hodgson, Keith O.; Soltis, S. Michael

    2013-05-01

    New software has been developed for automating the experimental and data-processing stages of fragment-based drug discovery at a macromolecular crystallography beamline. A new workflow-automation framework orchestrates beamline-control and data-analysis software while organizing results from multiple samples. AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallography steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully

  11. Rigid-cluster models of conformational transitions in macromolecular machines and assemblies.

    PubMed

    Kim, Moon K; Jernigan, Robert L; Chirikjian, Gregory S

    2005-07-01

    We present a rigid-body-based technique (called rigid-cluster elastic network interpolation) to generate feasible transition pathways between two distinct conformations of a macromolecular assembly. Many biological molecules and assemblies consist of domains which act more or less as rigid bodies during large conformational changes. These collective motions are thought to be strongly related with the functions of a system. This fact encourages us to simply model a macromolecule or assembly as a set of rigid bodies which are interconnected with distance constraints. In previous articles, we developed coarse-grained elastic network interpolation (ENI) in which, for example, only Calpha atoms are selected as representatives in each residue of a protein. We interpolate distance differences of two conformations in ENI by using a simple quadratic cost function, and the feasible conformations are generated without steric conflicts. Rigid-cluster interpolation is an extension of the ENI method with rigid-clusters replacing point masses. Now the intermediate conformations in an anharmonic pathway can be determined by the translational and rotational displacements of large clusters in such a way that distance constraints are observed. We present the derivation of the rigid-cluster model and apply it to a variety of macromolecular assemblies. Rigid-cluster ENI is then modified for a hybrid model represented by a mixture of rigid clusters and point masses. Simulation results show that both rigid-cluster and hybrid ENI methods generate sterically feasible pathways of large systems in a very short time. For example, the HK97 virus capsid is an icosahedral symmetric assembly composed of 60 identical asymmetric units. Its original Hessian matrix size for a Calpha coarse-grained model is >(300,000)(2). However, it reduces to (84)(2) when we apply the rigid-cluster model with icosahedral symmetry constraints. The computational cost of the interpolation no longer scales heavily with

  12. Time-resolved analysis of macromolecular structures during reactions by stopped-flow electrooptics.

    PubMed

    Porschke, D

    1998-07-01

    A stopped-flow field-jump instrument and its use for the analysis of macromolecular structure changes during reactions is described. The operation of the new instrument is simple and reliable, owing to a new type of cell construction with electrodes directly integrated in a quartz cuvette: major advantages are the relatively low demand on sample quantities and a high time resolution. The stopped flow is characterized by a dead time of approximately 0.5 ms. Electric field pulses with field strengths up to 20 kV/cm and rise times in the nanosecond range are applied at adjustable times after stop of the flow. The time resolution of the optical detection is up to the nanosecond time range. The instrument may be used for the combination of stopped flow with temperature-jump and field-jump experiments. A particularly useful new application is the analysis of macromolecular reactions by electrooptical measurements, because electrooptical data provide information about structures. This is demonstrated for the intercalation of ethidium into double-helical DNA. The transients, measured at 313 nm, where the signal is exclusively due to ethidium bound to the DNA, demonstrate a relatively high negative dichroism at 0.5 ms after mixing. The absolute value of this negative dichroism increases in the millisecond time range and approaches the equilibrium value within about a second. The dichroism decay time constants demonstrate a clear increase of the effective DNA length due to ethidium binding, already 0.5 ms after mixing; a further increase to the equilibrium value is found in the millisecond time range. The analysis of these data demonstrate the existence of up to three relaxation processes, depending on the conditions of the experiments. The dichroism amplitudes, together with the decay time constants, indicate that all the reaction states found in the present investigation are complexes with insertion of ethidium residues between basepairs. Moreover, the data clearly show

  13. Contrast agents for MRI.

    PubMed

    Shokrollahi, H

    2013-12-01

    Contrast agents are divided into two categories. The first one is paramagnetic compounds, including lanthanides like gadolinium, which mainly reduce the longitudinal (T1) relaxation property and result in a brighter signal. The second class consists of super-paramagnetic magnetic nanoparticles (SPMNPs) such as iron oxides, which have a strong effect on the transversal (T2) relaxation properties. SPMNPs have the potential to be utilized as excellent probes for magnetic resonance imaging (MRI). For instance, clinically benign iron oxide and engineered ferrite nanoparticles provide a good MRI probing capability for clinical applications. Furthermore, the limited magnetic property and inability to escape from the reticuloendothelial system (RES) of the used nanoparticles impede their further advancement. Therefore, it is necessary to develop the engineered magnetic nanoparticle probes for the next-generation molecular MRI. Considering the importance of MRI in diagnosing diseases, this paper presents an overview of recent scientific achievements in the development of new synthetic SPMNP probes whereby the sensitive and target-specific observation of biological events at the molecular and cellular levels is feasible.

  14. 08B1-1: an automated beamline for macromolecular crystallography experiments at the Canadian Light Source.

    PubMed

    Fodje, Michel; Grochulski, Pawel; Janzen, Kathryn; Labiuk, Shaunivan; Gorin, James; Berg, Russ

    2014-05-01

    Beamline 08B1-1 is a recently commissioned bending-magnet beamline at the Canadian Light Source. The beamline is designed for automation and remote access. Together with the undulator-based beamline 08ID-1, they constitute the Canadian Macromolecular Crystallography Facility. This paper describes the design, specifications, hardware and software of beamline 08B1-1. A few scientific results using data obtained at the beamline will be highlighted.

  15. Gordon Research Conference on Dynamics of Macromolecular and Polyelectrolyte Solutions Held in Oxnard, California on 12-16 February 1990

    DTIC Science & Technology

    1990-04-01

    Polyelectrolyte Solutions* was held IZ-16 February, 1990, at Casa Sirena Hotel, Oxnard, CA There were ninety-one scientists iarticipatIng. ’cludiq thirty-four...MACROMOLECULAR AND POLYELECT’ROLYTE 5’LUToNS Casa Sirena . Oxnard. CA Lee Magid - Chairman Peter Pusey - VIce-Chairman Monday. February 12 S.J. Candau...12-16, 1990 Casa Sirena Marina Hotel Oxnard, California REGISTRATION LIST Bruce J. Ackerson 149 Sauver Candau 136 Oklahoma State University Universit4

  16. Stationary Crystal Diffraction with a Monochromatic Convergent X-Ray Source and Application for Macromolecular Crystal Data Collection

    NASA Technical Reports Server (NTRS)

    Ho, Joseph X.; Snell, Eddie H.; Sisk, R. Charles; Ruble, John R.; Carter, Daniel C.; Owens, Scott M.; Gibson, Walter M.

    1998-01-01

    A diffraction geometry utilizing convergent X-rays from a polycapillary optic incident on a stationary crystal is described. A mathematical simulation of the resulting diffraction pattern (in terms of spot shape, position and intensity) is presented along with preliminary experimental results recorded from a lysozyme crystal. The effective source coverage factor is introduced to bring the reflection intensities onto the same scale. The feasibility of its application to macromolecular crystal data collection is discussed.

  17. Stationary Crystal Diffraction with a Monochromatic Convergent X-Ray Source and Application for Macromolecular Crystal Data Collection

    NASA Technical Reports Server (NTRS)

    Ho, J. X.; Snell, E. H.; Sisk, R. C.; Ruble, J. R.; Carter, D. C.; Owens, S. M.; Gibson, W. M.

    1997-01-01

    A diffraction geometry utilizing convergent X-rays from a polycapillary optic incident on a stationary crystal is described. A mathematical simulation of the resulting diffraction pattern (in terms of spot shape, position and intensity) is presented along with preliminary experimental results recorded from a lysozyme crystal. The effective source coverage factor is introduced to bring the reflection intensities onto the same scale. The feasibility of its application to macromolecular crystal data collection is discussed.

  18. Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay

    PubMed Central

    Bischoff, Iris; Hornburger, Michael C.; Mayer, Bettina A.; Beyerle, Andrea; Wegener, Joachim; Fürst, Robert

    2016-01-01

    The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer’s tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell® tracer assays and two commercial impedance devices (xCELLigence®, ECIS®). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments. PMID:27025965

  19. Effect of minimizing amount of template by addition of macromolecular crowding agent on preparation of molecularly imprinted monolith.

    PubMed

    Sun, Guang-Ying; Zhong, Dan-Dan; Li, Xiang-Jie; Luo, Yu-Qing; Ba, Hang; Liu, Zhao-Sheng; Aisa, Haji Akber

    2015-09-01

    One of the main challenges in the preparation of molecularly imprinted polymers (MIPs) is the substantial initial amount of template needed because of the requirement of high load capacities for most applications. A new strategy of macromolecular crowding was suggested to solve this problem by reducing the amount of template in the polymerization recipe. In a ternary porogenic system of polystyrene (PS) (crowding agent), tetrahydrofuran, and toluene, an imprinted monolithic column with high porosity and good permeability was synthesized using a mixture of ellagic acid (template), acrylamide, and ethylene glycol dimethacrylate. The effect of polymerization factors, including monomer-template molar ratio and the molecular weight and concentration of PS, on the imprinting effect of the resulting MIP monoliths was systematically investigated. At a high ratio of monomer-template (120:1), the greatest imprinting factor of 32.4 was obtained on the MIP monolith with the aid of macromolecular crowding agent. The PS-based imprinted monolith had imprinting even at the extremely high ratio of functional monomer to template of 1510:1. Furthermore, an off-line solid-phase extraction based on the ground MIP was conducted, and the purification recovery of ellagic acid from pomegranate-rind extract was up to 80 %. In conclusion, this approach based on macromolecular crowding is simple, and is especially valuable for those applications of MIP preparation for which a rare template is used.

  20. A decade of user operation on the macromolecular crystallography MAD beamline ID14-4 at the ESRF

    PubMed Central

    McCarthy, Andrew A.; Brockhauser, Sandor; Nurizzo, Didier; Theveneau, Pascal; Mairs, Trevor; Spruce, Darren; Guijarro, Matias; Lesourd, Marc; Ravelli, Raimond B. G.; McSweeney, Sean

    2009-01-01

    ID14-4 at the ESRF is the first tunable undulator-based macromolecular crystallography beamline that can celebrate a decade of user service. During this time ID14-4 has not only been instrumental in the determination of the structures of biologically important molecules but has also contributed significantly to the development of various instruments, novel data collection schemes and pioneering radiation damage studies on biological samples. Here, the evolution of ID14-4 over the last decade is presented, and some of the major improvements that were carried out in order to maintain its status as one of the most productive macromolecular crystallography beamlines are highlighted. The experimental hutch has been upgraded to accommodate a high-precision diffractometer, a sample changer and a large CCD detector. More recently, the optical hutch has been refurbished in order to improve the X-ray beam quality on ID14-4 and to incorporate the most modern and robust optical elements used at other ESRF beamlines. These new optical elements will be described and their effect on beam stability discussed. These studies may be useful in the design, construction and maintenance of future X-ray beamlines for macromolecular crystallography and indeed other applications, such as those planned for the ESRF upgrade. PMID:19844017

  1. Predictive Mechanical Characterization of Macro-Molecular Material Chemistry Structures of Cement Paste at Nano Scale - Two-phase Macro-Molecular Structures of Calcium Silicate Hydrate, Tri-Calcium Silicate, Di-Calcium Silicate and Calcium Hydroxide

    NASA Astrophysics Data System (ADS)

    Padilla Espinosa, Ingrid Marcela

    Concrete is a hierarchical composite material with a random structure over a wide range of length scales. At submicron length scale the main component of concrete is cement paste, formed by the reaction of Portland cement clinkers and water. Cement paste acts as a binding matrix for the other components and is responsible for the strength of concrete. Cement paste microstructure contains voids, hydrated and unhydrated cement phases. The main crystalline phases of unhydrated cement are tri-calcium silicate (C3S) and di-calcium silicate (C2S), and of hydrated cement are calcium silicate hydrate (CSH) and calcium hydroxide (CH). Although efforts have been made to comprehend the chemical and physical nature of cement paste, studies at molecular level have primarily been focused on individual components. Present research focuses on the development of a method to model, at molecular level, and analysis of the two-phase combination of hydrated and unhydrated phases of cement paste as macromolecular systems. Computational molecular modeling could help in understanding the influence of the phase interactions on the material properties, and mechanical performance of cement paste. Present work also strives to create a framework for molecular level models suitable for potential better comparisons with low length scale experimental methods, in which the sizes of the samples involve the mixture of different hydrated and unhydrated crystalline phases of cement paste. Two approaches based on two-phase cement paste macromolecular structures, one involving admixed molecular phases, and the second involving cluster of two molecular phases are investigated. The mechanical properties of two-phase macromolecular systems of cement paste consisting of key hydrated phase CSH and unhydrated phases C3S or C2S, as well as CSH with the second hydrated phase CH were calculated. It was found that these cement paste two-phase macromolecular systems predicted an isotropic material behavior. Also

  2. Phase Contrast Imaging

    SciTech Connect

    Menk, Ralf Hendrik

    2008-11-13

    All standard (medical) x-ray imaging technologies, rely primarily on the amplitude properties of the incident radiation, and do not depend on its phase. This is unchanged since the discovery by Roentgen that the intensity of an x-ray beam, as measured by the exposure on a film, was related to the relative transmission properties of an object. However, recently various imaging techniques have emerged which depend on the phase of the x-rays as well as the amplitude. Phase becomes important when the beam is coherent and the imaging system is sensitive to interference phenomena. Significant new advances have been made in coherent optic theory and techniques, which now promise phase information in medical imaging. The development of perfect crystal optics and the increasing availability of synchrotron radiation facilities have contributed to a significant increase in the application of phase based imaging in materials and life sciences. Unique source characteristics such as high intensity, monochromaticity, coherence and high collimating provide an ideal source for advanced imaging. Phase contrast imaging has been applied in both projection and computed tomography modes, and recent applications have been made in the field of medical imaging. Due to the underlying principle of X-ray detection conventional image receptors register only intensities of wave fields and not their phases. During the last decade basically five different methods were developed that translate the phase information into intensity variations. These methods are based on measuring the phase shift {phi} directly (using interference phenomena), the gradient {nabla}{sub {phi}}, or the Laplacian {nabla}{sup 2}{phi}. All three methods can be applied to polychromatic X-ray sources keeping in mind that the native source is synchrotron radiation, featuring monochromatic and reasonable coherent X-ray beams. Due to the vast difference in the coefficients that are driven absorption and phase effects (factor 1

  3. Effect of the macromolecular architecture of biodegradable polyurethanes on the controlled delivery of ocular drugs.

    PubMed

    da Silva, Gisele Rodrigues; da Silva Cunha, Armando; Ayres, Eliane; Oréfice, Rodrigo L

    2009-02-01

    Controlled delivery of drugs is a major issue in the treatment of ocular diseases, such as in the treatment of uveitis. In this study, dexamethasone acetate, an important type of corticoid used in the treatment of some uveitis, was incorporated into biodegradable polyurethanes (PU) having different macromolecular architectures. The biodegradable polyurethanes were obtained by preparing PU aqueous dispersions having poly(caprolactone) and/or poly(ethylene glycol) as soft segments. The drug was incorporated into the polymer by dissolving it in the PU aqueous dispersion. FTIR results showed the presence of the drug in the polymer with its original chemical structure. Small angle X-ray scattering (SAXS) results were explored to show that the incorporation of dexamethasone acetate led to the modification of the nanostructure of the polyurethane having only poly(caprolactone) as the soft segment, while the drug did not change significantly the microphase separated structure of PU having both poly(caprolactone) and poly(ethylene glycol) as soft segments. The evaluation of the release of the drug in vitro demonstrated that the obtained biodegradable polyurethanes were well succeeded in delivering dexamethasone acetate at an almost constant rate for 53 weeks. The presence of poly(ethylene glycol) together with poly(caprolactone) as soft segment in biodegradable PU was able to increase the rate of dexamethasone acetate release when compared to the rate of drug release from PU having only poly(caprolactone).

  4. Macromolecular dimensions and mechanical properties of monolayer films of Sonorean mesquite gum.

    PubMed

    López-Franco, Yolanda L; Valdez, Miguel A; Hernández, Javier; Calderón de la Barca, Ana M; Rinaudo, Marguerite; Goycoolea, Francisco M

    2004-09-16

    Mesquite gum sourced from Prosopis velutina trees and gum arabic (Acacia spp.) were characterized using light scattering and Langmuir isotherms. Both gum materials were fractionated by hydrophobic interaction chromatography, yielding four fractions for both gums: FI, FIIa, FIIb and FIII in mesquite gum and FI, FII, FIIIa and FIIIb in gum arabic. In mesquite gum, the obtained fractions had different protein content (7.18-38.60 wt.-%) and macromolecular dimensions (M approximately 3.89 x 10(5)-8.06 x 10(5) g.mol(-1), RG approximately 48.83-71.11 nm, RH approximately 9.61-24.06 nm) and architecture given by the structure factor (RG/RH ratio approximately 2.96-5.27). The mechanical properties of Langmuir monolayers at the air-water interface were very different on each gum and their fractions. For mesquite gum, the most active species at the interface were those comprised in Fractions IIa and IIb and III, while Fraction I the pi/A isotherm lied below that of the whole gum. In gum arabic only Fraction III developed greater surface pressure at the same surface per milligram of material than whole gum. This is rationalized in terms of structural differences in both materials. Mesquite gum tertiary structure seems to fit best with an elongated polydisperse macrocoil in agreement with the "twisted hairy rope" proposal for arabinogalactan proteoglycans.

  5. Macromolecular geometries determined with field-flow fractionation and their impact on the overlap concentration.

    PubMed

    Rojas, Cinthia Carola; Wahlund, Karl-Gustav; Bergenståhl, Björn; Nilsson, Lars

    2008-06-01

    In this paper we aim to understand the size/conformation relationship in waxy barley starch, a polydisperse and ultrahigh molar mass biomacromolecule. Characterizations are performed with asymmetrical flow field-flow fractionation (AsFlFFF). Furthermore, we study the effect of homogenization on the molar mass, rms radius (r rms) and hydrodynamic radius (r h). For the untreated sample, the macromolecules are elongated objects with low apparent density. As a result of homogenization, molar mass, and r rms decrease, while r h remains unaffected. The process also induces an increase, and scaling with size, of apparent density as well as changes in conformation, represented qualitatively by r rms/ r h. Finally, results from AsFlFFF are compared with viscosimetry and discussed in terms of concentration and close-packing in relation to macromolecular shape and conformation. Hence, the results show that AsFlFFF and our novel methodology enable the determination of several physical properties with high relevance for the solution behavior of polydisperse macromolecules.

  6. The status of the macromolecular crystallography beamlines at the European Synchrotron Radiation Facility

    NASA Astrophysics Data System (ADS)

    Mueller-Dieckmann, Christoph; Bowler, Matthew W.; Carpentier, Philippe; Flot, David; McCarthy, Andrew A.; Nanao, Max H.; Nurizzo, Didier; Pernot, Petra; Popov, Alexander; Round, Adam; Royant, Antoine; de Sanctis, Daniele; von Stetten, David; Leonard, Gordon A.

    2015-04-01

    The European Synchrotron Radiation Facility (ESRF) is the oldest and most powerful 3rd generation synchrotron in Europe, providing X-rays to more than 40 experimental stations welcoming several thousand researchers per year. A major success story has been the ESRF's facilities for macromolecular crystallography (MX). These are grouped around 3 straight sections: On ID23 canted undulators accommodate ID23-1, a mini-focus tuneable energy end station and ID23-2, the world's first micro-focus beamline dedicated to MX; ID29 houses a single, mini-focus, tuneable energy end station; ID30 will provide three end stations for MX due in operation from mid-2014 to early 2015. Here, one branch of a canted X-ray source feeds two fixed-energy end stations (MASSIF-1, MASSIF-3). The second feeds ID30B, a variable focus, tuneable energy beamline. MASSIF-1 is optimised for automatic high-throughput experiments requiring a relatively large beam size at the sample position, MASSIF-3 is a high-intensity, micro-focus facility designed to complement ID23-2. All end stations are highly automated, equipped with sample mounting robots and large area, fast-readout photon-counting detectors. Experiment control and tracking is achieved via a combination of the MXCuBE2 graphical user interface and the ISPyB database, the former allowing user-friendly control of all beamline components, the latter providing data tracking before, after and during experiments.

  7. Variations in macromolecular antifreeze levels in larvae of the darkling beetle, Meracantha contracta.

    PubMed

    Duman, J G

    1977-07-01

    Overwintering larvae of the darkling beetle, Meracantha contracta, produce a macromolecular antifreeze that is similar in activity to the glycoproteinaceous and proteinaceous antifreezes found in some cold-water, marine teleost fishes. The antifreeze is not present in the hemolymph of the Meracantha larvae in summer, but its production begins by late September in the wild population. The antifreeze reaches a maximum concentration in February, decreases slowly through spring, and disappears by early June. The supercooling points of the larvae are lowest in February, when the antifreeze levels are highest, and increase as the antifreeze concentrations in the hemolymph decrease in the spring. Larvae collected in mid-February and warm-acclimated lost the antifreeze with-in 12 days. Larvae collected in early September and cold-acclimated required nearly two months to produce concentrations of antifreeze comparable to those of overwintering larvae. Temperature seems to be the major environmental factor responsible for the control of antifreeze levels in Meracantha; however, other environmental factors may also be involved.

  8. Conservation of peptide structure of outer membrane protein-macromolecular complex from Neisseria gonorrhoeae.

    PubMed Central

    Hansen, M V; Wilde, C E

    1984-01-01

    The structural conservation of an outer membrane protein of Neisseria gonorrhoeae called OMP-MC (outer membrane protein-macromolecular complex) was investigated by determining the isoelectric point and amino-terminal amino acid sequence of the protein and by using high-performance liquid chromatography for comparative tryptic peptide mapping. The 76,000-dalton subunits generated by reduction and alkylation of the native 800,000-dalton complex from six test strains focused in ultrathin gels as bands of restricted heterogeneity at an approximate pI of 7.6. Dansyl chloride labeling indicated that all strains shared glycine as the amino-terminal amino acid. Sequence analysis of OMP-MC from two strains revealed no amino acid differences within the first 11 residues. Dual-label peptide maps revealed an extremely high degree of conservation of peptide structure. The results indicate that (i) OMP-MCs isolated from various strains of N. gonorrhoeae share structural homology and (ii) the 800,000-dalton complex is a homopolymer composed of 10 to 12 apparently identical 76,000-dalton subunits. Images PMID:6421738

  9. The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics

    PubMed Central

    Wang, Qian; Liang, Kao-Chen; Czader, Arkadiusz; Waxham, M. Neal; Cheung, Margaret S.

    2011-01-01

    The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells. PMID:21829336

  10. Multi-crystal Anomalous Diffraction for Low-resolution Macromolecular Phasing

    SciTech Connect

    Q Liu; Z Zhang; W Hendrickson

    2011-12-31

    Multiwavelength anomalous diffraction (MAD) and single-wavelength anomalous diffraction (SAD) are the two most commonly used methods for de novo determination of macromolecular structures. Both methods rely on the accurate extraction of anomalous signals; however, because of factors such as poor intrinsic order, radiation damage, inadequate anomalous scatterers, poor diffraction quality and other noise-causing factors, the anomalous signal from a single crystal is not always good enough for structure solution. In this study, procedures for extracting more accurate anomalous signals by merging data from multiple crystals are devised and tested. SAD phasing tests were made with a relatively large (1456 ordered residues) poorly diffracting (d{sub min} = 3.5 {angstrom}) selenomethionyl protein (20 Se). It is quantified that the anomalous signal, success in substructure determination and accuracy of phases and electron-density maps all improve with an increase in the number of crystals used in merging. Structure solutions are possible when no single crystal can support structural analysis. It is proposed that such multi-crystal strategies may be broadly useful when only weak anomalous signals are available.

  11. About Small Streams and Shiny Rocks: Macromolecular Crystal Growth in Microfluidics

    NASA Technical Reports Server (NTRS)

    vanderWoerd, Mark; Ferree, Darren; Spearing, Scott; Monaco, Lisa; Molho, Josh; Spaid, Michael; Brasseur, Mike; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    We are developing a novel technique with which we have grown diffraction quality protein crystals in very small volumes, utilizing chip-based, microfluidic ("LabChip") technology. With this technology volumes smaller than achievable with any laboratory pipette can be dispensed with high accuracy. We have performed a feasibility study in which we crystallized several proteins with the aid of a LabChip device. The protein crystals are of excellent quality as shown by X-ray diffraction. The advantages of this new technology include improved accuracy of dispensing for small volumes, complete mixing of solution constituents without bubble formation, highly repeatable recipe and growth condition replication, and easy automation of the method. We have designed a first LabChip device specifically for protein crystallization in batch mode and can reliably dispense and mix from a range of solution constituents. We are currently testing this design. Upon completion additional crystallization techniques, such as vapor diffusion and liquid-liquid diffusion will be accommodated. Macromolecular crystallization using microfluidic technology is envisioned as a fully automated system, which will use the 'tele-science' concept of remote operation and will be developed into a research facility aboard the International Space Station.

  12. Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection.

    PubMed

    Bowler, Matthew W; Guijarro, Matias; Petitdemange, Sebastien; Baker, Isabel; Svensson, Olof; Burghammer, Manfred; Mueller-Dieckmann, Christoph; Gordon, Elspeth J; Flot, David; McSweeney, Sean M; Leonard, Gordon A

    2010-08-01

    Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 microm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5-50 microm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.

  13. Temperature sensitivity of soil microbial communities: An application of macromolecular rate theory to microbial respiration

    NASA Astrophysics Data System (ADS)

    Alster, Charlotte J.; Koyama, Akihiro; Johnson, Nels G.; Wallenstein, Matthew D.; Fischer, Joseph C.

    2016-06-01

    There is compelling evidence that microbial communities vary widely in their temperature sensitivity and may adapt to warming through time. To date, this sensitivity has been largely characterized using a range of models relying on versions of the Arrhenius equation, which predicts an exponential increase in reaction rate with temperature. However, there is growing evidence from laboratory and field studies that observe nonmonotonic responses of reaction rates to variation in temperature, indicating that Arrhenius is not an appropriate model for quantitatively characterizing temperature sensitivity. Recently, Hobbs et al. (2013) developed macromolecular rate theory (MMRT), which incorporates thermodynamic temperature optima as arising from heat capacity differences between isoenzymes. We applied MMRT to measurements of respiration from soils incubated at different temperatures. These soils were collected from three grassland sites across the U.S. Great Plains and reciprocally transplanted, allowing us to isolate the effects of microbial community type from edaphic factors. We found that microbial community type explained roughly 30% of the variation in the CO2 production rate from the labile C pool but that temperature and soil type were most important in explaining variation in labile and recalcitrant C pool size. For six out of the nine soil × inoculum combinations, MMRT was superior to Arrhenius. The MMRT analysis revealed that microbial communities have distinct heat capacity values and temperature sensitivities sometimes independent of soil type. These results challenge the current paradigm for modeling temperature sensitivity of soil C pools and understanding of microbial enzyme dynamics.

  14. Reliable and efficient solution of genome-scale models of Metabolism and macromolecular Expression

    DOE PAGES

    Ma, Ding; Yang, Laurence; Fleming, Ronan M. T.; ...

    2017-01-18

    Currently, Constraint-Based Reconstruction and Analysis (COBRA) is the only methodology that permits integrated modeling of Metabolism and macromolecular Expression (ME) at genome-scale. Linear optimization computes steady-state flux solutions to ME models, but flux values are spread over many orders of magnitude. Data values also have greatly varying magnitudes. Furthermore, standard double-precision solvers may return inaccurate solutions or report that no solution exists. Exact simplex solvers based on rational arithmetic require a near-optimal warm start to be practical on large problems (current ME models have 70,000 constraints and variables and will grow larger). We also developed a quadrupleprecision version of ourmore » linear and nonlinear optimizer MINOS, and a solution procedure (DQQ) involving Double and Quad MINOS that achieves reliability and efficiency for ME models and other challenging problems tested here. DQQ will enable extensive use of large linear and nonlinear models in systems biology and other applications involving multiscale data.« less

  15. Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity

    PubMed Central

    Nogales, Eva; Scheres, Sjors H.W.

    2015-01-01

    Three-dimensional cryo-electron microscopy (cryo-EM) is an expanding structural biology technique that has recently undergone a quantum leap progression in its achievable resolution and its applicability to the study of challenging biological systems. Because crystallization is not required, only small amounts of sample are needed, and, because images can be classified in a computer, the technique has the potential to deal with compositional and conformational mixtures. Therefore, cryo-EM can be used to investigate complete and fully functional macromolecular complexes in different functional states, providing a richness of biological insight. In this review we underlie some of the principles behind the cryo-EM methodology of single particle analysis and discuss some recent results of its application to challenging systems of paramount biological importance. We place special emphasis on new methodological developments that are leading to an explosion of new studies, many of which are reaching resolutions that could only be dreamed of only a couple of years ago. PMID:26000851

  16. Discovering Free Energy Basins for Macromolecular Systems via Guided Multiscale Simulation

    PubMed Central

    Sereda, Yuriy V.; Singharoy, Abhishek B.; Jarrold, Martin F.; Ortoleva, Peter J.

    2012-01-01

    An approach for the automated discovery of low free energy states of macromolecular systems is presented. The method does not involve delineating the entire free energy landscape but proceeds in a sequential free energy minimizing state discovery, i.e., it first discovers one low free energy state and then automatically seeks a distinct neighboring one. These states and the associated ensembles of atomistic configurations are characterized by coarse-grained variables capturing the large-scale structure of the system. A key facet of our approach is the identification of such coarse-grained variables. Evolution of these variables is governed by Langevin dynamics driven by thermal-average forces and mediated by diffusivities, both of which are constructed by an ensemble of short molecular dynamics runs. In the present approach, the thermal-average forces are modified to account for the entropy changes following from our knowledge of the free energy basins already discovered. Such forces guide the system away from the known free energy minima, over free energy barriers, and to a new one. The theory is demonstrated for lactoferrin, known to have multiple energy-minimizing structures. The approach is validated using experimental structures and traditional molecular dynamics. The method can be generalized to enable the interpretation of nanocharacterization data (e.g., ion mobility – mass spectrometry, atomic force microscopy, chemical labeling, and nanopore measurements). PMID:22423635

  17. Quickly Getting the Best Data from Your Macromolecular Crystals with a New Generation of Beamline Instruments

    SciTech Connect

    Cipriani, Florent; Felisaz, Franck; Lavault, Bernard; Brockhauser, Sandor; Ravelli, Raimond; Launer, Ludovic

    2007-01-19

    While routine Macromolecular x-ray (MX) crystallography has relied on well established techniques for some years all the synchrotrons around the world are improving the throughput of their MX beamlines. Third generation synchrotrons provide small intense beams that make data collection of 5-10 microns sized crystals possible. The EMBL/ESRF MX Group in Grenoble has developed a new generation of instruments to easily collect data on 10 {mu}m size crystals in an automated environment. This work is part of the Grenoble automation program that enables FedEx like crystallography using fully automated data collection and web monitored experiments. Seven ESRF beamlines and the MRC BM14 ESRF/CRG beamline are currently equipped with these latest instruments. We describe here the main features of the MD2x diffractometer family and the SC3 sample changer robot. Although the SC3 was primarily designed to increase the throughput of MX beamlines, it has also been shown to be efficient in improving the quality of the data collected. Strategies in screening a large number of crystals, selecting the best, and collecting a full data set from several re-oriented micro-crystals can now be run with minimum time and effort. The MD2x and SC3 instruments are now commercialised by the company ACCEL GmbH.

  18. Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

    PubMed

    Lonhienne, T; Baise, E; Feller, G; Bouriotis, V; Gerday, C

    2001-02-09

    Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

  19. Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling

    PubMed Central

    2017-01-01

    Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling of solvent accessible residues followed by their MS-based identification yields modeling restraints that allow mapping the location and orientation of subunits within protein assemblies. Together with complementary restraints derived from cross-linking and native MS, we built native-like models of four heterocomplexes with known subunit structures and compared them with available X-ray crystal structures. The results demonstrated that covalent labeling followed by MS markedly increased the predictive power of the integrative modeling strategy enabling more accurate protein assembly models. We applied this strategy to the F-type ATP synthase from spinach chloroplasts (cATPase) providing a structural basis for its function as a nanomotor. By subjecting the models generated by our restraint-based strategy to molecular dynamics (MD) simulations, we revealed the conformational states of the peripheral stalk and assigned flexible regions in the enzyme. Our strategy can readily incorporate complementary chemical labeling strategies and we anticipate that it will be applicable to many other systems providing new insights into the structure and function of protein complexes. PMID:28208298

  20. Effects of sound exposure on the growth and intracellular macromolecular synthesis of E. coli k-12

    PubMed Central

    Zhang, Yongzhu; Wu, Ying

    2016-01-01

    Microbes, as one of the primary producers of the biosphere, play an important role in ecosystems. Exploring the mechanism of adaptation and resistance of microbial population to various environmental factors has come into focus in the fields of modern microbial ecology and molecular ecology. However, facing the increasingly serious problem of acoustic pollution, very few efforts have been put forth into studying the relation of single cell organisms and sound field exposure. Herein, we studied the biological effects of sound exposure on the growth of E. coli K-12 with different acoustic parameters. The effects of sound exposure on the intracellular macromolecular synthesis and cellular morphology of E. coli K-12 were also analyzed and discussed. Experimental results indicated that E. coli K-12 exposed to sound waves owned a higher biomass and a faster specific growth rate compared to the control group. Also, the average length of E. coli K-12 cells increased more than 27.26%. The maximum biomass and maximum specific growth rate of the stimulation group by 8000 Hz, 80dB sound wave was about 1.7 times and 2.5 times that of the control group, respectively. Moreover, it was observed that E. coli K-12 can respond rapidly to sound stress at both the transcriptional and posttranscriptional levels by promoting the synthesis of intracellular RNA and total protein. Some potential mechanisms may be involved in the responses of bacterial cells to sound stress. PMID:27077011

  1. Exploring the mechanistic bases of heterosis from the perspective of macromolecular complexes.

    PubMed

    Veitia, Reiner A; Vaiman, Daniel

    2011-02-01

    Heterosis is defined as greater biomass, fertility or other traits in heterozygotes, polyploids or hybrids compared to their genetically divergent (often homozygous) parents. Heterosis was noticed by various civilizations and scientifically reported by Darwin himself. Despite the importance of heterosis, its molecular bases are still enigmatic. Several genetic models have been proposed but fail to give mechanistic insights. Here we show how dominant negative interactions might give rise to heterotic responses. We also explore a biochemical model of gene dosage effects in macromolecular complexes in a similar context. With the help of heuristic examples and computer simulations we find that heterotic individuals display higher allelic diversity and smaller average multimer concentrations than nonheterotic ones. As intuitively expected, the existence of heterosis involving multimeric complexes arises when the inbred parents have on average smaller genetic values than the maximum possible. Despite its simplicity, the dosage model accounts for the puzzling phenomenon of "progressive heterosis" in which polyploids with increasing genetic diversity exhibit progressively greater heterosis.

  2. Implications of macromolecular crowding and reducing conditions for in vitro ribosome construction

    PubMed Central

    Fritz, Brian R.; Jamil, Osman K.; Jewett, Michael C.

    2015-01-01

    In vitro construction of Escherichia coli ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and ribosome assembly. Finally, we showed that iSAT ribosomes possess ∼70% of the protein synthesis activity of in vivo-assembled E. coli ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of ribosome biogenesis in the context of an in vitro transcription-translation system. PMID:25897121

  3. RoboDiff: combining a sample changer and goniometer for highly automated macromolecular crystallography experiments

    PubMed Central

    Nurizzo, Didier; Bowler, Matthew W.; Caserotto, Hugo; Dobias, Fabien; Giraud, Thierry; Surr, John; Guichard, Nicolas; Papp, Gergely; Guijarro, Matias; Mueller-Dieckmann, Christoph; Flot, David; McSweeney, Sean; Cipriani, Florent; Theveneau, Pascal; Leonard, Gordon A.

    2016-01-01

    Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically. PMID:27487827

  4. Assembly of the cochlear gap junction macromolecular complex requires connexin 26.

    PubMed

    Kamiya, Kazusaku; Yum, Sabrina W; Kurebayashi, Nagomi; Muraki, Miho; Ogawa, Kana; Karasawa, Keiko; Miwa, Asuka; Guo, Xueshui; Gotoh, Satoru; Sugitani, Yoshinobu; Yamanaka, Hitomi; Ito-Kawashima, Shioko; Iizuka, Takashi; Sakurai, Takashi; Noda, Tetsuo; Minowa, Osamu; Ikeda, Katsuhisa

    2014-04-01

    Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

  5. Effects of macromolecular crowding and DNA looping on gene regulation kinetics

    NASA Astrophysics Data System (ADS)

    Li, Gene-Wei; Berg, Otto G.; Elf, Johan

    2009-04-01

    DNA-binding proteins control how genomes function. The theory of facilitated diffusion explains how DNA-binding proteins can find targets apparently faster than the diffusion limit by using reduced dimensionality-combining three-dimensional (3D) diffusion through cytoplasm with 1D sliding along DNA (refs 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). However, it does not include a description of macromolecular crowding on DNA as observed in living cells. Here, we show that such a physical constraint to sliding greatly reduces the search speed, in agreement with single-molecule measurements. Interestingly, the generalized theory also reveals significant insights into the design principles of biology. First, it places a hard constraint on the total number of DNA-binding proteins per cell. Remarkably, the number measured for Escherichia coli fits within the optimal range. Secondly, it defines a new role for DNA looping, a ubiquitous topological motif in genomes. DNA looping can speed up the search process by bypassing proteins that block the sliding track close to the target.

  6. Macromolecular scaffolding: the relationship between nanoscale architecture and function in multichromophoric arrays for organic electronics.

    PubMed

    Palermo, Vincenzo; Schwartz, Erik; Finlayson, Chris E; Liscio, Andrea; Otten, Matthijs B J; Trapani, Sara; Müllen, Klaus; Beljonne, David; Friend, Richard H; Nolte, Roeland J M; Rowan, Alan E; Samorì, Paolo

    2010-02-23

    The optimization of the electronic properties of molecular materials based on optically or electrically active organic building blocks requires a fine-tuning of their self-assembly properties at surfaces. Such a fine-tuning can be obtained on a scale up to 10 nm by mastering principles of supramolecular chemistry, i.e., by using suitably designed molecules interacting via pre-programmed noncovalent forces. The control and fine-tuning on a greater length scale is more difficult and challenging. This Research News highlights recent results we obtained on a new class of macromolecules that possess a very rigid backbone and side chains that point away from this backbone. Each side chain contains an organic semiconducting moiety, whose position and electronic interaction with neighboring moieties are dictated by the central macromolecular scaffold. A combined experimental and theoretical approach has made it possible to unravel the physical and chemical properties of this system across multiple length scales. The (opto)electronic properties of the new functional architectures have been explored by constructing prototypes of field-effect transistors and solar cells, thereby providing direct insight into the relationship between architecture and function.

  7. Unveiling Contacts within Macromolecular Assemblies by Solving Minimum Weight Connectivity Inference (MWC) Problems.

    PubMed

    Agarwal, Deepesh; Caillouet, Christelle; Coudert, David; Cazals, Frederic

    2015-08-01

    Consider a set of oligomers listing the subunits involved in subcomplexes of a macromolecular assembly, obtained e.g. using native mass spectrometry or affinity purification. Given these oligomers, connectivity inference (CI) consists of finding the most plausible contacts between these subunits, and minimum connectivity inference (MCI) is the variant consisting of finding a set of contacts of smallest cardinality. MCI problems avoid speculating on the total number of contacts but yield a subset of all contacts and do not allow exploiting a priori information on the likelihood of individual contacts. In this context, we present two novel algorithms, MILP-W and MILP-WB. The former solves the minimum weight connectivity inference (MWCI), an optimization problem whose criterion mixes the number of contacts and their likelihood. The latter uses the former in a bootstrap fashion to improve the sensitivity and the specificity of solution sets.Experiments on three systems (yeast exosome, yeast proteasome lid, human eIF3), for which reference contacts are known (crystal structure, cryo electron microscopy, cross-linking), show that our algorithms predict contacts with high specificity and sensitivity, yielding a very significant improvement over previous work, typically a twofold increase in sensitivity.The software accompanying this paper is made available and should prove of ubiquitous interest whenever connectivity inference from oligomers is faced.

  8. Reliable and efficient solution of genome-scale models of Metabolism and macromolecular Expression

    PubMed Central

    Ma, Ding; Yang, Laurence; Fleming, Ronan M. T.; Thiele, Ines; Palsson, Bernhard O.; Saunders, Michael A.

    2017-01-01

    Constraint-Based Reconstruction and Analysis (COBRA) is currently the only methodology that permits integrated modeling of Metabolism and macromolecular Expression (ME) at genome-scale. Linear optimization computes steady-state flux solutions to ME models, but flux values are spread over many orders of magnitude. Data values also have greatly varying magnitudes. Standard double-precision solvers may return inaccurate solutions or report that no solution exists. Exact simplex solvers based on rational arithmetic require a near-optimal warm start to be practical on large problems (current ME models have 70,000 constraints and variables and will grow larger). We have developed a quadruple-precision version of our linear and nonlinear optimizer MINOS, and a solution procedure (DQQ) involving Double and Quad MINOS that achieves reliability and efficiency for ME models and other challenging problems tested here. DQQ will enable extensive use of large linear and nonlinear models in systems biology and other applications involving multiscale data. PMID:28098205

  9. Macromolecular Drug Targets in Cancer Treatment and Thiosemicarbazides as Anticancer Agents.

    PubMed

    Küçükgüzel, Ş Güniz; Coşkun, Göknil P

    2016-01-01

    Cancer is known as abnormal cell division and consisting of a group of diseases on various organ tissues. Many therapies are available in cancer treatment such as chemotherapy, radiotherapy etc. Without damaging normal tissue, there is a huge need for specified anticancer drugs which have effect only on abnormal cancer cells. Therefore, advances in anticancer drug discovery in treating cancer in the recent years, directed towards to the macromolecular targets. Heterocyclic molecules, such as fluconazole, acetazolamide, etc., have a significant role in health care and pharmaceutical drug design. Thiosemicarbazides (NH2-NH-CSNH2) are the simplest hydrazine derivatives of thiocarbamic acid and are not only transition compounds, but they are also very effective organic compounds. Thiosemicarbazides possess an amide and amine protons, carbonyl and thione carbons. These structures have attracted the attention of the researchers in the development of novel compounds with anticonvulsant, antiviral, anti-inflammatory, antibacterial, antimycobacterial, antifungal, antioxidant and anticancer activities. Recently, a number of thiosemicarbazides are available commercially as anticancer drugs for novel anticancer drug discovery. Antineoplastic or anticancer drugs prevent or inhibit the maturation and proliferation of neoplasms. These observations have been guiding the researchers for the development of new thiosemicarbazides that possess anticancer activity.

  10. Viral nanoparticles as macromolecular devices for new therapeutic and pharmaceutical approaches

    PubMed Central

    Grasso, Simone; Santi, Luca

    2010-01-01

    Viral nanoparticles are molecular cages derived from the assembly of viral structural proteins. They bear several peculiar features as proper dimensions for nanoscale applications, size homogeneity, an intrinsic robustness, a large surface area to mass ratio and a defined, repetitive and symmetric macromolecular organization. A number of expression strategies, using various biological systems, efficiently enable the production of significant quantities of viral nanoparticles, which can be easily purified. Genetic engineering and in vitro chemical modification consent to manipulate of the outer and inner surface of these nanocages, allowing specific changes of the original physico-chemical and biological properties. Moreover, several studies have focused on the in vitro disassembly/reassembly and gating of viral nanoparticles, with the aim of encapsulating exogenous molecules inside and therefore improving their potential as containment delivery devices. These technological progresses have led research to a growing variety of applications in different fields such as biomedicine, pharmacology, separation science, catalytic chemistry, crop pest control and material science. In this review we will focus on the strategies used to modify the characteristics of viral nanoparticles and on their use in biomedicine and pharmacology. PMID:21383892

  11. Biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm

    PubMed Central

    Yu, Isseki; Mori, Takaharu; Ando, Tadashi; Harada, Ryuhei; Jung, Jaewoon; Sugita, Yuji; Feig, Michael

    2016-01-01

    Biological macromolecules function in highly crowded cellular environments. The structure and dynamics of proteins and nucleic acids are well characterized in vitro, but in vivo crowding effects remain unclear. Using molecular dynamics simulations of a comprehensive atomistic model cytoplasm we found that protein-protein interactions may destabilize native protein structures, whereas metabolite interactions may induce more compact states due to electrostatic screening. Protein-protein interactions also resulted in significant variations in reduced macromolecular diffusion under crowded conditions, while metabolites exhibited significant two-dimensional surface diffusion and altered protein-ligand binding that may reduce the effective concentration of metabolites and ligands in vivo. Metabolic enzymes showed weak non-specific association in cellular environments attributed to solvation and entropic effects. These effects are expected to have broad implications for the in vivo functioning of biomolecules. This work is a first step towards physically realistic in silico whole-cell models that connect molecular with cellular biology. DOI: http://dx.doi.org/10.7554/eLife.19274.001 PMID:27801646

  12. The use of workflows in the design and implementation of complex experiments in macromolecular crystallography

    PubMed Central

    Brockhauser, Sandor; Svensson, Olof; Bowler, Matthew W.; Nanao, Max; Gordon, Elspeth; Leal, Ricardo M. F.; Popov, Alexander; Gerring, Matthew; McCarthy, Andrew A.; Gotz, Andy

    2012-01-01

    The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal re­orientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE. PMID:22868763

  13. Effects of sound exposure on the growth and intracellular macromolecular synthesis of E. coli k-12.

    PubMed

    Gu, Shaobin; Zhang, Yongzhu; Wu, Ying

    2016-01-01

    Microbes, as one of the primary producers of the biosphere, play an important role in ecosystems. Exploring the mechanism of adaptation and resistance of microbial population to various environmental factors has come into focus in the fields of modern microbial ecology and molecular ecology. However, facing the increasingly serious problem of acoustic pollution, very few efforts have been put forth into studying the relation of single cell organisms and sound field exposure. Herein, we studied the biological effects of sound exposure on the growth of E. coli K-12 with different acoustic parameters. The effects of sound exposure on the intracellular macromolecular synthesis and cellular morphology of E. coli K-12 were also analyzed and discussed. Experimental results indicated that E. coli K-12 exposed to sound waves owned a higher biomass and a faster specific growth rate compared to the control group. Also, the average length of E. coli K-12 cells increased more than 27.26%. The maximum biomass and maximum specific growth rate of the stimulation group by 8000 Hz, 80dB sound wave was about 1.7 times and 2.5 times that of the control group, respectively. Moreover, it was observed that E. coli K-12 can respond rapidly to sound stress at both the transcriptional and posttranscriptional levels by promoting the synthesis of intracellular RNA and total protein. Some potential mechanisms may be involved in the responses of bacterial cells to sound stress.

  14. Discovering free energy basins for macromolecular systems via guided multiscale simulation.

    PubMed

    Sereda, Yuriy V; Singharoy, Abhishek B; Jarrold, Martin F; Ortoleva, Peter J

    2012-07-26

    An approach for the automated discovery of low free energy states of macromolecular systems is presented. The method does not involve delineating the entire free energy landscape but proceeds in a sequential free energy minimizing state discovery; i.e., it first discovers one low free energy state and then automatically seeks a distinct neighboring one. These states and the associated ensembles of atomistic configurations are characterized by coarse-grained variables capturing the large-scale structure of the system. A key facet of our approach is the identification of such coarse-grained variables. Evolution of these variables is governed by Langevin dynamics driven by thermal-average forces and mediated by diffusivities, both of which are constructed by an ensemble of short molecular dynamics runs. In the present approach, the thermal-average forces are modified to account for the entropy changes following from our knowledge of the free energy basins already discovered. Such forces guide the system away from the known free energy minima, over free energy barriers, and to a new one. The theory is demonstrated for lactoferrin, known to have multiple energy-minimizing structures. The approach is validated using experimental structures and traditional molecular dynamics. The method can be generalized to enable the interpretation of nanocharacterization data (e.g., ion mobility-mass spectrometry, atomic force microscopy, chemical labeling, and nanopore measurements).

  15. Denatured state structural property determines protein stabilization by macromolecular crowding: a thermodynamic and structural approach.

    PubMed

    Mittal, Shruti; Singh, Laishram Rajendrakumar

    2013-01-01

    Understanding of protein structure and stability gained to date has been acquired through investigations made under dilute conditions where total macromolecular concentration never surpasses 10 g l(-1). However, biological macromolecules are known to evolve and function under crowded intracellular environments that comprises of proteins, nucleic acids, ribosomes and carbohydrates etc. Crowded environment is known to result in altered biological properties including thermodynamic, structural and functional aspect of macromolecules as compared to the macromolecules present in our commonly used experimental dilute buffers (for example, Tris HCl or phosphate buffer). In this study, we have investigated the thermodynamic and structural consequences of synthetic crowding agent (Ficoll 70) on three different proteins (Ribonuclease-A, lysozyme and holo α-lactalbumin) at different pH values. We report here that the effect of crowding is protein dependent in terms of protein thermal stability and structure. We also observed that the structural characteristics of the denatured state determines if crowding will have an effect or not on the protein stability.

  16. Biological Macromolecular Structures Data from the RCSB Protein Data Bank (RCSB PDB)

    DOE Data Explorer

    The Research Collaboratory for Structural Bioinformatics (RCSB) is a non-profit consortium that works to improve understanding of the function of biological systems through the study of the 3-D structure of biological macromolecules. The RCSB PDB is one of three sites serving as deposition, data processing, and distribution sites of the Protein Data Bank Archive. Each site provides its own view of the primary data, thus providing a variety of tools and resources for the global community. RCSB is also the official keeper for the PDB archive, with sole access authority to the PDB archive directory structure and contents. The RCSB PDB Information Portal for Biological Macromolecular Structures offers online tools for search and retrieval, for visualizing structures, for depositing, validating, or downloading data, news and highlights, a discussion forum, and links to other areas of related research. The PDB archive is a repository of atomic coordinates and other information describing proteins and other important biological macromolecules. Structural biologists use methods such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy to determine the location of each atom relative to each other in the molecule. They then deposit this information, which is then annotated and publicly released into the archive by the wwPDB. Results can be viewed as 3-D images or models.

  17. Effects of Macromolecular Crowding on Biochemical Reaction Equilibria: A Molecular Thermodynamic Perspective

    PubMed Central

    Hu, Zhongqiao; Jiang, Jianwen; Rajagopalan, Raj

    2007-01-01

    A molecular thermodynamic model is developed to investigate the effects of macromolecular crowding on biochemical reactions. Three types of reactions, representing protein folding/conformational isomerization, coagulation/coalescence, and polymerization/association, are considered. The reactants, products, and crowders are modeled as coarse-grained spherical particles or as polymer chains, interacting through hard-sphere interactions with or without nonbonded square-well interactions, and the effects of crowder size and chain length as well as product size are examined. The results predicted by this model are consistent with experimentally observed crowding effects based on preferential binding or preferential exclusion of the crowders. Although simple hard-core excluded-volume arguments do in general predict the qualitative aspects of the crowding effects, the results show that other intermolecular interactions can substantially alter the extent of enhancement or reduction of the equilibrium and can even change the direction of the shift. An advantage of the approach presented here is that competing reactions can be incorporated within the model. PMID:17513384

  18. Using support vector machines to improve elemental ion identification in macromolecular crystal structures

    DOE PAGES

    Morshed, Nader; Echols, Nathaniel; Adams, Paul D.

    2015-04-25

    In the process of macromolecular model building, crystallographers must examine electron density for isolated atoms and differentiate sites containing structured solvent molecules from those containing elemental ions. This task requires specific knowledge of metal-binding chemistry and scattering properties and is prone to error. A method has previously been described to identify ions based on manually chosen criteria for a number of elements. Here, the use of support vector machines (SVMs) to automatically classify isolated atoms as either solvent or one of various ions is described. Two data sets of protein crystal structures, one containing manually curated structures deposited with anomalousmore » diffraction data and another with automatically filtered, high-resolution structures, were constructed. On the manually curated data set, an SVM classifier was able to distinguish calcium from manganese, zinc, iron and nickel, as well as all five of these ions from water molecules, with a high degree of accuracy. Additionally, SVMs trained on the automatically curated set of high-resolution structures were able to successfully classify most common elemental ions in an independent validation test set. This method is readily extensible to other elemental ions and can also be used in conjunction with previous methods based on a priori expectations of the chemical environment and X-ray scattering.« less

  19. Macromolecular Concentrations in Bovine Nasal Cartilage by Fourier Transform Infrared Imaging and Principal Component Regression

    PubMed Central

    Yin, Jianhua; Xia, Yang

    2011-01-01

    Fourier transform infrared imaging (FT-IRI) technique and principal component regression (PCR) method were used to quantitatively determine collagen and proteoglycan concentrations in bovine nasal cartilage (BNC). An infrared spectral library was first established by obtaining eleven infrared spectra from a series of collagen and chondroitin 6-sulphate mixed in different ratios. FT-IR images were obtained from 6 μm thick sections of BNC specimens at 6.25 μm pixel size. The spectra from the FT-IR images were imported into a PCR program to obtain the relative concentrations of collagen and proteoglycan in BNC, based on the spectral library of pure chemicals. These PCR-determined concentrations agreed with the molecular concentrations determined biochemically using an enzyme digestion assay. Use of the imaging approach revealed that proteoglycan loss in the specimens occurs first at the surface of the tissue block when compared with the middle portion of the tissue block. The quantitative correlation of collagen and proteoglycan revealed that their infrared absorption peak-areas at 1338 and 1072-855 cm−1 can only be used as qualitative indicators of the molecular contents. The use of PCR in FT-IRI offers an accurate tool to spatially determine the distributions of macromolecular concentration in cartilage. PMID:21073787

  20. Characterization of PEG-Like Macromolecular Coatings on Plasma Modified NiTi Alloy

    NASA Astrophysics Data System (ADS)

    Yang, Jun; Gao, Jiacheng; Chang, Peng; Wang, Jianhua

    2008-04-01

    A poly (ethylene glycol) (PEG-like) coating was developed to improve the biocompatibility of Nickel-Titanium (NiTi) alloy implants. The PEG-like macromolecular coatings were deposited on NiTi substrates at a room temperature of 298 K through a ECR (electron-cyclotron resonance) cold-plasma enhanced chemical vapor deposition method using tetraglyme (CH3-O-(CH2-CH2-O)4-CH3) as a precursor. A power supply with a frequency of 2.45 GHz was applied to ignite the plasma with Ar(argon) used as the carrier gas. Based on the atomic force microscopy (AFM) studies, a thin smooth coating on NiTi substrates with highly amorphous functional groups on the modified NiTi surfaces were mainly the same accumulated stoichiometric ratio of C and O with PEG. The vitro studies showed that platelet-rich plasma (PRP) adsorption on the modified NiTi alloy surface was significantly reduced. This study indicated that plasma surface modification changes the surface components of NiTi alloy and subsequently improves its biocompatibility.

  1. One-Micron Beams for Macromolecular Crystallography at GM/CA-CAT

    SciTech Connect

    Yoder, D. W.; Sanishvili, R.; Xu, S.; Makarov, O.; Benn, R.; Corcoran, S.; Fischetti, R. F.; Vogt, S.

    2010-06-23

    GM/CA-CAT has developed a 1-{mu}m beam for challenging micro-diffraction experiments with macromolecular crystals (e.g. small crystals) and for radiation damage studies. Reflective (Kirkpatrick-Baez mirrors) and diffractive (Fresnel zone plates) optics have been used to focus the beam. Both cases are constrained by the need to maintain a small beam convergence. Using two different zone plates, 1.0x1.0 and 0.8x0.9 {mu}m{sup 2} (VxH,FWHM) beams were created at 15.2 keV and 18.5 keV, respectively. Additionally, by introducing a vertical focusing mirror upstream of the zone plate, a line focus at 15.2 keV was created (28x1.4 {mu}m{sup 2} VxH,FWHM) with the line oriented perpendicular to the X-ray polarization and the crystal rotation axis. Crystal-mounting stages with nanometer resolution have been assembled to profile these beams and to perform diffraction experiments.

  2. Distinct Contribution of Electrostatics, Initial Conformational Ensemble, and Macromolecular Stability in RNA Folding

    SciTech Connect

    Laederach,A.; Shcherbakova, I.; Jonikas, M.; Altman, R.; Brenowitz, M.

    2007-01-01

    We distinguish the contribution of the electrostatic environment, initial conformational ensemble, and macromolecular stability on the folding mechanism of a large RNA using a combination of time-resolved 'Fast Fenton' hydroxyl radical footprinting and exhaustive kinetic modeling. This integrated approach allows us to define the folding landscape of the L-21 Tetrahymena thermophila group I intron structurally and kinetically from its earliest steps with unprecedented accuracy. Distinct parallel pathways leading the RNA to its native form upon its Mg2+-induced folding are observed. The structures of the intermediates populating the pathways are not affected by variation of the concentration and type of background monovalent ions (electrostatic environment) but are altered by a mutation that destabilizes one domain of the ribozyme. Experiments starting from different conformational ensembles but folding under identical conditions show that whereas the electrostatic environment modulates molecular flux through different pathways, the initial conformational ensemble determines the partitioning of the flux. This study showcases a robust approach for the development of kinetic models from collections of local structural probes.

  3. Using support vector machines to improve elemental ion identification in macromolecular crystal structures

    SciTech Connect

    Morshed, Nader; Echols, Nathaniel; Adams, Paul D.

    2015-04-25

    In the process of macromolecular model building, crystallographers must examine electron density for isolated atoms and differentiate sites containing structured solvent molecules from those containing elemental ions. This task requires specific knowledge of metal-binding chemistry and scattering properties and is prone to error. A method has previously been described to identify ions based on manually chosen criteria for a number of elements. Here, the use of support vector machines (SVMs) to automatically classify isolated atoms as either solvent or one of various ions is described. Two data sets of protein crystal structures, one containing manually curated structures deposited with anomalous diffraction data and another with automatically filtered, high-resolution structures, were constructed. On the manually curated data set, an SVM classifier was able to distinguish calcium from manganese, zinc, iron and nickel, as well as all five of these ions from water molecules, with a high degree of accuracy. Additionally, SVMs trained on the automatically curated set of high-resolution structures were able to successfully classify most common elemental ions in an independent validation test set. This method is readily extensible to other elemental ions and can also be used in conjunction with previous methods based on a priori expectations of the chemical environment and X-ray scattering.

  4. Using support vector machines to improve elemental ion identification in macromolecular crystal structures

    SciTech Connect

    Morshed, Nader; Echols, Nathaniel; Adams, Paul D.

    2015-05-01

    A method to automatically identify possible elemental ions in X-ray crystal structures has been extended to use support vector machine (SVM) classifiers trained on selected structures in the PDB, with significantly improved sensitivity over manually encoded heuristics. In the process of macromolecular model building, crystallographers must examine electron density for isolated atoms and differentiate sites containing structured solvent molecules from those containing elemental ions. This task requires specific knowledge of metal-binding chemistry and scattering properties and is prone to error. A method has previously been described to identify ions based on manually chosen criteria for a number of elements. Here, the use of support vector machines (SVMs) to automatically classify isolated atoms as either solvent or one of various ions is described. Two data sets of protein crystal structures, one containing manually curated structures deposited with anomalous diffraction data and another with automatically filtered, high-resolution structures, were constructed. On the manually curated data set, an SVM classifier was able to distinguish calcium from manganese, zinc, iron and nickel, as well as all five of these ions from water molecules, with a high degree of accuracy. Additionally, SVMs trained on the automatically curated set of high-resolution structures were able to successfully classify most common elemental ions in an independent validation test set. This method is readily extensible to other elemental ions and can also be used in conjunction with previous methods based on a priori expectations of the chemical environment and X-ray scattering.

  5. Using support vector machines to improve elemental ion identification in macromolecular crystal structures.

    PubMed

    Morshed, Nader; Echols, Nathaniel; Adams, Paul D

    2015-05-01

    In the process of macromolecular model building, crystallographers must examine electron density for isolated atoms and differentiate sites containing structured solvent molecules from those containing elemental ions. This task requires specific knowledge of metal-binding chemistry and scattering properties and is prone to error. A method has previously been described to identify ions based on manually chosen criteria for a number of elements. Here, the use of support vector machines (SVMs) to automatically classify isolated atoms as either solvent or one of various ions is described. Two data sets of protein crystal structures, one containing manually curated structures deposited with anomalous diffraction data and another with automatically filtered, high-resolution structures, were constructed. On the manually curated data set, an SVM classifier was able to distinguish calcium from manganese, zinc, iron and nickel, as well as all five of these ions from water molecules, with a high degree of accuracy. Additionally, SVMs trained on the automatically curated set of high-resolution structures were able to successfully classify most common elemental ions in an independent validation test set. This method is readily extensible to other elemental ions and can also be used in conjunction with previous methods based on a priori expectations of the chemical environment and X-ray scattering.

  6. Interaction of wall shear stress magnitude and gradient in the prediction of arterial macromolecular permeability.

    PubMed

    LaMack, Jeffrey A; Himburg, Heather A; Li, Xue-Mei; Friedman, Morton H

    2005-04-01

    Large spatial shear stress gradients have anecdotally been associated with early atherosclerotic lesion susceptibility in vivo and have been proposed as promoters of endothelial cell dysfunction in vitro. Here, experiments are presented in which several measures of the fluid dynamic shear stress, including its gradient, at the walls of in vivo porcine iliac arteries, are correlated against the transendothelial macromolecular permeability of the vessels. The fluid dynamic measurements are based on postmortem vascular casts, and permeability is measured from Evans blue dye (EBD) uptake. Time-averaged wall shear stress (WSS), as well as a new parameter termed maximum gradient stress (MGS) that describes the spatial shear stress gradient due to flow acceleration at a given point, are mapped for each artery and compared on a point-by-point basis to the corresponding EBD patterns. While there was no apparent relation between MGS and EBD uptake, a composite parameter, WSS(-0.11) MGS(0.044), was highly correlated with permeability. Notwithstanding the small exponents, the parameter varied widely within the region of interest. The results suggest that sites exposed to low wall shear stresses are more likely to exhibit elevated permeability, and that this increase is exacerbated in the presence of large spatial shear stress gradients.

  7. Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching.

    PubMed Central

    Gribbon, P; Hardingham, T E

    1998-01-01

    Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically. PMID:9675204

  8. Polydimethylsiloxane as a Macromolecular Additive for Enhanced Performance of Molecular Bulk Heterojunction Organic Solar Cells

    SciTech Connect

    Graham, Kenneth R.; Mei, Jianguo; Stalder, Romain; Shim, Jae Won; Cheun, Hyeunseok; Steffy, Fred; So, Franky; Kippelen, Bernard; Reynolds, John R.

    2011-03-15

    The effect of the macromolecular additive, polydimethylsiloxane (PDMS), on the performance of solution processed molecular bulk heterojunction solar cells is investigated, and the addition of PDMS is shown to improve device power conversion efficiency by ~70% and significantly reduce cell-to-cell variation, from a power conversion efficiency of 1.25 ± 0.37% with no PDMS to 2.16 ± 0.09% upon the addition of 0.1 mg/mL PDMS to the casting solution. The cells are based on a thiophene and isoindigo containing oligomer as the electron donor and [6,6]-phenyl-C61 butyric acid methyl ester (PC61BM) as the electron acceptor. PDMS is shown to have a strong influence on film morphology, with a significant decrease in film roughness and feature size observed. The morphology change leads to improved performance parameters, most notably an increase in the short circuit current density from 4.3 to 6.8 mA/cm2 upon addition of 0.1 mg/mL PDMS. The use of PDMS is of particular interest, as this additive appears frequently as a lubricant in plastic syringes commonly used in device fabrication; therefore, PDMS may unintentionally be incorporated into device active layers.

  9. Recognition memory reveals just how CONTRASTIVE contrastive accenting really is

    PubMed Central

    Fraundorf, Scott H.; Watson, Duane G.; Benjamin, Aaron S.

    2010-01-01

    The effects of pitch accenting on memory were investigated in three experiments. Participants listened to short recorded discourses that contained contrast sets with two items (e.g. British scientists and French scientists); a continuation specified one item from the set. Pitch accenting on the critical word in the continuation was manipulated between non-contrastive (H* in the ToBI system) and contrastive (L+H*). On subsequent recognition memory tests, the L+H* accent increased hits to correct statements and correct rejections of the contrast item (Experiments 1–3), but did not impair memory for other parts of the discourse (Experiment 2). L+H* also did not facilitate correct rejections of lures not in the contrast set (Experiment 3), indicating that contrastive accents do not simply strengthen the representation of the target item. These results suggest comprehenders use pitch accenting to encode and update information about multiple elements in a contrast set. PMID:20835405

  10. Wave propagation in a strongly heterogeneous elastic porous medium: Homogenization of Biot medium with double porosities

    NASA Astrophysics Data System (ADS)

    Rohan, Eduard; Naili, Salah; Nguyen, Vu-Hieu

    2016-08-01

    We study wave propagation in an elastic porous medium saturated with a compressible Newtonian fluid. The porous network is interconnected whereby the pores are characterized by two very different characteristic sizes. At the mesoscopic scale, the medium is described using the Biot model, characterized by a high contrast in the hydraulic permeability and anisotropic elasticity, whereas the contrast in the Biot coupling coefficient is only moderate. Fluid motion is governed by the Darcy flow model extended by inertia terms and by the mass conservation equation. The homogenization method based on the asymptotic analysis is used to obtain a macroscopic model. To respect the high contrast in the material properties, they are scaled by the small parameter, which is involved in the asymptotic analysis and characterized by the size of the heterogeneities. Using the estimates of wavelengths in the double-porosity networks, it is shown that the macroscopic descriptions depend on the contrast in the static permeability associated with pores and micropores and on the frequency. Moreover, the microflow in the double porosity is responsible for fading memory effects via the macroscopic poroviscoelastic constitutive law. xml:lang="fr"

  11. Contrastive Analysis and Language Development.

    ERIC Educational Resources Information Center

    Ferguson, Charles A.

    Contrastive analysis is basic to all linguistics since only by this approach can a general theory of language (language universals) be constructed and only with at least implicit contrastive analysis can a particular language be fully characterized. Two kinds of contrastive analysis have been basic to diachronic linguistics: the comparison of…

  12. Endoscopy imaging intelligent contrast improvement.

    PubMed

    Sheraizin, S; Sheraizin, V

    2005-01-01

    In this paper, we present a medical endoscopy video contrast improvement method that provides intelligent automatic adaptive contrast control. The method fundamentals are video data clustering and video data histogram modification. The video data clustering allows an effective use the low noise two channel contrast enhancement processing. The histogram analysis permitted to determine the video exposure type for simple and complicated contrast distribution. We determined the needed gamma value for automatic local area contrast improvement for the following exposure types: dark, normal, light, dark light, dark normal etc. The experimental results of medical endoscopy video processing allow defining the automatic gamma control range from 0.5 to 2.0.

  13. Double contrast X-ray examination of stomach.

    PubMed

    Wiljasalo, M; Wiljasalo, S; Valle, M; Tallroth, K; Korhola, O; Somer, K

    1977-01-01

    A comparison was made between three double contrast methods and the conventional method of X-ray examination of the stomach. 106 patets received ordinary barium sulphate together with effervescent granules. 119 patients were examined with a special barium sulphate preparation containing dissolved carbon dioxide (Baritop). The third group, 100 patients, received Baritop and effervescent granules, and 100 patients a conventional barium meal. The films were analyzed as to the quality of mucosal pattern demonstration, mucosal affinity of the contrast medium, and the degree of dilatation of the stomach and duo8num. In addition, the degree of interference by gas bubbles was estimated. The best results were obtained with the contrast medium containing carbon dioxide (Baritop).

  14. The in vivo relaxivity of MRI contrast agents

    NASA Astrophysics Data System (ADS)

    Shuter, Borys

    1999-11-01

    Post-contrast clinical 1H Magnetic Resonance Images have to date been interpreted with little regard for possible variations in the in-vivo properties of injected magnetic pharmaceuticals (contrast agents), particularly in their relaxivity or ability to alter tissue relaxation rates, T2-1 and T 2-1, per unit concentration. The relaxivities of contrast agents have only rarely been measured in-vivo, measurements usually being performed on excised tissues and at magnetic field strengths lower than used in clinical practice. Some researchers have simply assumed that relaxivities determined in homogeneous tissue phantoms were applicable in-vivo. In this thesis, the relaxivities of two contrast agents, Gd-DTPA and Gd-EOB-DTPA, were measured in simple tissue phantoms and in the kidney and liver of intact, but sacrificed, Wistar rats using a clinical MR scanner with a magnetic field of 1.5 Tesla. T1 and T2 were determined from sets of images acquired using a standard clinical spin-echo pulse sequence. The contrast agent concentration in tissue was assessed by radioassay of 153Gd-DTPA or 153Gd-EOB-DTPA, mixed with the normal compound prior to injection. Relaxivity was taken as the slope of a linear regression fit of relaxation rate against Gd concentration. The relaxivities of Gd-EOB-DTPA were similarly determined in normal and biliary- obstructed guinea pigs. Relaxivities in tissue differed significantly from values obtained in simple phantoms. Kidney T1 relaxivity was reduced for both compounds in normal animals. Three days or more of biliary obstruction produced further reductions in kidney T1 relaxivity of Gd-EOB-DTPA, providing strong evidence that disease affects contrast agent relaxivity. Kidney T2 relaxivity was much greater than T1 relaxivity and was also depressed by biliary obstruction. Liver T1 and T 2 relaxivites were increased above phantom values, but were not affected by the biliary obstruction. Water compartmentalisation, macromolecular binding, proton

  15. The influence of turbid medium properties on object visibility in optical Kerr gated imaging

    NASA Astrophysics Data System (ADS)

    Zhan, Pingping; Si, Jinhai; Tan, Wenjiang; Liu, Xin; Wu, Bin; Xu, Shichao; Chen, Feng; Hou, Xun

    2014-01-01

    In this paper, we demonstrate femtosecond optical Kerr gated imaging of an object hidden behind a highly turbid medium. The influence of turbid medium properties on image contrast has been investigated. Experimental and Monte Carlo simulation results show that for a given optical density, the image contrast of direct imaging without an optical Kerr gate decreases with the increase of the scattering particle size or the decrease of the thickness of the turbid medium. Compared with direct imaging, optical Kerr gated imaging has a better image contrast as it eliminates more scattered photons effectively. Qualitative comparisons between experiments and simulations show good agreement.

  16. Chemico-biological interaction of Etravirine and its β-Cyclodextrin complex with macromolecular targets.

    PubMed

    Yousuf, Sameena; Natesan, Sudha; Enoch, Israel V M V

    2017-04-01

    The interaction of etravirine with β-cyclodextrin is analyzed by UV-visible absorption, infrared, fluorescence, nuclear magnetic resonance, two-dimensional rotational frame nuclear Overhauser effect spectroscopy, and molecular modeling studies. The 4-hydroxy-3, 5-dimethylbenzonitrile moiety is found to take part in the binding. The stoichiometry of the inclusion complex of ET with β-CD is 1:1 with the binding constant of 2.03 × 10(3) mol(-1) dm(3). The binding of ET with calf thymus DNA (ctDNA) and bovine serum albumin (BSA) protein is investigated in the presence and the absence of β-CD. Fluorescence enhancement is observed during the binding of ET with ctDNA in the absence of β-CD, whereas in the presence of β-CD, fluorescence quenching is observed. The binding constants of the binding of ET and ET-β-CD to ctDNA are 7.84 × 10(4) and 4.38 × 10(4) mol(-1) dm(3), respectively. The binding constant of the binding of ET and ET-β-CD to BSA are 3.14 × 10(4) and 1.6396 × 10(4) mol(-1) dm(3), respectively. The apparent binding constants between ET-β-CD complex and ctDNA or BSA protein decreases significantly. The numbers of binding sites of interaction of ET with BSA protein and the binding distance between BSA protein and ET the absence and the presence of β-CD differ. β-CD modulates the binding of ET with the macromolecular targets.

  17. Role of Heterogeneous Macromolecular Crowding and Geometrical Irregularity at Central Excitatory Synapses in Shaping Synaptic Transmission

    PubMed Central

    Gupta, Rahul; Reneaux, Melissa; Karmeshu

    2016-01-01

    Besides the geometrical tortousity due to the extrasynaptic structures, macromolecular crowding and geometrical irregularities constituting the cleft composition at central excitatory synapses has a major and direct role in retarding the glutamate diffusion within the cleft space. However, the cleft composition may not only coarsely reduce the overall diffusivity of the glutamate but may also lead to substantial spatial variation in the diffusivity across the cleft space. Decrease in the overall diffusivity of the glutamate may have straightforward consequences to the glutamate transients in the cleft. However, how spatial variation in the diffusivity may further affect glutamate transients is an intriguing aspect. Therefore, to understand the role of cleft heterogeneity, the present study adopts a novel approach of glutamate diffusion which considers a gamma statistical distribution of the diffusion coefficient of glutamate (Dglut) across the cleft space, such that its moments discernibly capture the dual impacts of the cleft composition, and further applies the framework of superstatistics. The findings reveal a power law behavior in the glutamate transients, akin to the long-range anomalous subdiffusion, which leads to slower decay profile of cleft glutamate at higher intensity of cleft heterogeneity. Moreover, increase in the cleft heterogeneity is seen to eventually cause slower-rising excitatory postsynaptic currents with higher amplitudes, lesser noise, and prolonged duration of charge transfer across the postsynaptic membrane. Further, with regard to the conventional standard diffusion approach, the study suggests that the effective Dglut essentially derives from the median of the Dglut distribution and does not necessarily need to be the mean Dglut. Together, the findings indicate a strong implication of cleft heterogeneity to the metabolically cost-effective tuning of synaptic response during the phenomenon of plasticity at individual synapses and also

  18. Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering

    PubMed Central

    Roedig, Philip; Duman, Ramona; Sanchez-Weatherby, Juan; Vartiainen, Ismo; Burkhardt, Anja; Warmer, Martin; David, Christian; Wagner, Armin; Meents, Alke

    2016-01-01

    Recent success at X-ray free-electron lasers has led to serial crystallography experiments staging a comeback at synchrotron sources as well. With crystal lifetimes typically in the millisecond range and the latest-generation detector technologies with high framing rates up to 1 kHz, fast sample exchange has become the bottleneck for such experiments. A micro-patterned chip has been developed from single-crystalline silicon, which acts as a sample holder for up to several thousand microcrystals at a very low background level. The crystals can be easily loaded onto the chip and excess mother liquor can be efficiently removed. Dehydration of the crystals is prevented by keeping them in a stream of humidified air during data collection. Further sealing of the sample holder, for example with Kapton, is not required. Room-temperature data collection from insulin crystals loaded onto the chip proves the applicability of the chip for macromolecular crystallography. Subsequent structure refinements reveal no radiation-damage-induced structural changes for insulin crystals up to a dose of 565.6 kGy, even though the total diffraction power of the crystals has on average decreased to 19.1% of its initial value for the same dose. A decay of the diffracting power by half is observed for a dose of D 1/2 = 147.5 ± 19.1 kGy, which is about 1/300 of the dose before crystals show a similar decay at cryogenic temperatures. PMID:27275143

  19. Oxytocin regulates gastrointestinal motility, inflammation, macromolecular permeability, and mucosal maintenance in mice

    PubMed Central

    Margolis, Kara G.; Li, Zhishan; Gershon, Michael D.

    2014-01-01

    Enteric neurons express oxytocin (OT); moreover, enteric neurons and enterocytes express developmentally regulated OT receptors (OTRs). Although OT (with secretin) opposes intestinal inflammation, physiological roles played by enteric OT/OTR signaling have not previously been determined. We tested hypotheses that OT/OTR signaling contributes to enteric nervous system (ENS)-related gastrointestinal (GI) physiology. GI functions and OT effects were compared in OTR-knockout (OTRKO) and wild-type (WT) mice. Stool mass and water content were greater in OTRKO mice than in WT. GI transit time in OTRKO animals was faster than in WT; OT inhibited in vitro generation of ENS-dependent colonic migrating motor complexes in WT but not in OTRKO mice. Myenteric neurons were hyperplastic in OTRKO animals, and mucosal exposure to cholera toxin (CTX) in vitro activated Fos in more myenteric neurons in OTRKO than WT than in WT mice; OT inhibited the CTX response in WT but not in OTRKO mice. Villi and crypts were shorter in OTRKO than in WT mice, and transit-amplifying cell proliferation in OTRKO crypts was deficient. Macromolecular intestinal permeability in OTRKO was greater than WT mice, and experimental colitis was more severe in OTRKO mice; moreover, OT protected WT animals from colitis. Observations suggest that OT/OTR signaling acts as a brake on intestinal motility, decreases mucosal activation of enteric neurons, and promotes enteric neuronal development and/or survival. It also regulates proliferation of crypt cells and mucosal permeability; moreover OT/OTR signaling is protective against inflammation. Oxytocinergic signaling thus appears to play an important role in multiple GI functions that are subject to neuronal regulation. PMID:25147234

  20. Temperature Sensitivity as a Microbial Trait Using Parameters from Macromolecular Rate Theory

    PubMed Central

    Alster, Charlotte J.; Baas, Peter; Wallenstein, Matthew D.; Johnson, Nels G.; von Fischer, Joseph C.

    2016-01-01

    The activity of soil microbial extracellular enzymes is strongly controlled by temperature, yet the degree to which temperature sensitivity varies by microbe and enzyme type is unclear. Such information would allow soil microbial enzymes to be incorporated in a traits-based framework to improve prediction of ecosystem response to global change. If temperature sensitivity varies for specific soil enzymes, then determining the underlying causes of variation in temperature sensitivity of these enzymes will provide fundamental insights for predicting nutrient dynamics belowground. In this study, we characterized how both microbial taxonomic variation as well as substrate type affects temperature sensitivity. We measured β-glucosidase, leucine aminopeptidase, and phosphatase activities at six temperatures: 4, 11, 25, 35, 45, and 60°C, for seven different soil microbial isolates. To calculate temperature sensitivity, we employed two models, Arrhenius, which predicts an exponential increase in reaction rate with temperature, and Macromolecular Rate Theory (MMRT), which predicts rate to peak and then decline as temperature increases. We found MMRT provided a more accurate fit and allowed for more nuanced interpretation of temperature sensitivity in all of the enzyme × isolate combinations tested. Our results revealed that both the enzyme type and soil isolate type explain variation in parameters associated with temperature sensitivity. Because we found temperature sensitivity to be an inherent and variable property of an enzyme, we argue that it can be incorporated as a microbial functional trait, but only when using the MMRT definition of temperature sensitivity. We show that the Arrhenius metrics of temperature sensitivity are overly sensitive to test conditions, with activation energy changing depending on the temperature range it was calculated within. Thus, we propose the use of the MMRT definition of tempera