Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

PubMed Central

Lew, Carolyn Ritterson; Tolan, Dean R.

2012-01-01

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer. PMID:23093405

A novel approach: high resolution inspection with wafer plane defect detection

NASA Astrophysics Data System (ADS)

Hess, Carl; Wihl, Mark; Shi, Rui-fang; Xiong, Yalin; Pang, Song

2008-05-01

High Resolution reticle inspection is well-established as a proven, effective, and efficient means of detecting yield-limiting mask defects as well as defects which are not immediately yield-limiting yet can enable manufacturing process improvements. Historically, RAPID products have enabled detection of both classes of these defects. The newly-developed Wafer Plane Inspection (WPI) detector technology meets the needs of some advanced mask manufacturers to identify the lithographically-significant defects while ignoring the other non-lithographically-significant defects. Wafer Plane Inspection accomplishes this goal by performing defect detection based on a modeled image of how the mask features would actually print in the photoresist. This has the effect of reducing sensitivity to non-printing defects while enabling higher sensitivity focused in high MEEF areas where small reticle defects still yield significant printing defects on wafers. WPI is a new inspection mode that has been developed by KLA-Tencor and is currently under test with multiple customers. It employs the same transmitted and reflected-light high-resolution images as the industry-standard high-resolution inspections, but with much more sophisticated processing involved. A rigorous mask pattern recovery algorithm is used to convert the transmitted and reflected light images into a modeled representation of the reticle. Lithographic modeling of the scanner is then used to generate an aerial image of the mask. This is followed by resist modeling to determine the exposure of the photoresist. The defect detectors are then applied on this photoresist plane so that only printing defects are detected. Note that no hardware modifications to the inspection system are required to enable this detector. The same tool will be able to perform both our standard High Resolution inspections and the Wafer Plane Inspection detector. This approach has several important features. The ability to ignore non-printing defects and to apply additional effective sensitivity in high MEEF areas enables advanced node development. In addition, the modeling allows the inclusion of important polarization effects that occur in the resist for high NA operation. This allows for the results to better match wafer print results compared to alternate approaches. Finally, the simulation easily allows for the application of arbitrary illumination profiles. With this approach, users of WPI can make use of unique or custom scanner illumination profiles. This allows the more precise modeling of profiles without inspection system hardware modification or loss of company intellectual property. This paper examines WPI in Die:Die mode. Future work includes a review of Die:Database WPI capability.

High-throughput automatic defect review for 300mm blank wafers with atomic force microscope

NASA Astrophysics Data System (ADS)

Zandiatashbar, Ardavan; Kim, Byong; Yoo, Young-kook; Lee, Keibock; Jo, Ahjin; Lee, Ju Suk; Cho, Sang-Joon; Park, Sang-il

2015-03-01

While feature size in lithography process continuously becomes smaller, defect sizes on blank wafers become more comparable to device sizes. Defects with nm-scale characteristic size could be misclassified by automated optical inspection (AOI) and require post-processing for proper classification. Atomic force microscope (AFM) is known to provide high lateral and the highest vertical resolution by mechanical probing among all techniques. However, its low throughput and tip life in addition to the laborious efforts for finding the defects have been the major limitations of this technique. In this paper we introduce automatic defect review (ADR) AFM as a post-inspection metrology tool for defect study and classification for 300 mm blank wafers and to overcome the limitations stated above. The ADR AFM provides high throughput, high resolution, and non-destructive means for obtaining 3D information for nm-scale defect review and classification.

Marvels of enzyme catalysis at true atomic resolution: distortions, bond elongations, hidden flips, protonation states and atom identities.

PubMed

Neumann, Piotr; Tittmann, Kai

2014-12-01

Although general principles of enzyme catalysis are fairly well understood nowadays, many important details of how exactly the substrate is bound and processed in an enzyme remain often invisible and as such elusive. In fortunate cases, structural analysis of enzymes can be accomplished at true atomic resolution thus making possible to shed light on otherwise concealed fine-structural traits of bound substrates, intermediates, cofactors and protein groups. We highlight recent structural studies of enzymes using ultrahigh-resolution X-ray protein crystallography showcasing its enormous potential as a tool in the elucidation of enzymatic mechanisms and in unveiling fundamental principles of enzyme catalysis. We discuss the observation of seemingly hyper-reactive, physically distorted cofactors and intermediates with elongated scissile substrate bonds, the detection of 'hidden' conformational and chemical equilibria and the analysis of protonation states with surprising findings. In delicate cases, atomic resolution is required to unambiguously disclose the identity of atoms as demonstrated for the metal cluster in nitrogenase. In addition to the pivotal structural findings and the implications for our understanding of enzyme catalysis, we further provide a practical framework for resolution enhancement through optimized data acquisition and processing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mass-transfer limitations for immobilized enzyme-catalyzed kinetic resolution of racemate in a fixed-bed reactor.

PubMed

Xiu, G H; Jiang, L; Li, P

2001-07-05

A mathematical model has been developed for immobilized enzyme-catalyzed kinetic resolution of racemate in a fixed-bed reactor in which the enzyme-catalyzed reaction (the irreversible uni-uni competitive Michaelis-Menten kinetics is chosen as an example) was coupled with intraparticle diffusion, external mass transfer, and axial dispersion. The effects of mass-transfer limitations, competitive inhibition of substrates, deactivation on the enzyme effective enantioselectivity, and the optical purity and yield of the desired product are examined quantitatively over a wide range of parameters using the orthogonal collocation method. For a first-order reaction, an analytical solution is derived from the mathematical model for slab-, cylindrical-, and spherical-enzyme supports. Based on the analytical solution for the steady-state resolution process, a new concise formulation is presented to predict quantitatively the mass-transfer limitations on enzyme effective enantioselectivity and optical purity and yield of the desired product for a continuous steady-state kinetic resolution process in a fixed-bed reactor. Copyright 2001 John Wiley & Sons, Inc.

Computational simulation of subatomic-resolution AFM and STM images for graphene/hexagonal boron nitride heterostructures with intercalated defects

NASA Astrophysics Data System (ADS)

Lee, Junsu; Kim, Minjung; Chelikowsky, James R.; Kim, Gunn

2016-07-01

Using ab initio density functional calculations, we predict subatomic-resolution atomic force microscopy (AFM) and scanning tunneling microscopy (STM) images of vertical heterostructures of graphene/hexagonal boron nitride (h-BN) with an intercalated metal atom (Li, K, Cr, Mn, Co, or Cu), and study the effects of the extrinsic metal defect on the interfacial coupling. We find that the structural deformation of the graphene/h-BN layer caused by the metal defect strongly affects the AFM images, whereas orbital hybridization between the metal defect and the graphene/h-BN layer characterizes the STM images.

High Resolution Eddy-Current Wire Testing Based on a Gmr Sensor-Array

NASA Astrophysics Data System (ADS)

Kreutzbruck, Marc; Allweins, Kai; Strackbein, Chris; Bernau, Hendrick

2009-03-01

Increasing demands in materials quality and cost effectiveness have led to advanced standards in manufacturing technology. Especially when dealing with high quality standards in conjunction with high throughput quantitative NDE techniques are vital to provide reliable and fast quality control systems. In this work we illuminate a modern electromagnetic NDE approach using a small GMR sensor array for testing superconducting wires. Four GMR sensors are positioned around the wire. Each GMR sensor provides a field sensitivity of 200 pT/√Hz and a spatial resolution of about 100 μm. This enables us to detect under surface defects of 100 μm in size in a depth of 200 μm with a signal-to-noise ratio of better than 400. Surface defects could be detected with a SNR of up to 10,000. Besides this remarkably SNR the small extent of GMR sensors results in a spatial resolution which offers new visualisation techniques for defect localisation, defect characterization and tomography-like mapping techniques. We also report on inverse algorithms based on either a Finite Element Method or an analytical approach. These allow for accurate defect localization on the urn scale and an estimation of the defect size.

Classification and printability of EUV mask defects from SEM images

NASA Astrophysics Data System (ADS)

Cho, Wonil; Price, Daniel; Morgan, Paul A.; Rost, Daniel; Satake, Masaki; Tolani, Vikram L.

2017-10-01

Classification and Printability of EUV Mask Defects from SEM images EUV lithography is starting to show more promise for patterning some critical layers at 5nm technology node and beyond. However, there still are many key technical obstacles to overcome before bringing EUV Lithography into high volume manufacturing (HVM). One of the greatest obstacles is manufacturing defect-free masks. For pattern defect inspections in the mask-shop, cutting-edge 193nm optical inspection tools have been used so far due to lacking any e-beam mask inspection (EBMI) or EUV actinic pattern inspection (API) tools. The main issue with current 193nm inspection tools is the limited resolution for mask dimensions targeted for EUV patterning. The theoretical resolution limit for 193nm mask inspection tools is about 60nm HP on masks, which means that main feature sizes on EUV masks will be well beyond the practical resolution of 193nm inspection tools. Nevertheless, 193nm inspection tools with various illumination conditions that maximize defect sensitivity and/or main-pattern modulation are being explored for initial EUV defect detection. Due to the generally low signal-to-noise in the 193nm inspection imaging at EUV patterning dimensions, these inspections often result in hundreds and thousands of defects which then need to be accurately reviewed and dispositioned. Manually reviewing each defect is difficult due to poor resolution. In addition, the lack of a reliable aerial dispositioning system makes it very challenging to disposition for printability. In this paper, we present the use of SEM images of EUV masks for higher resolution review and disposition of defects. In this approach, most of the defects detected by the 193nm inspection tools are first imaged on a mask SEM tool. These images together with the corresponding post-OPC design clips are provided to KLA-Tencor's Reticle Decision Center (RDC) platform which provides ADC (Automated Defect Classification) and S2A (SEM-to-Aerial printability) analysis of every defect. First, a defect-free or reference mask SEM is rendered from the post-OPC design, and the defective signature is detected from the defect-reference difference image. These signatures help assess the true nature of the defect as evident in e-beam imaging; for example, excess or missing absorber, line-edge roughness, contamination, etc. Next, defect and reference contours are extracted from the grayscale SEM images and fed into the simulation engine with an EUV scanner model to generate corresponding EUV defect and reference aerial images. These are then analyzed for printability and dispositioned using an Aerial Image Analyzer (AIA) application to automatically measure and determine the amount of CD errors. Thus by integrating EUV ADC and S2A applications together, every defect detection is characterized for its type and printability which is essential for not only determining which defects to repair, but also in monitoring the performance of EUV mask process tools. The accuracy of the S2A print modeling has been verified with other commercially-available simulators, and will also be verified with actual wafer print results. With EUV lithography progressing towards volume manufacturing at 5nm technology, and the likelihood of EBMI inspectors approaching the horizon, the EUV ADC-S2A system will continue serving an essential role of dispositioning defects off e-beam imaging.

A view on sphingolipids and disease.

PubMed

Kolter, Thomas

2011-09-01

Sphingolipid and glycosphingolipid levels and expression of sphingolipid metabolizing enzymes are altered in a variety of diseases or in response to drug treatment. Inherited defects of enzymes and other proteins required for the lysosomal degradation of these lipids lead to human sphingolipidoses. Also genetic defects that affect sphingolipid biosynthesis are known. Although the molecular details are often far from clear, (glyco)sphingolipids have been implicated to play a role in atherosclerosis, insulin resistance, cancer, and infections by pathogens. More general aspects of selected diseases are discussed. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Collimator optimization in myocardial perfusion SPECT using the ideal observer and realistic background variability for lesion detection and joint detection and localization tasks

NASA Astrophysics Data System (ADS)

Ghaly, Michael; Du, Yong; Links, Jonathan M.; Frey, Eric C.

2016-03-01

In SPECT imaging, collimators are a major factor limiting image quality and largely determine the noise and resolution of SPECT images. In this paper, we seek the collimator with the optimal tradeoff between image noise and resolution with respect to performance on two tasks related to myocardial perfusion SPECT: perfusion defect detection and joint detection and localization. We used the Ideal Observer (IO) operating on realistic background-known-statistically (BKS) and signal-known-exactly (SKE) data. The areas under the receiver operating characteristic (ROC) and localization ROC (LROC) curves (AUCd, AUCd+l), respectively, were used as the figures of merit for both tasks. We used a previously developed population of 54 phantoms based on the eXtended Cardiac Torso Phantom (XCAT) that included variations in gender, body size, heart size and subcutaneous adipose tissue level. For each phantom, organ uptakes were varied randomly based on distributions observed in patient data. We simulated perfusion defects at six different locations with extents and severities of 10% and 25%, respectively, which represented challenging but clinically relevant defects. The extent and severity are, respectively, the perfusion defect’s fraction of the myocardial volume and reduction of uptake relative to the normal myocardium. Projection data were generated using an analytical projector that modeled attenuation, scatter, and collimator-detector response effects, a 9% energy resolution at 140 keV, and a 4 mm full-width at half maximum (FWHM) intrinsic spatial resolution. We investigated a family of eight parallel-hole collimators that spanned a large range of sensitivity-resolution tradeoffs. For each collimator and defect location, the IO test statistics were computed using a Markov Chain Monte Carlo (MCMC) method for an ensemble of 540 pairs of defect-present and -absent images that included the aforementioned anatomical and uptake variability. Sets of test statistics were computed for both tasks and analyzed using ROC and LROC analysis methodologies. The results of this study suggest that collimators with somewhat poorer resolution and higher sensitivity than those of a typical low-energy high-resolution (LEHR) collimator were optimal for both defect detection and joint detection and localization tasks in myocardial perfusion SPECT for the range of defect sizes investigated. This study also indicates that optimizing instrumentation for a detection task may provide near-optimal performance on the more challenging detection-localization task.

Eddy Current Testing for Detecting Small Defects in Thin Films

NASA Astrophysics Data System (ADS)

Obeid, Simon; Tranjan, Farid M.; Dogaru, Teodor

2007-03-01

Presented here is a technique of using Eddy Current based Giant Magneto-Resistance sensor (GMR) to detect surface and sub-layered minute defects in thin films. For surface crack detection, a measurement was performed on a copper metallization of 5-10 microns thick. It was done by scanning the GMR sensor on the surface of the wafer that had two scratches of 0.2 mm, and 2.5 mm in length respectively. In another experiment, metal coatings were deposited over the layers containing five defects with known lengths such that the defects were invisible from the surface. The limit of detection (resolution), in terms of defect size, of the GMR high-resolution Eddy Current probe was studied using this sample. Applications of Eddy Current testing include detecting defects in thin film metallic layers, and quality control of metallization layers on silicon wafers for integrated circuits manufacturing.

Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii1

PubMed Central

Dauvillée, David; Colleoni, Christophe; Shaw, Eudean; Mouille, Gregory; D'Hulst, Christophe; Morell, Matthew; Samuel, Michael S.; Bouchet, Brigitte; Gallant, Daniel J.; Sinskey, Anthony; Ball, Steven

1999-01-01

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii. PMID:9880375

Atomic Structure and Properties of Extended Defects in Silicon

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

Buczko, R.; Chisholm, M.F.; Kaplan, T.

1998-10-15

The Z-contrast technique represents a new approach to high-resolution electron microscopy allowing for the first time incoherent imaging of materials on the atomic scale. The key advantages of the technique, an intrinsically higher resolution limit and directly interpretable, compositionally sensitive imaging, allow a new level of insight into the atomic configurations of extended defects in silicon. This experimental technique has been combined with theoretical calculations (a combination of first principles, tight binding, and classical methods) to extend this level of insight by obtaining the energetic and electronic structure of the defects.

Lipoic acid biosynthesis defects.

PubMed

Mayr, Johannes A; Feichtinger, René G; Tort, Frederic; Ribes, Antonia; Sperl, Wolfgang

2014-07-01

Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metabolism, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metabolism, branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metabolism with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulphur-cluster-dependent enzyme, a number of recently identified defects in mitochondrial iron-sulphur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clinical spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.

Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding. alpha. -mannosidase II

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

Fukuda, M.N.; Masri, K.A.; Dell, A.

1990-10-01

Congenital dyserythropoietic anemia type II, or hereditary erythroblastic multinuclearity with a positive acidified-serum-lysis test (HEMPAS), is a genetic anemia in humans inherited by an autosomally recessive mode. The enzyme defect in most HEMPAS patients has previously been proposed as a lowered activity of N-acetylglucosaminyltransferase II, resulting in a lack of polylactosamine on proteins and leading to the accumulation of polylactosaminyl lipids. A recent HEMPAS case, G.C., has now been analyzed by cell-surface labeling, fast-atom-bombardment mass spectrometry of glycopeptides, and activity assay of glycosylation enzymes. Significantly decreased glycosylation of polylactosaminoglycan proteins and incompletely processed asparagine-linked oligosaccharides were detected in the erythrocytemore » membranes of G.C. These results suggest that G.C. cells contain a mutation in {alpha}-ManII-encoding gene that results in inefficient expression of {alpha}-ManII mRNA, either through reduced transcription or message instability. This report demonstrates that HEMPAS is caused by a defective gene encoding an enzyme necessary for the synthesis of asparagine-linked oligosaccharides.« less

Challenges in Resolution for IC Failure Analysis

NASA Astrophysics Data System (ADS)

Martinez, Nick

1999-10-01

Resolution is becoming more and more of a challenge in the world of Failure Analysis in integrated circuits. This is a result of the ongoing size reduction in microelectronics. Determining the cause of a failure depends upon being able to find the responsible defect. The time it takes to locate a given defect is extremely important so that proper corrective actions can be taken. The limits of current microscopy tools are being pushed. With sub-micron feature sizes and even smaller killing defects, optical microscopes are becoming obsolete. With scanning electron microscopy (SEM), the resolution is high but the voltage involved can make these small defects transparent due to the large mean-free path of incident electrons. In this presentation, I will give an overview of the use of inspection methods in Failure Analysis and show example studies of my work as an Intern student at Texas Instruments. 1. Work at Texas Instruments, Stafford, TX, was supported by TI. 2. Work at Texas Tech University, was supported by NSF Grant DMR9705498.

Actinic inspection of EUV reticles with arbitrary pattern design

NASA Astrophysics Data System (ADS)

Mochi, Iacopo; Helfenstein, Patrick; Rajeev, Rajendran; Fernandez, Sara; Kazazis, Dimitrios; Yoshitake, Shusuke; Ekinci, Yasin

2017-10-01

The re ective-mode EUV mask scanning lensless imaging microscope (RESCAN) is being developed to provide actinic mask inspection capabilities for defects and patterns with high resolution and high throughput, for 7 nm node and beyond. Here we, will report on our progress and present the results on programmed defect detection on random, logic-like patterns. The defects we investigated range from 200 nm to 50 nm size on the mask. We demonstrated the ability of RESCAN to detect these defects in die-to-die and die-to-database mode with a high signal to noise ratio. We also describe future plans for the upgrades to increase the resolution, the sensitivity, and the inspection speed of the demo tool.

Molecular Diagnostics of Copper-Transporting Protein Mutations Allows Early Onset Individual Therapy of Menkes Disease.

PubMed

Králík, L; Flachsová, E; Hansíková, H; Saudek, V; Zeman, J; Martásek, P

2017-01-01

Menkes disease is a severe X-linked recessive disorder caused by a defect in the ATP7A gene, which encodes a membrane copper-transporting ATPase. Deficient activity of the ATP7A protein results in decreased intestinal absorption of copper, low copper level in serum and defective distribution of copper in tissues. The clinical symptoms are caused by decreased activities of copper-dependent enzymes and include neurodegeneration, connective tissue disorders, arterial changes and hair abnormalities. Without therapy, the disease is fatal in early infancy. Rapid diagnosis of Menkes disease and early start of copper therapy is critical for the effectiveness of treatment. We report a molecular biology-based strategy that allows early diagnosis of copper transport defects and implementation of individual therapies before the full development of pathological symptoms. Low serum copper and decreased activity of copperdependent mitochondrial cytochrome c oxidase in isolated platelets found in three patients indicated a possibility of functional defects in copper-transporting proteins, especially in the ATPA7 protein, a copper- transporting P-type ATPase. Rapid mutational screening of the ATP7A gene using high-resolution melting analysis of DNA indicated presence of mutations in the patients. Molecular investigation for mutations in the ATP7A gene revealed three nonsense mutations: c.2170C>T (p.Gln724Ter); c.3745G>T (p.Glu1249Ter); and c.3862C>T (p.Gln1288Ter). The mutation c.3745G>T (p.Glu1249Ter) has not been identified previously. Molecular analysis of the ATOX1 gene as a possible modulating factor of Menkes disease did not reveal presence of pathogenic mutations. Molecular diagnostics allowed early onset of individual therapies, adequate genetic counselling and prenatal diagnosis in the affected families.

Improved depth profiling with slow positrons of ion implantation-induced damage in silicon

NASA Astrophysics Data System (ADS)

Fujinami, M.; Miyagoe, T.; Sawada, T.; Akahane, T.

2003-10-01

Variable-energy positron annihilation spectroscopy (VEPAS) has been extensively applied to study defects in near-surface regions and buried interfaces, but there is an inherent limit for depth resolution due to broadening of the positron implantation profile. In order to overcome this limit and obtain optimum depth resolution, iterative chemical etching of the sample surface and VEPAS measurement are employed. This etch-and-measure technique is described in detail and the capabilities are illustrated by investigating the depth profile of defects in Si after B and P implantations with 2×1014/cm2 at 100 keV followed by annealing. Defect tails can be accurately examined and the extracted defect profile is proven to extend beyond the implanted ion range predicted by the Monte Carlo code TRIM. This behavior is more remarkable for P ion implantation than B, and the mass difference of the implanted ions is strongly related to it. No significant difference is recognized in the annealing behavior between B and P implantations. After annealing at 300 °C, the defect profile is hardly changed, but the ratio of the characteristic Doppler broadening, S, a parameter for defects, to that for the bulk Si rises by 0.01, indicating that divacancies, V2, are transformed into V4. Annealing at more than 500 °C causes diffusion of the defects toward the surface and positron traps are annealed out at 800 °C. It is proved that this resolution-enhanced VEPAS can eliminate some discrepancies in defect profiles extracted by conventional means.

Phonons, defects and optical damage in crystalline acetanilide

NASA Astrophysics Data System (ADS)

Kosic, Thomas J.; Hill, Jeffrey R.; Dlott, Dana D.

1986-04-01

Intense picosecond pulses cause accumulated optical damage in acetanilide crystals at low temperature. Catastrophic damage to the irradiated volume occurs after an incubation period where defects accumulate. The optical damage is monitored with subanosecond time resolution. The generation of defects is studied with damage-detected picosecond spectroscopy. The accumulation of defects is studied by time-resolved coherent Raman scattering, which is used to measure optical phonon scattering from the accumulating defects.

X-Ray Topography of Tetragonal Lysozyme Grown by the Temperature-Controlled Technique

NASA Technical Reports Server (NTRS)

Stojanoff, V.; Siddons, D. P.; Monaco, Lisa A.; Vekilov, Peter; Rosenberger, Franz

1997-01-01

Growth-induced defects in lysozyme crystals were observed by white-beam and monochromatic X-ray topography at the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory (BNL). The topographic methods were non-destructive to the extent that traditional diffraction data collection could be performed to high resolution after topography. It was found that changes in growth parameters, defect concentration as detected by X-ray topography, and the diffraction quality obtainable from the crystals were all strongly correlated. In addition, crystals with fewer defects showed lower mosaicity and higher diffraction resolution as expected.

Measurement Variability of Vertical Scanning Interferometry Tool Used for Orbiter Window Defect Assessment

NASA Technical Reports Server (NTRS)

Padula, Santo, II

2009-01-01

The ability to sufficiently measure orbiter window defects to allow for window recertification has been an ongoing challenge for the orbiter vehicle program. The recent Columbia accident has forced even tighter constraints on the criteria that must be met in order to recertify windows for flight. As a result, new techniques are being investigated to improve the reliability, accuracy and resolution of the defect detection process. The methodology devised in this work, which is based on the utilization of a vertical scanning interferometric (VSI) tool, shows great promise for meeting the ever increasing requirements for defect detection. This methodology has the potential of a 10-100 fold greater resolution of the true defect depth than can be obtained from the currently employed micrometer based methodology. An added benefit is that it also produces a digital elevation map of the defect, thereby providing information about the defect morphology which can be utilized to ascertain the type of debris that induced the damage. However, in order to successfully implement such a tool, a greater understanding of the resolution capability and measurement repeatability must be obtained. This work focused on assessing the variability of the VSI-based measurement methodology and revealed that the VSI measurement tool was more repeatable and more precise than the current micrometer based approach, even in situations where operator variation could affect the measurement. The analysis also showed that the VSI technique was relatively insensitive to the hardware and software settings employed, making the technique extremely robust and desirable

A graphical automated detection system to locate hardwood log surface defects using high-resolution three-dimensional laser scan data

Treesearch

Liya Thomas; R. Edward Thomas

2011-01-01

We have developed an automated defect detection system and a state-of-the-art Graphic User Interface (GUI) for hardwood logs. The algorithm identifies defects at least 0.5 inch high and at least 3 inches in diameter on barked hardwood log and stem surfaces. To summarize defect features and to build a knowledge base, hundreds of defects were measured, photographed, and...

US Army Institute of Dental Research Annual Progress Report FY80.

DTIC Science & Technology

1980-10-01

indicates that use of the laser technique does result in increased connectiv tissue regeneration and improved resolution of the periodontal defects...connectiv tissue regeneration and improved resolution of the periodontal defects. Final analysis of histologic data on the use of the neodymium laser for in...and 21 Bone Regeneration in Trnumatic Wounds (Pathology) DA OH 6038 Development of Endodontic Procedures for 22 Military Dentistry ( Oral Biology) DA OK

Photoluminescence Imaging and LBIC Characterization of Defects in mc-Si Solar Cells

NASA Astrophysics Data System (ADS)

Sánchez, L. A.; Moretón, A.; Guada, M.; Rodríguez-Conde, S.; Martínez, O.; González, M. A.; Jiménez, J.

2018-05-01

Today's photovoltaic market is dominated by multicrystalline silicon (mc-Si) based solar cells with around 70% of worldwide production. In order to improve the quality of the Si material, a proper characterization of the electrical activity in mc-Si solar cells is essential. A full-wafer characterization technique such as photoluminescence imaging (PLi) provides a fast inspection of the wafer defects, though at the expense of the spatial resolution. On the other hand, a study of the defects at a microscopic scale can be achieved through the light-beam induced current technique. The combination of these macroscopic and microscopic resolution techniques allows a detailed study of the electrical activity of defects in mc-Si solar cells. In this work, upgraded metallurgical-grade Si solar cells are studied using these two techniques.

Defects in oxide surfaces studied by atomic force and scanning tunneling microscopy

PubMed Central

König, Thomas; Simon, Georg H; Heinke, Lars; Lichtenstein, Leonid

2011-01-01

Summary Surfaces of thin oxide films were investigated by means of a dual mode NC-AFM/STM. Apart from imaging the surface termination by NC-AFM with atomic resolution, point defects in magnesium oxide on Ag(001) and line defects in aluminum oxide on NiAl(110), respectively, were thoroughly studied. The contact potential was determined by Kelvin probe force microscopy (KPFM) and the electronic structure by scanning tunneling spectroscopy (STS). On magnesium oxide, different color centers, i.e., F0, F+, F2+ and divacancies, have different effects on the contact potential. These differences enabled classification and unambiguous differentiation by KPFM. True atomic resolution shows the topography at line defects in aluminum oxide. At these domain boundaries, STS and KPFM verify F2+-like centers, which have been predicted by density functional theory calculations. Thus, by determining the contact potential and the electronic structure with a spatial resolution in the nanometer range, NC-AFM and STM can be successfully applied on thin oxide films beyond imaging the topography of the surface atoms. PMID:21977410

A Portable, High Resolution, Surface Measurement Device

NASA Technical Reports Server (NTRS)

Ihlefeld, Curtis M.; Burns, Bradley M.; Youngquist, Robert C.

2012-01-01

A high resolution, portable, surface measurement device has been demonstrated to provide micron-resolution topographical plots. This device was specifically developed to allow in-situ measurements of defects on the Space Shuttle Orbiter windows, but is versatile enough to be used on a wide variety of surfaces. This paper discusses the choice of an optical sensor and then the decisions required to convert a lab bench optical measurement device into an ergonomic portable system. The necessary trade-offs between performance and portability are presented along with a description of the device developed to measure Orbiter window defects.

Toward reducing immunogenicity of enzyme replacement therapy: altering the specificity of human β-glucuronidase to compensate for α-iduronidase deficiency.

PubMed

Chuang, Huai-Yao; Suen, Ching-Shu; Hwang, Ming-Jing; Roffler, Steve R

2015-11-01

Enzyme replacement therapy (ERT) is an effective treatment for many patients with lysosomal storage disorders caused by deficiency in enzymes involved in cell metabolism. However, immune responses that develop against the administered enzyme in some patients can hinder therapeutic efficacy and cause serious side effects. Here we investigated the feasibility of a general approach to decrease ERT immunogenicity by altering the specificity of a normal endogenous enzyme to compensate for a defective enzyme. We sought to identify human β-glucuronidase variants that display α-iduronidase activity, which is defective in mucopolysaccharidosis (MPS) type I patients. A human β-glucuronidase library was screened by the Enzyme Cleavable Surface-Tethered All-purpose Screen sYstem to isolate variants that exhibited 100-290-fold greater activity against the α-iduronidase substrate 4-methylumbelliferyl α-l-iduronide and 7900-24 500-fold enzymatic specificity shift when compared with wild-type β-glucuronidase. In vitro treatment of MPS I cells with the β-glucuronidase variants significantly restored lysosome appearance similar to treatment with α-iduronidase. Our study suggests that β-glucuronidase variants can be isolated to display α-iduronidase activity and produce significant phenotype improvement of MPS I cells. This strategy may represent a possible approach to produce enzymes that display therapeutic benefits with potentially less immunogenicity. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cryo-EM structure of the gasdermin A3 membrane pore.

PubMed

Ruan, Jianbin; Xia, Shiyu; Liu, Xing; Lieberman, Judy; Wu, Hao

2018-05-01

Gasdermins mediate inflammatory cell death after cleavage by caspases or other, unknown enzymes. The cleaved N-terminal fragments bind to acidic membrane lipids to form pores, but the mechanism of pore formation remains unresolved. Here we present the cryo-electron microscopy structures of the 27-fold and 28-fold single-ring pores formed by the N-terminal fragment of mouse GSDMA3 (GSDMA3-NT) at 3.8 and 4.2 Å resolutions, and of a double-ring pore at 4.6 Å resolution. In the 27-fold pore, a 108-stranded anti-parallel β-barrel is formed by two β-hairpins from each subunit capped by a globular domain. We identify a positively charged helix that interacts with the acidic lipid cardiolipin. GSDMA3-NT undergoes radical conformational changes upon membrane insertion to form long, membrane-spanning β-strands. We also observe an unexpected additional symmetric ring of GSDMA3-NT subunits that does not insert into the membrane in the double-ring pore, which may represent a pre-pore state of GSDMA3-NT. These structures provide a basis that explains the activities of several mutant gasdermins, including defective mutants that are associated with cancer.

Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

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

Krungkrai, Sudaratana R.; Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871; Tokuoka, Keiji

Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 asmore » a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V{sub M} = 2.3 Å{sup 3} Da{sup −1})« less

Improving depth resolutions in positron beam spectroscopy by concurrent ion-beam sputtering

NASA Astrophysics Data System (ADS)

John, Marco; Dalla, Ayham; Ibrahim, Alaa M.; Anwand, Wolfgang; Wagner, Andreas; Böttger, Roman; Krause-Rehberg, Reinhard

2018-05-01

The depth resolution of mono-energetic positron annihilation spectroscopy using a positron beam is shown to improve by concurrently removing the sample surface layer during positron beam spectroscopy. During ion-beam sputtering with argon ions, Doppler-broadening spectroscopy is performed with energies ranging from 3 keV to 5 keV allowing for high-resolution defect studies just below the sputtered surface. With this technique, significantly improved depth resolutions could be obtained even at larger depths when compared to standard positron beam experiments which suffer from extended positron implantation profiles at higher positron energies. Our results show that it is possible to investigate layered structures with a thickness of about 4 microns with significantly improved depth resolution. We demonstrated that a purposely generated ion-beam induced defect profile in a silicon sample could be resolved employing the new technique. A depth resolution of less than 100 nm could be reached.

Mass Defect from Nuclear Physics to Mass Spectral Analysis.

PubMed

Pourshahian, Soheil

2017-09-01

Mass defect is associated with the binding energy of the nucleus. It is a fundamental property of the nucleus and the principle behind nuclear energy. Mass defect has also entered into the mass spectrometry terminology with the availability of high resolution mass spectrometry and has found application in mass spectral analysis. In this application, isobaric masses are differentiated and identified by their mass defect. What is the relationship between nuclear mass defect and mass defect used in mass spectral analysis, and are they the same? Graphical Abstract ᅟ.

Autolytic defective mutant of Streptococcus faecalis.

PubMed Central

Cornett, J B; Redman, B E; Shockman, G D

1978-01-01

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme. PMID:415045

Absorption, autoionization, and predissociation in molecular hydrogen: High-resolution spectroscopy and multichannel quantum defect theory.

PubMed

Sommavilla, M; Merkt, F; Mezei, J Zs; Jungen, Ch

2016-02-28

Absorption and photoionization spectra of H2 have been recorded at a resolution of 0.09 and 0.04 cm(-1), respectively, between 125,600 cm(-1) and 126,000 cm(-1). The observed Rydberg states belong to series (n = 10 - 14) converging on the first vibrationally excited level of the X (2)Σ(g)(+) state of H2(+), and of lower members of series converging on higher vibrational levels. The observed resonances are characterized by the competition between autoionization, predissociation, and fluorescence. The unprecedented resolution of the present experimental data leads to a full characterization of the predissociation/autoionization profiles of many resonances that had not been resolved previously. Multichannel quantum defect theory is used to predict the line positions, widths, shapes, and intensities of the observed spectra and is found to yield quantitative agreement using previously determined quantum defect functions as the unique set of input parameters.

Efficacy of platelet-rich fibrin vs. enamel matrix derivative in the treatment of periodontal intrabony defects: a clinical and cone beam computed tomography study.

PubMed

Gupta, Swyeta Jain; Jhingran, Rajesh; Gupta, Vivek; Bains, Vivek Kumar; Madan, Rohit; Rizvi, Iram

2014-07-01

To evaluate and compare the efficacy of platelet-rich fibrin (PRF) with enamel matrix derivative (EMD; Emdogain) in the treatment of periodontal intrabony defects in patients with chronic periodontitis, six months after surgery. Forty-four (44) intrabony defects in 30 patients (15 males) were randomly allocated into two treatment groups: EMD (n = 22) and PRF (n = 22). Measurement of the defects was done using clinical and cone beam computed tomography at baseline and 6 months. Clinical and radiographic parameters such as probing depth, clinical attachment level, intrabony defect depth and defect angle, were recorded at baseline and 6 months post-operatively. Within group change was evaluated using the Wilcoxon signed rank test. Intergroup comparisons were made using the Mann-Whitney U test. Postsurgical measurements revealed that there was an equal reduction in probing depth and a greater but statistically non-significant attachment gain for the Emdogain group when compared to the platelet-rich fibrin group. The Emdogain group presented with significantly greater percentage defect resolution (43.07% ± 12.21) than did the platelet-rich fibrin group (32.41% ± 14.61). Post-operatively the changes in defect width and defect angle were significant in both groups, but upon intergroup comparison they were found to be statistically non-significantly different. Both Emdogain and platelet-rich fibrin were effective in the regeneration of intrabony defects. Emdogain was significantly superior in terms of percentage defect resolution.

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

PubMed

Sriram, V; Krishnan, K S; Mayor, Satyajit

2003-05-12

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Influence of aspect ratio and surface defect density on hydrothermally grown ZnO nanorods towards amperometric glucose biosensing applications

NASA Astrophysics Data System (ADS)

Shukla, Mayoorika; Pramila; Dixit, Tejendra; Prakash, Rajiv; Palani, I. A.; Singh, Vipul

2017-11-01

In this work, hydrothermally grown ZnO Nanorods Array (ZNA) has been synthesized over Platinum (Pt) coated glass substrate, for biosensing applications. In-situ addition of strong oxidizing agent viz KMnO4 during hydrothermal growth was found to have profound effect on the physical properties of ZNA. Glucose oxidase (GOx) was later immobilized over ZNA by means of physical adsorption process. Further influence of varying aspect ratio, enzyme loading and surface defects on amperometric glucose biosensor has been analyzed. Significant variation in biosensor performance was observed by varying the amount of KMnO4 addition during the growth. Moreover, investigations revealed that the suppression of surface defects and aspect ratio variation of the ZNA played key role towards the observed improvement in the biosensor performance, thereby significantly affecting the sensitivity and response time of the fabricated biosensor. Among different biosensors fabricated having varied aspect ratio and surface defect density of ZNA, the best electrode resulted into sensitivity and response time to be 18.7 mA cm-2 M-1 and <5 s respectively. The observed results revealed that apart from high aspect ratio nanostructures and the extent of enzyme loading, surface defect density also hold a key towards ZnO nanostructures based bio-sensing applications.

Defect Clustering and Nano-Phase Structure Characterization of Multi-Component Rare Earth Oxide Doped Zirconia-Yttria Thermal Barrier Coatings

NASA Technical Reports Server (NTRS)

Zhu, Dongming; Chen, Yuan L.; Miller, Robert A.

2003-01-01

Advanced oxide thermal barrier coatings have been developed by incorporating multi-component rare earth oxide dopants into zirconia-yttria to effectively promote the creation of the thermodynamically stable, immobile oxide defect clusters and/or nano-scale phases within the coating systems. The presence of these nano-sized defect clusters has found to significantly reduce the coating intrinsic thermal conductivity, improve sintering resistance, and maintain long-term high temperature stability. In this paper, the defect clusters and nano-structured phases, which were created by the addition of multi-component rare earth dopants to the plasma-sprayed and electron-beam physical vapor deposited thermal barrier coatings, were characterized by high-resolution transmission electron microscopy (TEM). The defect cluster size, distribution, crystallographic and compositional information were investigated using high-resolution TEM lattice imaging, selected area diffraction (SAD), electron energy-loss spectroscopy (EELS) and energy dispersive spectroscopy (EDS) analysis techniques. The results showed that substantial defect clusters were formed in the advanced multi-component rare earth oxide doped zirconia- yttria systems. The size of the oxide defect clusters and the cluster dopant segregation was typically ranging from 5 to 50 nm. These multi-component dopant induced defect clusters are an important factor for the coating long-term high temperature stability and excellent performance.

Defect Clustering and Nano-Phase Structure Characterization of Multi-Component Rare Earth Oxide Doped Zirconia-Yttria Thermal Barrier Coatings

NASA Technical Reports Server (NTRS)

Zhu, Dongming; Chen, Yuan L.; Miller, Robert A.

1990-01-01

Advanced oxide thermal barrier coatings have been developed by incorporating multi- component rare earth oxide dopants into zirconia-yttria to effectively promote the creation of the thermodynamically stable, immobile oxide defect clusters and/or nano-scale phases within the coating systems. The presence of these nano-sized defect clusters has found to significantly reduce the coating intrinsic thermal conductivity, improve sintering resistance, and maintain long-term high temperature stability. In this paper, the defect clusters and nano-structured phases, which were created by the addition of multi-component rare earth dopants to the plasma- sprayed and electron-beam physical vapor deposited thermal barrier coatings, were characterized by high-resolution transmission electron microscopy (TEM). The defect cluster size, distribution, crystallographic and compositional information were investigated using high-resolution TEM lattice imaging, selected area diffraction (SAD), and energy dispersive spectroscopy (EDS) analysis techniques. The results showed that substantial defect clusters were formed in the advanced multi-component rare earth oxide doped zirconia-yttria systems. The size of the oxide defect clusters and the cluster dopant segregation was typically ranging fiom 5 to 50 nm. These multi-component dopant induced defect clusters are an important factor for the coating long-term high temperature stability and excellent performance.

Automatic Fabric Defect Detection with a Multi-Scale Convolutional Denoising Autoencoder Network Model.

PubMed

Mei, Shuang; Wang, Yudan; Wen, Guojun

2018-04-02

Fabric defect detection is a necessary and essential step of quality control in the textile manufacturing industry. Traditional fabric inspections are usually performed by manual visual methods, which are low in efficiency and poor in precision for long-term industrial applications. In this paper, we propose an unsupervised learning-based automated approach to detect and localize fabric defects without any manual intervention. This approach is used to reconstruct image patches with a convolutional denoising autoencoder network at multiple Gaussian pyramid levels and to synthesize detection results from the corresponding resolution channels. The reconstruction residual of each image patch is used as the indicator for direct pixel-wise prediction. By segmenting and synthesizing the reconstruction residual map at each resolution level, the final inspection result can be generated. This newly developed method has several prominent advantages for fabric defect detection. First, it can be trained with only a small amount of defect-free samples. This is especially important for situations in which collecting large amounts of defective samples is difficult and impracticable. Second, owing to the multi-modal integration strategy, it is relatively more robust and accurate compared to general inspection methods (the results at each resolution level can be viewed as a modality). Third, according to our results, it can address multiple types of textile fabrics, from simple to more complex. Experimental results demonstrate that the proposed model is robust and yields good overall performance with high precision and acceptable recall rates.

Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

PubMed Central

Deng, Hua

2017-01-01

Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

Pattern centric design based sensitive patterns and process monitor in manufacturing

NASA Astrophysics Data System (ADS)

Hsiang, Chingyun; Cheng, Guojie; Wu, Kechih

2017-03-01

When design rule is mitigating to smaller dimension, process variation requirement is tighter than ever and challenges the limits of device yield. Masks, lithography, etching and other processes have to meet very tight specifications in order to keep defect and CD within the margins of the process window. Conventionally, Inspection and metrology equipments are utilized to monitor and control wafer quality in-line. In high throughput optical inspection, nuisance and review-classification become a tedious labor intensive job in manufacturing. Certain high-resolution SEM images are taken to validate defects after optical inspection. These high resolution SEM images catch not only optical inspection highlighted point, also its surrounding patterns. However, this pattern information is not well utilized in conventional quality control method. Using this complementary design based pattern monitor not only monitors and analyzes the variation of patterns sensitivity but also reduce nuisance and highlight defective patterns or killer defects. After grouping in either single or multiple layers, systematic defects can be identified quickly in this flow. In this paper, we applied design based pattern monitor in different layers to monitor process variation impacts on all kinds of patterns. First, the contour of high resolutions SEM image is extracted and aligned to design with offset adjustment and fine alignment [1]. Second, specified pattern rules can be applied on design clip area, the same size as SEM image, and form POI (pattern of interest) areas. Third, the discrepancy of contour and design measurement at different pattern types in measurement blocks. Fourth, defective patterns are reported by discrepancy detection criteria and pattern grouping [4]. Meanwhile, reported pattern defects are ranked by number and severity by discrepancy. In this step, process sensitive high repeatable systematic defects can be identified quickly Through this design based process pattern monitor method, most of optical inspection nuisances can be filtered out at contour to design discrepancy measurement. Daily analysis results are stored at database as reference to compare with incoming data. Defective pattern library contains existing and known systematic defect patterns which help to catch and identify new pattern defects or process impacts. On the other hand, this defect pattern library provides extra valuable information for mask, pattern and defects verification, inspection care area generation, further OPC fix and process enhancement and investigation.

In-process fault detection for textile fabric production: onloom imaging

NASA Astrophysics Data System (ADS)

Neumann, Florian; Holtermann, Timm; Schneider, Dorian; Kulczycki, Ashley; Gries, Thomas; Aach, Til

2011-05-01

Constant and traceable high fabric quality is of high importance both for technical and for high-quality conventional fabrics. Usually, quality inspection is carried out by trained personal, whose detection rate and maximum period of concentration are limited. Low resolution automated fabric inspection machines using texture analysis were developed. Since 2003, systems for the in-process inspection on weaving machines ("onloom") are commercially available. With these defects can be detected, but not measured quantitative precisely. Most systems are also prone to inevitable machine vibrations. Feedback loops for fault prevention are not established. Technology has evolved since 2003: Camera and computer prices dropped, resolutions were enhanced, recording speeds increased. These are the preconditions for real-time processing of high-resolution images. So far, these new technological achievements are not used in textile fabric production. For efficient use, a measurement system must be integrated into the weaving process; new algorithms for defect detection and measurement must be developed. The goal of the joint project is the development of a modern machine vision system for nondestructive onloom fabric inspection. The system consists of a vibration-resistant machine integration, a high-resolution machine vision system, and new, reliable, and robust algorithms with quality database for defect documentation. The system is meant to detect, measure, and classify at least 80 % of economically relevant defects. Concepts for feedback loops into the weaving process will be pointed out.

Automatic optical inspection of regular grid patterns with an inspection camera used below the Shannon-Nyquist criterion for optical resolution

NASA Astrophysics Data System (ADS)

Ferreira, Flávio P.; Forte, Paulo M. F.; Felgueiras, Paulo E. R.; Bret, Boris P. J.; Belsley, Michael S.; Nunes-Pereira, Eduardo J.

2017-02-01

An Automatic Optical Inspection (AOI) system for optical inspection of imaging devices used in automotive industry using an inspecting optics of lower spatial resolution than the device under inspection is described. This system is robust and with no moving parts. The cycle time is small. Its main advantage is that it is capable of detecting and quantifying defects in regular patterns, working below the Shannon-Nyquist criterion for optical resolution, using a single low resolution image sensor. It is easily scalable, which is an important advantage in industrial applications, since the same inspecting sensor can be reused for increasingly higher spatial resolutions of the devices to be inspected. The optical inspection is implemented with a notch multi-band Fourier filter, making the procedure especially fitted for regular patterns, like the ones that can be produced in image displays and Head Up Displays (HUDs). The regular patterns are used in production line only, for inspection purposes. For image displays, functional defects are detected at the level of a sub-image display grid element unit. Functional defects are the ones impairing the function of the display, and are preferred in AOI to the direct geometric imaging, since those are the ones directly related with the end-user experience. The shift in emphasis from geometric imaging to functional imaging is critical, since it is this that allows quantitative inspection, below Shannon-Nyquist. For HUDs, the functional detect detection addresses defects resulting from the combined effect of the image display and the image forming optics.

Bioenergetic Defects and Oxidative Damage in Transgenic Mouse Models of Neurodegenerative Disorders

DTIC Science & Technology

2003-05-01

transport chain enzyme activities in G93A ALS mice at 60 and 120d. A^^; The laboratory moved from Massachussetts General Hospital, Boston, MA, to...glucose use changes by measurement at 42d, 56d and 84d of age. 7) Measurement of electron transport chain enzyme activities in R6/2 HD mice. 8) NMR...lactate imaging; in vitro spectrophotometric oxidative phosphorylation enzyme assays; HPLC detection of metabolites), and to investigate the

High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhibitors and antihypertensive drugs.

PubMed

Akif, Mohd; Georgiadis, Dimitris; Mahajan, Aman; Dive, Vincent; Sturrock, Edward D; Isaac, R Elwyn; Acharya, K Ravi

2010-07-16

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE. 2010 Elsevier Ltd. All rights reserved.

Development of an optical inspection platform for surface defect detection in touch panel glass

NASA Astrophysics Data System (ADS)

Chang, Ming; Chen, Bo-Cheng; Gabayno, Jacque Lynn; Chen, Ming-Fu

2016-04-01

An optical inspection platform combining parallel image processing with high resolution opto-mechanical module was developed for defect inspection of touch panel glass. Dark field images were acquired using a 12288-pixel line CCD camera with 3.5 µm per pixel resolution and 12 kHz line rate. Key features of the glass surface were analyzed by parallel image processing on combined CPU and GPU platforms. Defect inspection of touch panel glass, which provided 386 megapixel image data per sample, was completed in roughly 5 seconds. High detection rate of surface scratches on the touch panel glass was realized with minimum defects size of about 10 µm after inspection. The implementation of a custom illumination source significantly improved the scattering efficiency on the surface, therefore enhancing the contrast in the acquired images and overall performance of the inspection system.

Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase

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

Bacik, John -Paul; Mekasha, Sophanit; Forsberg, Zarah

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1–3 mm 3) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected andmore » processed to 1.1 Å resolution in space group P2 12 12 1. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. As a result, joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.« less

Neutron and high-resolution room-temperature X-ray data collection from crystallized lytic polysaccharide monooxygenase

DOE PAGES

Bacik, John -Paul; Mekasha, Sophanit; Forsberg, Zarah; ...

2015-01-01

Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1–3 mm 3) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected andmore » processed to 1.1 Å resolution in space group P2 12 12 1. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. As a result, joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.« less

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes*

PubMed Central

Chan, Yvonne Gar-Yun; Kim, Hwan Keun; Schneewind, Olaf; Missiakas, Dominique

2014-01-01

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan. PMID:24753256

The very-long-chain hydroxy fatty acyl-CoA dehydratase PASTICCINO2 is essential and limiting for plant development

PubMed Central

Bach, Liên; Michaelson, Louise V.; Haslam, Richard; Bellec, Yannick; Gissot, Lionel; Marion, Jessica; Da Costa, Marco; Boutin, Jean-Pierre; Miquel, Martine; Tellier, Frédérique; Domergue, Frederic; Markham, Jonathan E.; Beaudoin, Frederic; Napier, Johnathan A.; Faure, Jean-Denis

2008-01-01

Very-long-chain fatty acids (VLCFAs) are synthesized as acyl-CoAs by the endoplasmic reticulum-localized elongase multiprotein complex. Two Arabidopsis genes are putative homologues of the recently identified yeast 3-hydroxy-acyl-CoA dehydratase (PHS1), the third enzyme of the elongase complex. We showed that Arabidopsis PASTICCINO2 (PAS2) was able to restore phs1 cytokinesis defects and sphingolipid long chain base overaccumulation. Conversely, the expression of PHS1 was able to complement the developmental defects and the accumulation of long chain bases of the pas2–1 mutant. The pas2–1 mutant was characterized by a general reduction of VLCFA pools in seed storage triacylglycerols, cuticular waxes, and complex sphingolipids. Most strikingly, the defective elongation cycle resulted in the accumulation of 3-hydroxy-acyl-CoA intermediates, indicating premature termination of fatty acid elongation and confirming the role of PAS2 in this process. We demonstrated by in vivo bimolecular fluorescence complementation that PAS2 was specifically associated in the endoplasmic reticulum with the enoyl-CoA reductase CER10, the fourth enzyme of the elongase complex. Finally, complete loss of PAS2 function is embryo lethal, and the ectopic expression of PHS1 led to enhanced levels of VLCFAs associated with severe developmental defects. Altogether these results demonstrate that the plant 3-hydroxy-acyl-CoA dehydratase PASTICCINO2 is an essential and limiting enzyme in VLCFA synthesis but also that PAS2-derived VLCFA homeostasis is required for specific developmental processes. PMID:18799749

Defect imaging and detection of precipitates using a new scanning positron microbeam

NASA Astrophysics Data System (ADS)

Gigl, T.; Beddrich, L.; Dickmann, M.; Rienäcker, B.; Thalmayr, M.; Vohburger, S.; Hugenschmidt, C.

2017-12-01

We report on a newly developed scanning positron microbeam based on threefold moderation of positrons provided by the high intensity positron source NEPOMUC. For brightness enhancement a remoderation unit with a 100 nm thin Ni(100) foil and 9.6% efficiency is applied to reduce the area of the beam spot by a factor of 60. In this way, defect spectroscopy is enabled with a lateral resolution of 33 μm over a large scanning range of 19 × 19 mm2. Moreover, 2D defect imaging using Doppler broadening spectroscopy (DBS) is demonstrated to be performed within exceptional short measurement times of less than two minutes for an area of 1 × 1 mm2 (100 × 100 μm2) with a resolution of 250 μm (50 μm). We studied the defect structure in laser beam welds of the high-strength age-hardened Al alloy (AlCu6Mn, EN AW-2219 T87) by applying (coincident) DBS with unprecedented spatial resolution. The visualization of the defect distribution revealed a sharp transition between the raw material and the welded zone as well as a very small heat affected zone. Vacancy-like defects and Cu rich precipitates are detected in the as-received material and, to a lesser extent, in the transition zone of the weld. Most notably, in the center of the weld vacancies without forming Cu-vacancy complexes, and the dissolution of the Cu atoms in the crystal lattice, i.e. formation of a supersaturated solution, could be clearly identified.

Ammonia toxicity and its prevention in inherited defects of the urea cycle.

PubMed

Walker, V

2009-09-01

The urea cycle is the final pathway for removal of surplus nitrogen from the body, and the major route in humans for detoxification of ammonia. The full complement of enzymes is expressed only in liver. Inherited deficiencies of urea cycle enzymes lead to hyperammonaemia, which causes brain damage. Severe defects present with hyperammonaemic crises in neonates. Equally devastating episodes may occur in previously asymptomatic adults with mild defects, most often X-linked ornithine transcarbamylase (OTC) deficiency. Several mechanisms probably contribute to pathogenesis. Treatment aims to reduce plasma ammonia quickly, reduce production of waste nitrogen, dispose of waste nitrogen using alternative pathways to the urea cycle and replace arginine. These therapies have increased survival and probably improve the neurological outcome. Arginine, sodium benzoate, sodium phenylbutyrate and, less often, sodium phenylacetate are used. Long-term correction is achieved by liver transplantation. Gene therapy for OTC deficiency is effective in animals, and work is ongoing to improve persistence and safety.

Magnetic resonance imaging study of eye congenital birth defects in mouse model

PubMed Central

Tucker, Zachary; Mongan, Maureen; Meng, Qinghang; Xia, Ying

2017-01-01

Purpose Embryonic eyelid closure is a well-documented morphogenetic episode in mammalian eye development. Detection of eyelid closure defect in humans is a major challenge because eyelid closure and reopen occur entirely in utero. As a consequence, congenital eye defects that are associated with failure of embryonic eyelid closure remain unknown. To fill the gap, we developed a mouse model of defective eyelid closure. This preliminary work demonstrates that the magnetic resonance imaging (MRI) approach can be used for the detection of extraocular muscle abnormalities in the mouse model. Methods Mice with either normal (Map3k1+/−) or defective (Map3k1−/−) embryonic eyelid closure were used in this study. Images of the extraocular muscles were obtained with a 9.4 T high resolution microimaging MRI system. The extraocular muscles were identified, segmented, and measured in each imaging slice using an in-house program. Results In agreement with histological findings, the imaging data show that mice with defective embryonic eyelid closure develop less extraocular muscle than normal mice. In addition, the size of the eyeballs was noticeably reduced in mice with defective embryonic eyelid closure. Conclusions We demonstrated that MRI can potentially be used for the study of extraocular muscle in the mouse model of the eye open-at-birth defect, despite the lack of specificity of muscle group provided by the current imaging resolution. PMID:28848319

Using parallel computing methods to improve log surface defect detection methods

Treesearch

R. Edward Thomas; Liya Thomas

2013-01-01

Determining the size and location of surface defects is crucial to evaluating the potential yield and value of hardwood logs. Recently a surface defect detection algorithm was developed using the Java language. This algorithm was developed around an earlier laser scanning system that had poor resolution along the length of the log (15 scan lines per foot). A newer...

Comprehensive characterization of C-glycosyl flavones in wheat (Triticum aestivum L.) germ using UPLC-PDA-ESI/HRMSn and mass defect filtering

USDA-ARS?s Scientific Manuscript database

A comprehensive characterization of C-glycosyl flavones in wheat germ has been conducted using multi-stage high resolution mass spectrometry (HRMS) combined with mass defect filter (MDF). MDF performed the initial search of raw data with defined mass ranges and mass defect windows to generate the n...

Structural defects in cubic semiconductors characterized by aberration-corrected scanning transmission electron microscopy.

PubMed

Arroyo Rojas Dasilva, Yadira; Kozak, Roksolana; Erni, Rolf; Rossell, Marta D

2017-05-01

The development of new electro-optical devices and the realization of novel types of transistors require a profound understanding of the structural characteristics of new semiconductor heterostructures. This article provides a concise review about structural defects which occur in semiconductor heterostructures on the basis of micro-patterned Si substrates. In particular, one- and two-dimensional crystal defects are being discussed which are due to the plastic relaxation of epitaxial strain caused by the misfit of crystal lattices. Besides a few selected examples from literature, we treat in particular crystal defects occurring in GaAs/Si, Ge/Si and β-SiC/Si structures which are studied by high-resolution annular dark-field scanning transmission electron microscopy. The relevance of this article is twofold; firstly, it should provide a collection of data which are of help for the identification and characterization of defects in cubic semiconductors by means of atomic-resolution imaging, and secondly, the experimental data shall provide a basis for advancing the understanding of device characteristics with the aid of theoretical modelling by considering the defective nature of strained semiconductor heterostructures. Copyright © 2016 Elsevier B.V. All rights reserved.

Subangstrom Measurements of Enzyme Function Using a Biological Nanopore, SPRNT.

PubMed

Laszlo, A H; Derrrington, I M; Gundlach, J H

2017-01-01

Nanopores are emerging as new single-molecule tools in the study of enzymes. Based on the progress in nanopore sequencing of DNA, a tool called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) was developed to measure the movement of enzymes along DNA in real time. In this new method, an enzyme is loaded onto a DNA (or RNA) molecule. A single-stranded DNA end of this complex is drawn into a nanopore by an electrostatic potential that is applied across the pore. The single-stranded DNA passes through the pore's constriction until the enzyme comes into contact with the pore. Further progression of the DNA through the pore is then controlled by the enzyme. An ion current that flows through the pore's constriction is modulated by the DNA in the constriction. Analysis of ion current changes reveals the advance of the DNA with high spatiotemporal precision, thereby providing a real-time record of the enzyme's activity. Using an engineered version of the protein nanopore MspA, SPRNT has spatial resolution as small as 40pm at millisecond timescales, while simultaneously providing the DNA's sequence within the enzyme. In this chapter, SPRNT is introduced and its extraordinary potential is exemplified using the helicase Hel308. Two distinct substates are observed for each one-nucleotide advance; one of these about half-nucleotide long steps is ATP dependent and the other is ATP independent. The spatiotemporal resolution of this low-cost single-molecule technique lifts the study of enzymes to a new level of precision, enabling exploration of hitherto unobservable enzyme dynamics in real time. © 2017 Elsevier Inc. All rights reserved.

A study of irradiation-induced defects in silicon using low temperature photoluminescence

NASA Technical Reports Server (NTRS)

Streetman, B. G.

1971-01-01

Irradiation-induced defects in silicon, using low temperature photoluminescence as a probe of defect properties were investigated. The goal of this research was to gain new understanding of defects which degrade solar cell characteristics in a radiation environment. In this regard, an important aspect of this program was a study of radiation damage and annealing in lithium doped silicon, which is useful in reducing solar cell degradation. Luminescence was used to study defects because this property reveals electron transitions through a number of defect energy levels at any given annealing stage; the luminescence spectra give excellent resolution of many defect energy levels, and these measurements can be used to give defect symmetry in the lattice, impurity dependence, and annealing properties.

Nano-size defects in arsenic-implanted HgCdTe films: a HRTEM study

NASA Astrophysics Data System (ADS)

Bonchyk, O. Yu.; Savytskyy, H. V.; Swiatek, Z.; Morgiel, Y.; Izhnin, I. I.; Voitsekhovskii, A. V.; Korotaev, A. G.; Mynbaev, K. D.; Fitsych, O. I.; Varavin, V. S.; Dvoretsky, S. A.; Marin, D. V.; Yakushev, M. V.

2018-02-01

Radiation damage and its transformation under annealing were studied with bright-field and high-resolution transmission electron microscopy for arsenic-implanted HgCdTe films with graded-gap surface layers. In addition to typical highly defective layers in as-implanted material, a 50 nm-thick sub-surface layer with very low defect density was observed. The main defects in other layers after implantation were dislocation loops, yet after arsenic activation annealing, the dominating defects were single dislocations. Transport (from depth to surface), transformation and annihilation of radiation-induced defects were observed as a result of annealing, with the depth with the maximum defect density decreasing from 110 to 40 nm.

Retrovirally mediated correction of bone marrow-derived mesenchymal stem cells from patients with mucopolysaccharidosis type I.

PubMed

Baxter, Melissa A; Wynn, Robert F; Deakin, Jonathan A; Bellantuono, Ilaria; Edington, Kirsten G; Cooper, Alan; Besley, Guy T N; Church, Heather J; Wraith, J Ed; Carr, Trevor F; Fairbairn, Leslie J

2002-03-01

We have investigated the utility of bone marrow-derived mesenchymal stem cells (MSCs) as targets for gene therapy of the autosomal recessive disorder mucopolysaccharidosis type IH (MPS-IH, Hurler syndrome). Cultures of MSCs were initially exposed to a green fluorescent protein-expressing retrovirus. Green fluorescent protein-positive cells maintained their proliferative and differentiation capacity. Next we used a vector encoding alpha-L-iduronidase (IDUA), the enzyme that is defective in MPS-IH. Following transduction, MPS-IH MSCs expressed high levels of IDUA and secreted supernormal levels of this enzyme into the extracellular medium. Exogenous IDUA expression led to a normalization of glycosaminoglycan storage in MPS-IH cells, as evidenced by a dramatic decrease in the amount of (35)SO(4) sequestered within the heparan sulfate and dermatan sulfate compartments of these cells. Finally, gene-modified MSCs were able to cross-correct the enzyme defect in untransduced MPS-IH fibroblasts via protein transfer.

Helium bubbles aggravated defects production in self-irradiated copper

NASA Astrophysics Data System (ADS)

Wu, FengChao; Zhu, YinBo; Wu, Qiang; Li, XinZhu; Wang, Pei; Wu, HengAn

2017-12-01

Under the environment of high radiation, materials used in fission and fusion reactors will internally accumulate numerous lattice defects and bubbles. With extensive studies focused on bubble resolution under irradiation, the mutually effects between helium bubbles and displacement cascades in irradiated materials remain unaddressed. Therefore, the defects production and microstructure evolution under self-irradiation events in vicinity of helium bubbles are investigated by preforming large scale molecular dynamics simulations in single-crystal copper. When subjected to displacement cascades, distinguished bubble resolution categories dependent on bubble size are observed. With the existence of bubbles, radiation damage is aggravated with the increasing bubble size, represented as the promotion of point defects and dislocations. The atomic mechanisms of heterogeneous dislocation structures are attributed to different helium-vacancy cluster modes, transforming from the resolved gas trapped with vacancies to the biased absorption of vacancies by the over-pressured bubble. In both cases, helium impedes the recombination of point defects, leading to the accelerated formation of interstitial loops. The results and insight obtained here might contribute to understand the underlying mechanism of transmutant solute on the long-term evolution of irradiated materials.

Effect of defects on reaction of NiO surface with Pb-contained solution

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

Kim, Jongjin; Hou, Binyang; Park, Changyong

In order to understand the role of defects in chemical reactions, we used two types of samples, which are molecular beam epitaxy (MBE) grown NiO(001) film on Mg(001) substrate as the defect free NiO prototype and NiO grown on Ni(110) single crystal as the one with defects. In-situ observations for oxide-liquid interfacial structure and surface morphology were performed for both samples in water and Pb-contained solution using high-resolution X-ray reflectivity and atomic force microscopy. For the MBE grown NiO, no significant changes were detected in the high-resolution X-ray reflectivity data with monotonic increase in roughness. Meanwhile, in the case ofmore » native grown NiO on Ni(110), significant changes in both the morphology and atomistic structure at the interface were observed when immersed in water and Pb-contained solution. Our results provide simple and direct experimental evidence of the role of the defects in chemical reaction of oxide surfaces with both water and Pb-contained solution.« less

RBS/C, HRTEM and HRXRD study of damage accumulation in irradiated SrTiO3

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

Jagielski, Jacek; Jozwik, Przemyslaw A.; Jozwik Biala, Iwona

2013-05-14

Damage accumulation in argon-irradiated SrTiO3 single crystals has been studied by using combination of Rutherford Backscattering/Channeling (RBS/C), High Resolution Transmission Electron Microscopy (HRTEM) and High Resolution X-Ray Diffraction (HRXRD) techniques. The RBS/C spectra were fitted using McChasy, a Monte Carlo simulation code allowing the quantitative analysis of amorphous-like and dislocation-like types of defects. The results were interpreted by using a Multi-Step Damage Accumulation model which assumes, that the damage accumulation occurs in a series of structural transformations, the defect transformations are triggered by a stress caused by formation of a free volume in the irradiated crystal. This assumption has beenmore » confirmed by High Resolution Transmission Electron Microscopy and High Resolution X-Ray Diffraction analysis.« less

Increased nuclear sphingoid base-1-phosphates and HDAC inhibition after fumonisin and FTY720-treatment: the link between epigenomic modifications and neural tube defects?

USDA-ARS?s Scientific Manuscript database

Introduction: Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs). FB1 inhibits the enzyme ceramide synthase in de novo sphingolipid biosynthes...

Wafer plane inspection for advanced reticle defects

NASA Astrophysics Data System (ADS)

Nagpal, Rajesh; Ghadiali, Firoz; Kim, Jun; Huang, Tracy; Pang, Song

2008-05-01

Readiness of new mask defect inspection technology is one of the key enablers for insertion & transition of the next generation technology from development into production. High volume production in mask shops and wafer fabs demands a reticle inspection system with superior sensitivity complemented by a low false defect rate to ensure fast turnaround of reticle repair and defect disposition (W. Chou et al 2007). Wafer Plane Inspection (WPI) is a novel approach to mask defect inspection, complementing the high resolution inspection capabilities of the TeraScanHR defect inspection system. WPI is accomplished by using the high resolution mask images to construct a physical mask model (D. Pettibone et al 1999). This mask model is then used to create the mask image in the wafer aerial plane. A threshold model is applied to enhance the inspectability of printing defects. WPI can eliminate the mask restrictions imposed on OPC solutions by inspection tool limitations in the past. Historically, minimum image restrictions were required to avoid nuisance inspection stops and/or subsequent loss of sensitivity to defects. WPI has the potential to eliminate these limitations by moving the mask defect inspections to the wafer plane. This paper outlines Wafer Plane Inspection technology, and explores the application of this technology to advanced reticle inspection. A total of twelve representative critical layers were inspected using WPI die-to-die mode. The results from scanning these advanced reticles have shown that applying WPI with a pixel size of 90nm (WPI P90) captures all the defects of interest (DOI) with low false defect detection rates. In validating CD predictions, the delta CDs from WPI are compared against Aerial Imaging Measurement System (AIMS), where a good correlation is established between WPI and AIMSTM.

Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse

PubMed Central

Chao, Yu-Hsin; Giagtzoglou, Nikolaos; Putluri, Nagireddy; Coarfa, Cristian; Donti, Taraka; Faust, Joseph E.; McNew, James A.; Sardiello, Marco; Baes, Myriam; Bellen, Hugo J.

2017-01-01

Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD. PMID:28640802

Residue Phe112 of the Human-Type Corrinoid Adenosyltransferase (PduO) Enzyme of Lactobacillus reuteri Is Critical to the Formation of the Four-Coordinate Co(II) Corrinoid Substrate and to the Activity of the Enzyme

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

Mera, Paola E.; St. Maurice, Martin; Rayment, Ivan

2009-06-08

ATP:Corrinoid adenosyltransferases (ACAs) catalyze the transfer of the adenosyl moiety from ATP to cob(I)alamin via a four-coordinate cob(II)alamin intermediate. At present, it is unknown how ACAs promote the formation of the four-coordinate corrinoid species needed for activity. The published high-resolution crystal structure of the ACA from Lactobacillus reuteri (LrPduO) in complex with ATP and cob(II)alamin shows that the environment around the alpha face of the corrin ring consists of bulky hydrophobic residues. To understand how these residues promote the generation of the four-coordinate cob(II)alamin, variants of the human-type ACA enzyme from L. reuteri (LrPduO) were kinetically and structurally characterized. Thesemore » studies revealed that residue Phe112 is critical in the displacement of 5,6-dimethylbenzimidazole (DMB) from its coordination bond with the Co ion of the ring, resulting in the formation of the four-coordinate species. An F112A substitution resulted in a 80% drop in the catalytic efficiency of the enzyme. The explanation for this loss of activity was obtained from the crystal structure of the mutant protein, which showed cob(II)alamin bound in the active site with DMB coordinated to the cobalt ion. The crystal structure of an LrPduO(F112H) variant showed a DMB-off/His-on interaction between the corrinoid and the enzyme, whose catalytic efficiency was 4 orders of magnitude lower than that of the wild-type protein. The analysis of the kinetic parameters of LrPduO(F112H) suggests that the F112H substitution negatively impacts product release. Substitutions of other hydrophobic residues in the Cbl binding pocket did not result in significant defects in catalytic efficiency in vitro; however, none of the variant enzymes analyzed in this work supported AdoCbl biosynthesis in vivo.« less

Printability of 1 x reticle defects for submicron design rules

NASA Astrophysics Data System (ADS)

Schurz, Dan L.; Flack, Warren W.; Newman, Gary

1997-02-01

As the push for improved resolution in wafer lithography intensifies and 0.18 micrometer devices are nearing production, the potential impact of subhalf micron reticle defects has become a growing concern. There have been several studies on the printability of subhalf-micron defects on high resolution reduction photolithography equipment. These studies have been extended to 1X lithography systems and more recently to advanced sub-micron 1X steppers. Previous studies have indicated that 0.20 micrometer opaque and 0.25 micrometer clear pinhole defects were at the margins of adversely impacting 0.65 micrometer lithography on a 1X stepper. However, due to the limited number of defects at these sizes on the reticle, definitive conclusions on printability could not be drawn. An additional study, using a three dimensional (3D) optical lithography simulation program, has shown defect size, proximity to an adjacent feature, and feature pitch to be significant factors contributing to reticle defect printability. Using the simulation findings as a guide, a new reticle was designed to contain an increased number of clear pinhole and opaque defects in the 0.15 to 0.30 micrometer range located in multiple pitches of both horizontal and vertical line/space pairs. Defect printability was determined using a 1X i-line projection stepper with focus and exposure optimized for nominal critical dimensions of 0.65 micrometer. The reticle and wafer defects were measured using low voltage SEM metrology. Simulation and experimental results have shown that pitch is the most significant contributor in the printability of clear pinhole, opaque, square and aspect ratio defects. In general, the impact of defect proximity to an adjacent feature is less extreme than the effect of pitch, but is more pronounced for clear pinhole defects. This study suggests that simulation can be a useful tool to help lithographers understand the behavior of reticle defects for particular layout design parameters. Consequently, simulation can be used to develop realistic reticle defect specifications with mask vendors, and improve cost-effectiveness. Defect printability simulation can also be used to predict the effect of known defects on existing reticles to determine if these reticles should be used for manufacturing.

Surface defects evaluation system based on electromagnetic model simulation and inverse-recognition calibration method

NASA Astrophysics Data System (ADS)

Yang, Yongying; Chai, Huiting; Li, Chen; Zhang, Yihui; Wu, Fan; Bai, Jian; Shen, Yibing

2017-05-01

Digitized evaluation of micro sparse defects on large fine optical surfaces is one of the challenges in the field of optical manufacturing and inspection. The surface defects evaluation system (SDES) for large fine optical surfaces is developed based on our previously reported work. In this paper, the electromagnetic simulation model based on Finite-Difference Time-Domain (FDTD) for vector diffraction theory is firstly established to study the law of microscopic scattering dark-field imaging. Given the aberration in actual optical systems, point spread function (PSF) approximated by a Gaussian function is introduced in the extrapolation from the near field to the far field and the scatter intensity distribution in the image plane is deduced. Analysis shows that both diffraction-broadening imaging and geometrical imaging should be considered in precise size evaluation of defects. Thus, a novel inverse-recognition calibration method is put forward to avoid confusion caused by diffraction-broadening effect. The evaluation method is applied to quantitative evaluation of defects information. The evaluation results of samples of many materials by SDES are compared with those by OLYMPUS microscope to verify the micron-scale resolution and precision. The established system has been applied to inspect defects on large fine optical surfaces and can achieve defects inspection of surfaces as large as 850 mm×500 mm with the resolution of 0.5 μm.

Deconvolution imaging of weak reflective pipe defects using guided-wave signals captured by a scanning receiver.

PubMed

Sun, Zeqing; Sun, Anyu; Ju, Bing-Feng

2017-02-01

Guided-wave echoes from weak reflective pipe defects are usually interfered by coherent noise and difficult to interpret. In this paper, a deconvolution imaging method is proposed to reconstruct defect images from synthetically focused guided-wave signals, with enhanced axial resolution. A compact transducer, circumferentially scanning around the pipe, is used to receive guided-wave echoes from discontinuities at a distance. This method achieves a higher circumferential sampling density than arrayed transducers-up to 72 sampling spots per lap for a pipe with a diameter of 180 mm. A noise suppression technique is used to enhance the signal-to-noise ratio. The enhancement in both signal-to-noise ratio and axial resolution of the method is experimentally validated by the detection of two kinds of artificial defects: a pitting defect of 5 mm in diameter and 0.9 mm in maximum depth, and iron pieces attached to the pipe surface. A reconstructed image of the pitting defect is obtained with a 5.87 dB signal-to-noise ratio. It is revealed that a high circumferential sampling density is important for the enhancement of the inspection sensitivity, by comparing the images reconstructed with different down-sampling ratios. A modified full width at half maximum is used as the criterion to evaluate the circumferential extent of the region where iron pieces are attached, which is applicable for defects with inhomogeneous reflection intensity.

Nondestructive Superresolution Imaging of Defects and Nonuniformities in Metals, Semiconductors, Dielectrics, Composites, and Plants Using Evanescent Microwaves

NASA Technical Reports Server (NTRS)

Tabib-Azar, M.; Pathak, P. S.; Ponchak, G.; LeClair, S.

1999-01-01

We have imaged and mapped material nonuniformities and defects using microwaves generated at the end of a microstripline resonator with 0.4 micrometer lateral spatial resolution at 1 GHz. Here we experimentally examine the effect of microstripline substrate permittivity, the feedline-to-resonator coupling strength, and probe tip geometry on the spatial resolution of the probe. Carbon composites, dielectrics, semiconductors, metals, and botanical samples were scanned for defects, residual stresses, subsurface features, areas of different film thickness, and moisture content. The resulting evanescent microwave probe (EMP) images are discussed. The main objective of this work is to demonstrate the overall capabilities of the EMP imaging technique as well as to discuss various probe parameters that can be used to design EMPs for different applications.

High resolution, monochromatic x-ray topography capability at CHESS

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

Finkelstein, K. D., E-mail: kdf1@cornell.edu; Pauling, A.; Brown, Z.

2016-07-27

CHESS has a monochromatic x-ray topography capability serving continually expanding user interest. The setup consists of a beam expanding monochromator, 6-circle diffactometer, and CHESS designed CMOS camera with real time sample-alignment capability. This provides rocking curve mapping with angle resolution as small as 2 µradians, spatial resolution to 3 microns, and field of view up to 7mm. Thus far the capability has been applied for: improving CVD-diamond growth, evaluating perfection of ultra-thin diamond membranes, correlating performance of diamond-based electronics with crystal defect structure, and defect analysis of single crystal silicon carbide. This paper describes our topography system, explains its capabilities,more » and presents experimental results from several applications.« less

cDNA cloning of the human peroxisomal enoyl-CoA hydratase: 3-Hydroxyacyl-CoA dehydrogenase bifunctional enzyme and localization to chromosome 3q26. 3-3q28: A free left Alu arm is inserted in the 3[prime] noncoding region

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

Hoefler, G.; Forstner, M.; Hulla, W.

1994-01-01

Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme is one of the four enzymes of the peroxisomal, [beta]-oxidation pathway. Here, the authors report the full-length human cDNA sequence and the localization of the corresponding gene on chromosome 3q26.3-3q28. The cDNA sequence spans 3779 nucleotides with an open reading frame of 2169 nucleotides. The tripeptide SKL at the carboxy terminus, known to serve as a peroxisomal targeting signal, is present. DNA sequence comparison of the coding region showed an 80% homology between human and rat bifunctional enzyme cDNA. The 3[prime] noncoding sequence contains 117 nucleotides homologous to an Alu repeat. Based on sequence comparison,more » they propose that these nucleotides are a free left Alu arm with 86% homology to the Alu-J family. RNA analysis shows one band with highest intensity in liver and kidney. This cDNA will allow in-depth studies of molecular defects in patients with defective peroxisomal bifunctional enzyme. Moreover, it will also provide a means for studying the regulation of peroxisomal [beta]-oxidation in humans. 33 refs., 5 figs.« less

Repair of localized defects in multilayer-coated reticle blanks for extreme ultraviolet lithography

DOEpatents

Stearns, Daniel G [Los Altos, CA; Sweeney, Donald W [San Ramon, CA; Mirkarimi, Paul B [Sunol, CA

2004-11-23

A method is provided for repairing defects in a multilayer coating layered onto a reticle blank used in an extreme ultraviolet lithography (EUVL) system. Using high lateral spatial resolution, energy is deposited in the multilayer coating in the vicinity of the defect. This can be accomplished using a focused electron beam, focused ion beam or a focused electromagnetic radiation. The absorbed energy will cause a structural modification of the film, producing a localized change in the film thickness. The change in film thickness can be controlled with sub-nanometer accuracy by adjusting the energy dose. The lateral spatial resolution of the thickness modification is controlled by the localization of the energy deposition. The film thickness is adjusted locally to correct the perturbation of the reflected field. For example, when the structural modification is a localized film contraction, the repair of a defect consists of flattening a mound or spreading out the sides of a depression.

Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

PubMed Central

Ding, Jiaxi; Jiang, DeChen; Kurczy, Michael; Nalepka, Jennifer; Dudley, Brian; Merkel, Erin I; Porter, Forbes D; Ewing, Andrew G; Winograd, Nicholas; Burgess, James; Molyneaux, Kathleen

2008-01-01

Background Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival. PMID:19117526

Transmission/Scanning Transmission Electron Microscopy | Materials Science

Science.gov Websites

imaging such as high resolution TEM. Transmission electron diffraction patterns help to determine the microstructure of a material and its defects. Phase-contrast imaging or high-resolution (HR) TEM imaging gives high scattering angle can be collected to form high-resolution, chemically sensitive, atomic number (Z

Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva.

PubMed

Minniti, Alicia N; Labarca, Mariana; Hurtado, Claudia; Brandan, Enrique

2004-10-01

In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. In the present study, we show that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis probably corresponds to SDN-1. In addition, we show that SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 (ok449) deletion mutants. These mutants show a defective egg-laying phenotype. Our results show for the first time, the identification, localization and some functional aspects of syndecan in the nematode C. elegans.

Evolution of a Major Drug Metabolizing Enzyme Defect in the Domestic Cat and Other Felidae: Phylogenetic Timing and the Role of Hypercarnivory

PubMed Central

Shrestha, Binu; Reed, J. Michael; Starks, Philip T.; Kaufman, Gretchen E.; Goldstone, Jared V.; Roelke, Melody E.; O'Brien, Stephen J.; Koepfli, Klaus-Peter; Frank, Laurence G.; Court, Michael H.

2011-01-01

The domestic cat (Felis catus) shows remarkable sensitivity to the adverse effects of phenolic drugs, including acetaminophen and aspirin, as well as structurally-related toxicants found in the diet and environment. This idiosyncrasy results from pseudogenization of the gene encoding UDP-glucuronosyltransferase (UGT) 1A6, the major species-conserved phenol detoxification enzyme. Here, we established the phylogenetic timing of disruptive UGT1A6 mutations and explored the hypothesis that gene inactivation in cats was enabled by minimal exposure to plant-derived toxicants. Fixation of the UGT1A6 pseudogene was estimated to have occurred between 35 and 11 million years ago with all extant Felidae having dysfunctional UGT1A6. Out of 22 additional taxa sampled, representative of most Carnivora families, only brown hyena (Parahyaena brunnea) and northern elephant seal (Mirounga angustirostris) showed inactivating UGT1A6 mutations. A comprehensive literature review of the natural diet of the sampled taxa indicated that all species with defective UGT1A6 were hypercarnivores (>70% dietary animal matter). Furthermore those species with UGT1A6 defects showed evidence for reduced amino acid constraint (increased dN/dS ratios approaching the neutral selection value of 1.0) as compared with species with intact UGT1A6. In contrast, there was no evidence for reduced amino acid constraint for these same species within UGT1A1, the gene encoding the enzyme responsible for detoxification of endogenously generated bilirubin. Our results provide the first evidence suggesting that diet may have played a permissive role in the devolution of a mammalian drug metabolizing enzyme. Further work is needed to establish whether these preliminary findings can be generalized to all Carnivora. PMID:21464924

Clinical significance of enzymatic deficiencies in the gastrointestinal tract with particular reference to lactase deficiency.

PubMed

Rossi, E; Lentze, M J

1984-12-01

The study of deficiencies of small intestinal brush-border hydrolases increased our knowledge about the specific functions of hydrolases in the digestion of smaller molecules on the microvillus surface of the absorptive cells. The sucrase-isomaltase (SI) complex has been shown to be synthesized as a precursor (pro-sucrase-isomaltase) which is then incorporated into the membrane. The hydrophobic N-terminal end of the molecule is anchored in the lipid bilayer. In SI deficiency the molecular base of the disease is still not clear. Absence of SI activity could be due to complete lack of precursor synthesis or to structural changes within the N-terminal end of the SI-complex. Deficiencies of peptide hydrolases have not been reported with the exception of enteropeptidase (EP). Here a congenital deficiency of the enzyme was observed as the primary defect in enzyme synthesis within the enterocytes and as a secondary defect due to exocrine pancreatic insufficiency. In contrast to the primary EP deficiency, the activity of EP can be restored in the cases of exocrine pancreatic insufficiency by treatment with pancreatic extracts. Primary lactase deficiency exists in various forms. Besides congenital lactase deficiency, the late onset or adult type of lactase deficiency has been observed. The latter occurs in many different ethnic groups around the world. Here, using gel electrophoresis and immunoelectrophoresis, the lack of enzyme activity could be shown to be a primary defect in enzyme protein synthesis. In man and in the rat, two different lactases have been identified. In contrast to adult lactase, fetal lactase contains sialic acid at the end of carbohydrate side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of composite/difference field scattering properties between a slightly rough optical surface and multi-body defects.

PubMed

Gong, Lei; Wu, Zhensen; Gao, Ming; Qu, Tan

2018-03-20

The effective extraction of optical surface roughness and defect characteristic provide important realistic values to improve optical system efficiency. Based on finite difference time domain/multi-resolution time domain (FDTD/MRTD) mixed approach, composite scattering between a slightly rough optical surface and multi-body defect particles with different positions is investigated. The scattering contribution of defect particles or the slightly rough optical surface is presented. Our study provides a theoretical and technological basis for the nondestructive examination and optical performance design of nanometer structures.

Biochemical and genetic analysis of Leigh syndrome patients in Korea.

PubMed

Chae, Jong-Hee; Lee, Jin Sook; Kim, Ki Joong; Hwang, Yong Seung; Hirano, Michio

2008-06-01

Sixteen Korean patients with Leigh syndrome were identified at the Seoul National University Children's Hospital in 2001-2006. Biochemical or molecular defects were identified in 14 patients (87.5%). Thirteen patients had respiratory chain enzyme defects; 9 had complex I deficiency, and 4 had combined defects of complex I+III+IV. Based on the biochemical defects, targeted genetic studies in 4 patients with complex I deficiency revealed two heteroplasmic mitochondrial DNA mutations in ND genes. One patient had the mitochondrial DNA T8993G point mutation. No mitochondrial DNA defects were identified in 11 (68.7%) of our LS patients, who probably have mutations in nuclear DNA. Although a limited study based in a single tertiary medical center, our findings suggest that isolated complex I deficiency may be the most common cause of Leigh syndrome in Korea.

Mucopolysaccharidosis type III

MedlinePlus

... the enzymes needed to break down the heparan sulfate sugar chain are missing or defective. There are ... a large amount of a mucopolysaccharide called heparan sulfate in the urine. Other tests may include: Blood ...

Classical late infantile neuronal ceroid lipofuscinosis fibroblasts are deficient in lysosomal tripeptidyl peptidase I.

PubMed

Vines, D J; Warburton, M J

1999-01-25

Tripeptidyl peptidase I (TPP-I) is a lysosomal enzyme that cleaves tripeptides from the N-terminus of polypeptides. A comparison of TPP-I amino acid sequences with sequences derived from an EST database suggested that TPP-I is identical to a pepstatin-insensitive carboxyl proteinase of unknown specificity which is mutated in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal storage disease. Both TPP-I and the carboxyl proteinase have an M(r) of about 46 kDa and are, or are predicted to be, resistant to inhibitors of the four major classes of proteinases. Fibroblasts from LINCL patients have less than 5% of the normal TPP-I activity. The activities of other lysosomal enzymes, including proteinases, are in the normal range. LINCL fibroblasts are also defective at degrading short polypeptides and this defect can be induced in normal fibroblasts by treatment with a specific inhibitor or TPP-I. These results suggest that the cell damage, especially neuronal, observed in LINCL results from the defective degradation and consequent lysosomal storage of small peptides.

Photomask quality assessment solution for 90-nm technology node

NASA Astrophysics Data System (ADS)

Ohira, Katsumi; Chung, Dong Hoon P.; Nobuyuki, Yoshioka; Tateno, Motonari; Matsumura, Kenichi; Chen, Jiunn-Hung; Luk-Pat, Gerard T.; Fukui, Norio; Tanaka, Yoshio

2004-08-01

As 90 nm LSI devices are about to enter pre-production, the cost and turn-around time of photomasks for such devices will be key factors for success in device production. Such devices will be manufactured with state-of-the-art 193nm photolithography systems. Photomasks for these devices are being produced with the most advanced equipment, material and processing technologies and yet, quality assurance still remains an issue for volume production. These issues include defect classification and disposition due to the insufficient resolution of the defect inspection system at conventional review and classification processes and to aggressive RETs, uncertainty of the impact the defects have on the printed feature as well as inconsistencies of classical defect specifications as applied in the sub-wavelength era are becoming a serious problem. Simulation-based photomask qualification using the Virtual Stepper System is widely accepted today as a reliable mask quality assessment tool of mask defects for both the 180 nm and 130 nm technology nodes. This study examines the extendibility of the Virtual Stepper System to 90nm technology node. The proposed method of simulation-based mask qualification uses aerial image defect simulation in combination with a next generation DUV inspection system with shorter wavelength (266nm) and small pixel size combined with DUV high-resolution microscope for some defect cases. This paper will present experimental results that prove the applicability for enabling 90nm technology nodes. Both contact and line/space patterns with varies programmed defects on ArF Attenuated PSM will be used. This paper will also address how to make the strategy production-worthy.

Comparison of binary mask defect printability analysis using virtual stepper system and aerial image microscope system

NASA Astrophysics Data System (ADS)

Phan, Khoi A.; Spence, Chris A.; Dakshina-Murthy, S.; Bala, Vidya; Williams, Alvina M.; Strener, Steve; Eandi, Richard D.; Li, Junling; Karklin, Linard

1999-12-01

As advanced process technologies in the wafer fabs push the patterning processes toward lower k1 factor for sub-wavelength resolution printing, reticles are required to use optical proximity correction (OPC) and phase-shifted mask (PSM) for resolution enhancement. For OPC/PSM mask technology, defect printability is one of the major concerns. Current reticle inspection tools available on the market sometimes are not capable of consistently differentiating between an OPC feature and a true random defect. Due to the process complexity and high cost associated with the making of OPC/PSM reticles, it is important for both mask shops and lithography engineers to understand the impact of different defect types and sizes to the printability. Aerial Image Measurement System (AIMS) has been used in the mask shops for a number of years for reticle applications such as aerial image simulation and transmission measurement of repaired defects. The Virtual Stepper System (VSS) provides an alternative method to do defect printability simulation and analysis using reticle images captured by an optical inspection or review system. In this paper, pre- programmed defects and repairs from a Defect Sensitivity Monitor (DSM) reticle with 200 nm minimum features (at 1x) will be studied for printability. The simulated resist lines by AIMS and VSS are both compared to SEM images of resist wafers qualitatively and quantitatively using CD verification.Process window comparison between unrepaired and repaired defects for both good and bad repair cases will be shown. The effect of mask repairs to resist pattern images for the binary mask case will be discussed. AIMS simulation was done at the International Sematech, Virtual stepper simulation at Zygo and resist wafers were processed at AMD-Submicron Development Center using a DUV lithographic process for 0.18 micrometer Logic process technology.

Defect inspection of actuator lenses using swept-source optical coherence tomography

NASA Astrophysics Data System (ADS)

Lee, Jaeyul; Shirazi, Muhammad Faizan; Park, Kibeom; Jeon, Mansik; Kim, Jeehyun

2017-12-01

Actuator lens industries have gained an enormous interest with the enhancement of various latest communication devices, such as mobile phone and notebooks. The quality of the aforementioned devices can be degraded due to the internal defects of actuator lenses. Therefore, in this study, we implemented swept-source optical coherence tomography (SS-OCT) system to inspect defects of actuator lenses. Owing to the high-resolution of the SS-OCT system, defected foreign substances between the actuator lenses, defective regions of lenses and surface stains were more clearly distinguished through three-dimensional (3D) and two-dimensional (2D) cross-sectional OCT images. Therefore, the implemented SS-OCT system can be considered as a potential application to defect inspection of actuator lens.

Excision of thymine dimers in vitro by extracts of bacteriophage-infected Escherichia coli

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

Friedberg, E.C.; Minton, K.; Pawl, G.

1974-05-01

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from uv- irradiated DNA previously nicked with T4 uv endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 uv-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. Amore » similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7. (auth)« less

Identifying Defects with Guided Algorithms in Bragg Coherent Diffractive Imaging

DOE PAGES

Ulvestad, A.; Nashed, Y.; Beutier, G.; ...

2017-08-30

In this study, crystallographic defects such as dislocations can significantly alter material properties and functionality. However, imaging these imperfections during operation remains challenging due to the short length scales involved and the reactive environments of interest. Bragg coherent diffractive imaging (BCDI) has emerged as a powerful tool capable of identifying dislocations, twin domains, and other defects in 3D detail with nanometer spatial resolution within nanocrystals and grains in reactive environments. However, BCDI relies on phase retrieval algorithms that can fail to accurately reconstruct the defect network. Here, we use numerical simulations to explore different guided phase retrieval algorithms for imagingmore » defective crystals using BCDI. We explore different defect types, defect densities, Bragg peaks, and guided algorithm fitness metrics as a function of signal-to-noise ratio. Based on these results, we offer a general prescription for phasing of defective crystals with no a prior knowledge.« less

The use of platelet-rich plasma combined with demineralized freeze-dried bone allograft in the treatment of periodontal endosseous defects: a report of two clinical cases.

PubMed

Markou, Nikolaos; Pepelassi, Eudoxie; Kotsovilis, Sotirios; Vrotsos, Ioannis; Vavouraki, Helen; Stamatakis, Harry Charalabos

2010-08-01

On the basis of systematic reviews and randomized controlled trials, the authors provide reports of two cases in which platelet-rich plasma (PRP) combined with demineralized freeze-dried bone allograft (DFDBA) was used to treat periodontal endosseous defects. Clinicians treated two circumferential endosseous defects with a probing pocket depth of 5 and 8 millimeters, respectively (case 1), and a combined 1-2-3-wall endosseous defect with a probing pocket depth of 6 mm (case 2) by using the combination of PRP and DFDBA. At six months, complete periodontal pocket resolution occurred in all defects, and clinical attachment level and radiographic defect fill in all defects exhibited significant improvement compared with presurgical values. The combination of PRP and DFDBA may be clinically and radiographically efficacious in the treatment of periodontal endosseous defects.

Identifying Defects with Guided Algorithms in Bragg Coherent Diffractive Imaging

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

Ulvestad, A.; Nashed, Y.; Beutier, G.

In this study, crystallographic defects such as dislocations can significantly alter material properties and functionality. However, imaging these imperfections during operation remains challenging due to the short length scales involved and the reactive environments of interest. Bragg coherent diffractive imaging (BCDI) has emerged as a powerful tool capable of identifying dislocations, twin domains, and other defects in 3D detail with nanometer spatial resolution within nanocrystals and grains in reactive environments. However, BCDI relies on phase retrieval algorithms that can fail to accurately reconstruct the defect network. Here, we use numerical simulations to explore different guided phase retrieval algorithms for imagingmore » defective crystals using BCDI. We explore different defect types, defect densities, Bragg peaks, and guided algorithm fitness metrics as a function of signal-to-noise ratio. Based on these results, we offer a general prescription for phasing of defective crystals with no a prior knowledge.« less

Nonlinear air-coupled emission: The signature to reveal and image microdamage in solid materials

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

Solodov, Igor; Busse, Gerd

2007-12-17

It is shown that low-frequency elastic vibrations of near-surface planar defects cause high-frequency ultrasonic radiation in surrounding air. The frequency conversion mechanism is concerned with contact nonlinearity of the defect vibrations and provides efficient generation of air-coupled higher-order ultraharmonics, ultrasubharmonics, and combination frequencies. The nonlinear air-coupled ultrasonic emission is applied for location and high-resolution imaging of damage-induced defects in a variety of solid materials.

Improving Bone Formation in a Rat Femur Segmental Defect by Controlling Bone Morphogenetic Protein-2 Release

DTIC Science & Technology

2011-04-01

tissue and polymer: mineralized tissue stained dark green, osteoid and collagen bright red, soft tissue pink to light green, and erythrocytes bright...of bone, soft tissue , and polymer, high-resolution digital images were acquired at 1.25 · or 20 · . The area of interest comprising the bone defect...bone, soft tissue , and polymer (when present) within the defect were quantified using Metamorph software (Molecular Devices, Inc.) and were calculated

Characterization of Local Carrier Dynamics in AlN and AlGaN Films using High Spatial- and Time-resolution Cathodoluminescence Spectroscopy

DTIC Science & Technology

2012-10-12

21/2012 Abstract: In order to assess the impacts of structural and point defects on the local carrier (exciton) recombination dynamics in...quantitatively understood as functions of structural / point defect and impurity concentrations (crystal imperfections). However, only few papers [5...NOTES 14. ABSTRACT In order to assess the impacts of structural and point defects on the local carrier (exciton) recombination dynamics in wide bandgap

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 Å resolution

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

Maraite, Andy; Schmidt, Thomas; Ansörge-Schumacher, Marion B.

2007-07-01

The X-ray crystal structure of the ThDP-dependent enzyme benzaldehyde lyase has been refined to 1.65 Å. Benzaldehyde lyase (BAL; EC 4.1.2.38) is a thiamine diphosphate (ThDP) dependent enzyme that catalyses the enantioselective carboligation of two molecules of benzaldehyde to form (R)-benzoin. BAL has hence aroused interest for its potential in the industrial synthesis of optically active benzoins and derivatives. The structure of BAL was previously solved to a resolution of 2.6 Å using MAD experiments on a selenomethionine derivative [Mosbacher et al. (2005 ▶), FEBS J.272, 6067–6076]. In this communication of parallel studies, BAL was crystallized in an alternative spacemore » group (P2{sub 1}2{sub 1}2{sub 1}) and its structure refined to a resolution of 1.65 Å, allowing detailed observation of the water structure, active-site interactions with ThDP and also the electron density for the co-solvent 2-methyl-2,4-pentanediol (MPD) at hydrophobic patches of the enzyme surface.« less

Resolution of Toll-like receptor 4-mediated acute lung injury is linked to eicosanoids and suppressor of cytokine signaling 3

PubMed Central

Hilberath, Jan N.; Carlo, Troy; Pfeffer, Michael A.; Croze, Roxanne H.; Hastrup, Frantz; Levy, Bruce D.

2011-01-01

The purpose of this study was to investigate roles for Toll-like receptor 4 (TLR4) in host responses to sterile tissue injury. Hydrochloric acid was instilled into the left mainstem bronchus of TLR4-defective (both C3H/HeJ and congenic C.C3-Tlr4Lps-d/J) and control mice to initiate mild, self-limited acute lung injury (ALI). Outcome measures included respiratory mechanics, barrier integrity, leukocyte accumulation, and levels of select soluble mediators. TLR4-defective mice were more resistant to ALI, with significantly decreased perturbations in lung elastance and resistance, resulting in faster resolution of these parameters [resolution interval (Ri); ∼6 vs. 12 h]. Vascular permeability changes and oxidative stress were also decreased in injured HeJ mice. These TLR4-defective mice paradoxically displayed increased lung neutrophils [(HeJ) 24×103 vs. (control) 13×103 cells/bronchoalveolar lavage]. Proresolving mechanisms for TLR4-defective animals included decreased eicosanoid biosynthesis, including cysteinyl leukotrienes (80% mean decrease) that mediated CysLT1 receptor-dependent vascular permeability changes; and induction of lung suppressor of cytokine signaling 3 (SOCS3) expression that decreased TLR4-driven oxidative stress. Together, these findings indicate pivotal roles for TLR4 in promoting sterile ALI and suggest downstream provocative roles for cysteinyl leukotrienes and protective roles for SOCS3 in the intensity and duration of host responses to ALI.—Hilberath, J N., Carlo, T., Pfeffer, M. A., Croze, R. H., Hastrup, F., Levy, B. D. Resolution of Toll-like receptor 4-mediated acute lung injury is linked to eicosanoids and suppressor of cytokine signaling 3. PMID:21321188

The Roles of β-Oxidation and Cofactor Homeostasis in Peroxisome Distribution and Function in Arabidopsis thaliana

PubMed Central

Rinaldi, Mauro A.; Patel, Ashish B.; Park, Jaeseok; Lee, Koeun; Strader, Lucia C.; Bartel, Bonnie

2016-01-01

Key steps of essential metabolic pathways are housed in plant peroxisomes. We conducted a microscopy-based screen for anomalous distribution of peroxisomally targeted fluorescence in Arabidopsis thaliana. This screen uncovered 34 novel alleles in 15 genes affecting oil body mobilization, fatty acid β-oxidation, the glyoxylate cycle, peroxisome fission, and pexophagy. Partial loss-of-function of lipid-mobilization enzymes conferred peroxisomes clustered around retained oil bodies without other notable defects, suggesting that this microscopy-based approach was sensitive to minor perturbations, and that fatty acid β-oxidation rates in wild type are higher than required for normal growth. We recovered three mutants defective in PECTIN METHYLESTERASE31, revealing an unanticipated role in lipid mobilization for this cytosolic enzyme. Whereas mutations reducing fatty acid import had peroxisomes of wild-type size, mutations impairing fatty acid β-oxidation displayed enlarged peroxisomes, possibly caused by excess fatty acid β-oxidation intermediates in the peroxisome. Several fatty acid β-oxidation mutants also displayed defects in peroxisomal matrix protein import. Impairing fatty acid import reduced the large size of peroxisomes in a mutant defective in the PEROXISOMAL NAD+ TRANSPORTER (PXN), supporting the hypothesis that fatty acid accumulation causes pxn peroxisome enlargement. The diverse mutants isolated in this screen will aid future investigations of the roles of β-oxidation and peroxisomal cofactor homeostasis in plant development. PMID:27605050

Enzyme replacement prevents enamel defects in hypophosphatasia mice

PubMed Central

Yadav, Manisha C.; de Oliveira, Rodrigo Cardoso; Foster, Brian L.; Fong, Hanson; Cory, Esther; Narisawa, Sonoko; Sah, Robert L.; Somerman, Martha; Whyte, Michael P.; Millán, José Luis

2012-01-01

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl−/−, a.k.a. Akp2−/−) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl−/− mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl−/− mice, histological, μCT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP (sALP-FcD10, a.k.a. ENB-0040) at 8.2 mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization, and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP. PMID:22461224

Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

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

O'Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and themore » production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.« less

In situ high temperature microwave microscope for nondestructive detection of surface and sub-surface defects.

PubMed

Wang, Peiyu; Li, Zhencheng; Pei, Yongmao

2018-04-16

An in situ high temperature microwave microscope was built for detecting surface and sub-subsurface structures and defects. This system was heated with a self-designed quartz lamp radiation module, which is capable of heating to 800°C. A line scanning of a metal grating showed a super resolution of 0.5 mm (λ/600) at 1 GHz. In situ scanning detections of surface hole defects on an aluminium plate and a glass fiber reinforced plastic (GFRP) plate were conducted at different high temperatures. A post processing algorithm was proposed to remove the background noises induced by high temperatures and the 3.0 mm-spaced hole defects were clearly resolved. Besides, hexagonal honeycomb lattices were in situ detected and clearly resolved under a 1.0 mm-thick face panel at 20°C and 50°C, respectively. The core wall positions and bonding width were accurately detected and evaluated. In summary, this in situ microwave microscope is feasible and effective in sub-surface detection and super resolution imaging at different high temperatures.

Digestion of peptidoglycan near the cross-link is necessary for the growth of Bacillus subtilis.

PubMed

Hashimoto, Masayuki; Matsushima, Hiroaki; Suparthana, I Putu; Ogasawara, Hiroshi; Yamamoto, Hiroki; Teng, ChingHao; Sekiguchi, Junichi

2018-03-01

Bacterial cells are covered with peptidoglycan (PG) layer(s), serving as the cellular exoskeleton. The PG sacculus changes its shape during cell growth, and thus both the synthesis and disassembly of PG are important for cell proliferation. In Bacillus subtilis, four dl-endopeptidases (DLEPases; LytE, LytF, CwlO and CwlS) are involved in the maintenance of cell morphology. The lytE cwlO double mutant exhibits synthetic lethality and defective cell elongation, while the lytE lytF cwlS triple mutant exhibits defective cell separation, albeit with septum formation. LytE is involved in both cell separation and elongation. We propose that DLEPases have varied roles in cell separation and elongation. To determine these roles, the catalytic domain of LytE was substituted with another catalytic domain that digests the other bonds in PG. By using the chimeric enzymes, we assessed the suppression of the synthetic lethality by the cell elongation defect and the disruption of chain morphology by the cell separation defect. All the constructed chimeric enzymes suppressed the cell separation defect, restoring the chain morphology. Digestion at any position of PG broke the linkage between two daughter cells, releasing them from each other. However, only d,d-endopeptidases suppressed the lack of DLEPase in the lytE cwlO double mutant. This indicated that the release of tension on the expanding PG sacculus is not the sole essential function of DLEPases. Considering that the structure of the digested PG is important for cell elongation, the digested product might be reused in the growth process in some way.

Heparan Sulfate Expression in the Neural Crest is Essential for Mouse Cardiogenesis

PubMed Central

Pan, Yi; Carbe, Christian; Pickhinke, Ute; Kupich, Sabine; Ohlig, Stefanie; Frye, Maike; Seelige, Ruth; Pallerla, Srinivas R.; Moon, Anne M.; Lawrence, Roger; Esko, Jeffrey D.; Zhang, Xin; Grobe, Kay

2015-01-01

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-Deacetylase/GlcN N-Sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1−/− embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect. PMID:24200809

CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation.

PubMed

Baresova, Veronika; Krijt, Matyas; Skopova, Vaclava; Souckova, Olga; Kmoch, Stanislav; Zikanova, Marie

2016-11-01

Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential therapeutic agents. Copyright © 2016 Elsevier Inc. All rights reserved.

Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

PubMed

Scollar, M P; Sigal, G; Klibanov, A M

1985-03-01

Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

Chronic Invasive Fungal Sinusitis Presenting as Inferior Altitudinal Visual Field Defect.

PubMed

Bansal, Reema; Takkar, Aastha; Lal, Vivek; Bal, Amanjit; Bansal, Sandeep

2017-06-01

A young male with acute blurring of vision (6/9) complained of an inferior altitudinal field defect in right eye. Clinical ophthalmological examination was normal. Magnetic resonance imaging (MRI) of the brain revealed the expansion and mucosal thickening of right posterior ethmoid and sphenoid sinuses and opacified right maxillary sinus. Surgical intervention (transethmoidal sphenoidotomy) and histopathological examination revealed chronic invasive granulomatous fungal sinusitis. Anti-fungal therapy led to resolution of visual complaints and restoration of visual field defects.

Middle cranial fossa approach to repair tegmen defects assisted by three-dimensionally printed temporal bone models.

PubMed

Ahmed, Sameer; VanKoevering, Kyle K; Kline, Stephanie; Green, Glenn E; Arts, H Alexander

2017-10-01

To explore the perioperative utility of three-dimensionally (3D)-printed temporal bone models of patients undergoing repair of lateral skull base defects and spontaneous cerebrospinal fluid leaks with the middle cranial fossa approach. Case series. 3D-printed temporal bone models-based on patient-specific, high-resolution computed tomographic imaging-were constructed using inexpensive polymer materials. Preoperatively, the models demonstrated the extent of temporal lobe retraction necessary to visualize the proposed defects in the lateral skull base. Also preoperatively, Silastic sheeting was arranged across the modeled tegmen, marked, and cut to cover all of the proposed defect sites. The Silastic sheeting was then sterilized and subsequently served as a precise intraoperative template for a synthetic dural replacement graft. Of note, these grafts were customized without needing to retract the temporal lobe. Five patients underwent the middle cranial fossa approach assisted by 3D-printed temporal bone models to repair tegmen defects and spontaneous cerebrospinal fluid leaks. No complications were encountered. The prefabricated dural repair grafts were easily placed and fit precisely onto the middle fossa floor without any additional modifications. All defects were covered as predicted by the 3D temporal bone models. At their postoperative visits, all five patients maintained resolution of their spontaneous cerebrospinal fluid leaks. Inexpensive 3D-printed temporal bone models of tegmen defects can serve as beneficial adjuncts during lateral skull base repair. The models provide a panoramic preoperative view of all tegmen defects and allow for custom templating of dural grafts without temporal lobe retraction. 4 Laryngoscope, 127:2347-2351, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Can three-dimensional high-resolution anorectal manometry detect anal sphincter defects in patients with faecal incontinence?

PubMed

Rezaie, A; Iriana, S; Pimentel, M; Murrell, Z; Fleshner, P; Zaghiyan, K

2017-05-01

Endoanal ultrasound (EAUS) is the gold standard for detecting anal sphincter defects in patients with faecal incontinence (FI), while anorectal manometry evaluates sphincter function. Three-dimensional high-resolution anorectal manometry (3D HRAM) is a newer modality with the potential to assess both sphincter function and anatomy. The purpose of the present study was to compare 3D HRAM with 3D EAUS for the detection of anal sphincter defects in patients with FI. A linkage analysis was performed between the 3D HRAM and 3D EAUS databases of a tertiary referral centre to identify patients with FI who underwent both 3D EAUS and 3D HRAM. With 3D HRAM, a defect was defined as any pressure measurement below 25 mmHg at rest with at least 18° of continuous expansion. The 3D HRAM findings were compared with those of 3D EAUS. The study cohort included 39 patients with a mean age of 64.7 ± 15.2 years (SD); and 31 (79%) were female. Eight (21%) patients had an anal sphincter defect on EAUS with a median size of 93° (range 40°-136°). Fourteen (36%) had a defect shown by 3D HRAM with a median size of 144° (36°-180°). The sensitivity, specificity and positive and negative predictive values of 3D HRAM in detecting a sphincter defect were 75%, 74%, 43% and 92%, respectively. With a negative predictive value of 92%, 3D HRAM may be a useful screening method for ruling out a sphincter defect in patients with FI, thereby avoiding both EAUS and manometry in selected patients. Colorectal Disease © 2016 The Association of Coloproctology of Great Britain and Ireland.

Novel dental dynamic depth profilometric imaging using simultaneous frequency-domain infrared photothermal radiometry and laser luminescence

NASA Astrophysics Data System (ADS)

Nicolaides, Lena; Mandelis, Andreas

2000-01-01

A high-spatial-resolution dynamic experimental imaging setup, which can provide simultaneous measurements of laser- induced frequency-domain infrared photothermal radiometric and luminescence signals from defects in teeth, has been developed for the first time. The major findings of this work are: (1) radiometric images are complementary to (anticorrelated with) luminescence images, as a result of the nature of the two physical signal generation processes; (2) the radiometric amplitude exhibits much superior dynamic (signal resolution) range to luminescence in distinguishing between intact and cracked sub-surface structures in the enamel; (3) the radiometric signal (amplitude and phase) produces dental images with much better defect localization, delineation, and resolution; (4) radiometric images (amplitude and phase) at a fixed modulation frequency are depth profilometric, whereas luminescence images are not; and (5) luminescence frequency responses from enamel and hydroxyapatite exhibit two relaxation lifetimes, the longer of which (approximately ms) is common to all and is not sensitive to the defect state and overall quality of the enamel. Simultaneous radiometric and luminescence frequency scans for the purpose of depth profiling were performed and a quantitative theoretical two-lifetime rate model of dental luminescence was advanced.

The chaperone role of the pyridoxal 5'-phosphate and its implications for rare diseases involving B6-dependent enzymes.

PubMed

Cellini, Barbara; Montioli, Riccardo; Oppici, Elisa; Astegno, Alessandra; Voltattorni, Carla Borri

2014-02-01

The biologically active form of the B6 vitamers is pyridoxal 5'-phosphate (PLP), which plays a coenzymatic role in several distinct enzymatic activities ranging from the synthesis, interconversion and degradation of amino acids to the replenishment of one-carbon units, synthesis and degradation of biogenic amines, synthesis of tetrapyrrolic compounds and metabolism of amino-sugars. In the catalytic process of PLP-dependent enzymes, the substrate amino acid forms a Schiff base with PLP and the electrophilicity of the PLP pyridine ring plays important roles in the subsequent catalytic steps. While the essential role of PLP in the acquisition of biological activity of many proteins is long recognized, the finding that some PLP-enzymes require the coenzyme for refolding in vitro points to an additional role of PLP as a chaperone in the folding process. Mutations in the genes encoding PLP-enzymes are causative of several rare inherited diseases. Patients affected by some of these diseases (AADC deficiency, cystathionuria, homocystinuria, gyrate atrophy, primary hyperoxaluria type 1, xanthurenic aciduria, X-linked sideroblastic anaemia) can benefit, although at different degrees, from the administration of pyridoxine, a PLP precursor. The effect of the coenzyme is not limited to mutations that affect the enzyme-coenzyme interaction, but also to those that cause folding defects, reinforcing the idea that PLP could play a chaperone role and improve the folding efficiency of misfolded variants. In this review, recent biochemical and cell biology studies highlighting the chaperoning activity of the coenzyme on folding-defective variants of PLP-enzymes associated with rare diseases are presented and discussed. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy.

PubMed

Murthy, Vaibhav; Dacus, Dalton; Gamez, Monica; Hu, Changkun; Wendel, Sebastian O; Snow, Jazmine; Kahn, Andrew; Walterhouse, Stephen H; Wallace, Nicholas A

2018-06-08

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

The molecular defect of ferrochelatase in a patient with erythropoietic protoporphyria.

PubMed Central

Nakahashi, Y; Fujita, H; Taketani, S; Ishida, N; Kappas, A; Sassa, S

1992-01-01

The molecular basis of an inherited defect of ferrochelatase in a patient with erythropoietic protoporphyria (EPP) was investigated. Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway and catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. In Epstein-Barr virus-transformed lymphoblastoid cells from a proband with EPP, enzyme activity, an immunochemically quantifiable protein, and mRNA content of ferrochelatase were about one-half the normal level. In contrast, the rate of transcription of ferrochelatase mRNA in the proband's cells was normal, suggesting that decreased ferrochelatase mRNA is due to an unstable transcript. cDNA clones encoding ferrochelatase in the proband, isolated by amplification using the polymerase chain reaction, were found to be classified either into those encoding the normal protein or into those encoding an abnormal protein that lacked exon 2 of the ferrochelatase gene, indicating that the proband is heterozygous for the ferrochelatase defect. Genomic DNA analysis revealed that the abnormal allele had a point mutation, C----T, near the acceptor site of intron 1. This point mutation appears to be responsible for the post-transcriptional splicing abnormality resulting in an aberrant transcript of ferrochelatase in this patient. Images PMID:1729699

Age-related defects in erythrocyte 2,3-diphosphoglycerate metabolism in dementia.

PubMed

Kaminsky, Yury G; Reddy, V Prakash; Ashraf, Ghulam Md; Ahmad, Ausaf; Benberin, Valery V; Kosenko, Elena A; Aliev, Gjumrakch

2013-01-01

Alzheimer disease (AD) is the most common dementing illness. Metabolic defects in the brain with aging contribute to the pathogenesis of AD. These changes can be found systematically and thus can be used as potential biomarkers. Erythrocytes (RBCs) are passive "reporter cells" that are not well studied in AD. In the present study, we analyzed an array of glycolytic and related enzymes and intermediates in RBCs from patients with AD and non-Alzheimer dementia (NA), age-matched controls (AC) and young adult controls (YC). AD is characterized by higher activities of hexokinase, phosphofructokinase, and bisphosphoglycerate mutase and bisphosphoglycerate phosphatase in RBCs. In our study, we observed that glycolytic and related enzymes displayed significantly lower activities in AC. However, similar or significantly higher activities were observed in AD and NA groups as compared to YC group. 2,3-diphosphoglycerate (2,3-DPG) levels were significantly decreased in AD and NA patients. The pattern of changes between groups in the above indices strongly correlates with each other. Collectively, our data suggested that AD and NA patients are associated with chronic disturbance of 2,3-DPG metabolism in RBCs. These defects may play a pivotal role in physiological processes, which predispose elderly subjects to AD and NA.

Effect of dose and size on defect engineering in carbon cluster implanted silicon wafers

NASA Astrophysics Data System (ADS)

Okuyama, Ryosuke; Masada, Ayumi; Shigematsu, Satoshi; Kadono, Takeshi; Hirose, Ryo; Koga, Yoshihiro; Okuda, Hidehiko; Kurita, Kazunari

2018-01-01

Carbon-cluster-ion-implanted defects were investigated by high-resolution cross-sectional transmission electron microscopy toward achieving high-performance CMOS image sensors. We revealed that implantation damage formation in the silicon wafer bulk significantly differs between carbon-cluster and monomer ions after implantation. After epitaxial growth, small and large defects were observed in the implanted region of carbon clusters. The electron diffraction pattern of both small and large defects exhibits that from bulk crystalline silicon in the implanted region. On the one hand, we assumed that the silicon carbide structure was not formed in the implanted region, and small defects formed because of the complex of carbon and interstitial silicon. On the other hand, large defects were hypothesized to originate from the recrystallization of the amorphous layer formed by high-dose carbon-cluster implantation. These defects are considered to contribute to the powerful gettering capability required for high-performance CMOS image sensors.

Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes.

PubMed Central

Mittl, P. R.; Schulz, G. E.

1994-01-01

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel beta-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones. PMID:8061609

Resolution of Toll-like receptor 4-mediated acute lung injury is linked to eicosanoids and suppressor of cytokine signaling 3.

PubMed

Hilberath, Jan N; Carlo, Troy; Pfeffer, Michael A; Croze, Roxanne H; Hastrup, Frantz; Levy, Bruce D

2011-06-01

The purpose of this study was to investigate roles for Toll-like receptor 4 (TLR4) in host responses to sterile tissue injury. Hydrochloric acid was instilled into the left mainstem bronchus of TLR4-defective (both C3H/HeJ and congenic C.C3-Tlr4(Lps-d)/J) and control mice to initiate mild, self-limited acute lung injury (ALI). Outcome measures included respiratory mechanics, barrier integrity, leukocyte accumulation, and levels of select soluble mediators. TLR4-defective mice were more resistant to ALI, with significantly decreased perturbations in lung elastance and resistance, resulting in faster resolution of these parameters [resolution interval (R(i)); ∼6 vs. 12 h]. Vascular permeability changes and oxidative stress were also decreased in injured HeJ mice. These TLR4-defective mice paradoxically displayed increased lung neutrophils [(HeJ) 24×10(3) vs. (control) 13×10(3) cells/bronchoalveolar lavage]. Proresolving mechanisms for TLR4-defective animals included decreased eicosanoid biosynthesis, including cysteinyl leukotrienes (80% mean decrease) that mediated CysLT1 receptor-dependent vascular permeability changes; and induction of lung suppressor of cytokine signaling 3 (SOCS3) expression that decreased TLR4-driven oxidative stress. Together, these findings indicate pivotal roles for TLR4 in promoting sterile ALI and suggest downstream provocative roles for cysteinyl leukotrienes and protective roles for SOCS3 in the intensity and duration of host responses to ALI.

A Role for Cytosolic Fumarate Hydratase in Urea Cycle Metabolism and Renal Neoplasia

PubMed Central

Adam, Julie; Yang, Ming; Bauerschmidt, Christina; Kitagawa, Mitsuhiro; O’Flaherty, Linda; Maheswaran, Pratheesh; Özkan, Gizem; Sahgal, Natasha; Baban, Dilair; Kato, Keiko; Saito, Kaori; Iino, Keiko; Igarashi, Kaori; Stratford, Michael; Pugh, Christopher; Tennant, Daniel A.; Ludwig, Christian; Davies, Benjamin; Ratcliffe, Peter J.; El-Bahrawy, Mona; Ashrafian, Houman; Soga, Tomoyoshi; Pollard, Patrick J.

2013-01-01

Summary The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target. PMID:23643539

A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

PubMed

Adam, Julie; Yang, Ming; Bauerschmidt, Christina; Kitagawa, Mitsuhiro; O'Flaherty, Linda; Maheswaran, Pratheesh; Özkan, Gizem; Sahgal, Natasha; Baban, Dilair; Kato, Keiko; Saito, Kaori; Iino, Keiko; Igarashi, Kaori; Stratford, Michael; Pugh, Christopher; Tennant, Daniel A; Ludwig, Christian; Davies, Benjamin; Ratcliffe, Peter J; El-Bahrawy, Mona; Ashrafian, Houman; Soga, Tomoyoshi; Pollard, Patrick J

2013-05-30

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Proteoliposomes as matrix vesicles’ biomimetics to study the initiation of skeletal mineralization

PubMed Central

Simão, A.M.S.; Yadav, M.C.; Ciancaglini, P.; Millán, J.L.

2017-01-01

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants. PMID:20401430

Proteoliposomes as matrix vesicles' biomimetics to study the initiation of skeletal mineralization.

PubMed

Simão, A M S; Yadav, M C; Ciancaglini, P; Millán, J L

2010-03-01

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.

Studying post-etching silicon crystal defects on 300mm wafer by automatic defect review AFM

NASA Astrophysics Data System (ADS)

Zandiatashbar, Ardavan; Taylor, Patrick A.; Kim, Byong; Yoo, Young-kook; Lee, Keibock; Jo, Ahjin; Lee, Ju Suk; Cho, Sang-Joon; Park, Sang-il

2016-03-01

Single crystal silicon wafers are the fundamental elements of semiconductor manufacturing industry. The wafers produced by Czochralski (CZ) process are very high quality single crystalline materials with known defects that are formed during the crystal growth or modified by further processing. While defects can be unfavorable for yield for some manufactured electrical devices, a group of defects like oxide precipitates can have both positive and negative impacts on the final device. The spatial distribution of these defects may be found by scattering techniques. However, due to limitations of scattering (i.e. light wavelength), many crystal defects are either poorly classified or not detected. Therefore a high throughput and accurate characterization of their shape and dimension is essential for reviewing the defects and proper classification. While scanning electron microscopy (SEM) can provide high resolution twodimensional images, atomic force microscopy (AFM) is essential for obtaining three-dimensional information of the defects of interest (DOI) as it is known to provide the highest vertical resolution among all techniques [1]. However AFM's low throughput, limited tip life, and laborious efforts for locating the DOI have been the limitations of this technique for defect review for 300 mm wafers. To address these limitations of AFM, automatic defect review AFM has been introduced recently [2], and is utilized in this work for studying DOI on 300 mm silicon wafer. In this work, we carefully etched a 300 mm silicon wafer with a gaseous acid in a reducing atmosphere at a temperature and for a sufficient duration to decorate and grow the crystal defects to a size capable of being detected as light scattering defects [3]. The etched defects form a shallow structure and their distribution and relative size are inspected by laser light scattering (LLS). However, several groups of defects couldn't be properly sized by the LLS due to the very shallow depth and low light scattering. Likewise, SEM cannot be used effectively for post-inspection defect review and classification of these very shallow types of defects. To verify and obtain accurate shape and three-dimensional information of those defects, automatic defect review AFM (ADR AFM) is utilized for accurate locating and imaging of DOI. In ADR AFM, non-contact mode imaging is used for non-destructive characterization and preserving tip sharpness for data repeatability and reproducibility. Locating DOI and imaging are performed automatically with a throughput of many defects per hour. Topography images of DOI has been collected and compared with SEM images. The ADR AFM has been shown as a non-destructive metrology tool for defect review and obtaining three-dimensional topography information.

Mice lacking the USP2 deubiquitinating enzyme have severe male subfertility associated with defects in fertilization and sperm motility.

PubMed

Bedard, Nathalie; Yang, Yaoming; Gregory, Mary; Cyr, Daniel G; Suzuki, João; Yu, Xiaomin; Chian, Ri-Cheng; Hermo, Louis; O'Flaherty, Cristian; Smith, Charles E; Clarke, Hugh J; Wing, Simon S

2011-09-01

The ubiquitin-proteasome system plays an important role in spermatogenesis. However, the functions of deubiquitinating enzymes in this process remain poorly characterized. We previously showed that the deubiquitinating enzyme USP2 is induced in late elongating spermatids. To identify its function, we generated mice lacking USP2. Usp2 -/- mice appeared normal, and the weights of major organs, including the testis, did not differ from wild type (Usp2 +/+). However, although the numbers of testicular spermatids and epididymal spermatozoa were normal in Usp2 -/- males, these animals had a severe defect in fertility, yielding only 12% as many offspring as Usp2 +/+ littermates. Spermatogenesis in Usp2 -/- mice was morphologically normal except for the presence of abnormal aggregations of elongating spermatids and formation of multinucleated cells in some tubules. The epididymal epithelium was morphologically normal in Usp2 -/- mice, but some abnormal cells other than sperm were present in the lumen. Usp2 -/- epididymal spermatozoa manifested normal motility when incubated in culture media, but rapidly became immotile when incubated in PBS in contrast to Usp2 +/+ spermatozoa, which largely maintained motility under this condition. Usp2 -/- and +/+ spermatozoa underwent acrosome reactions in vitro with similar frequency. In vitro fertilization assays demonstrated a severe defect in the ability of Usp2 -/- spermatozoa to fertilize eggs. This could be bypassed by intracytoplasmic sperm injection or removal of the zona pellucida, which resulted in fertilization rates similar to that of Usp2 +/+ mice. We demonstrate for the first time, using mouse transgenic approaches, a role for the ubiquitin system in fertilization.

Treatment of Refractory Filamentary Keratitis With Autologous Serum Tears.

PubMed

Read, Sarah P; Rodriguez, Marianeli; Dubovy, Sander; Karp, Carol L; Galor, Anat

2017-09-01

To report a case of filamentary keratitis (FK) successfully treated with autologous serum tears and to review the pathogenesis and management of FK. Case report including high-resolution anterior segment optical coherence tomography and filament histopathology. A 61-year-old Hispanic man presented with pain and photophobia of the right eye. He was found to have a corneal epithelial defect and a small peripheral infiltrate 4 months after Laser Assisted in situ Keratomileusis. After resolution of the epithelial defect, he developed FK. Over a 4-month period, conservative management with aggressive lubrication, lid hygiene, topical corticosteroids, topical cyclosporine, bandage contact lenses, and oral doxycycline failed to resolve the corneal filaments. Notably, treatment with 20% autologous serum tears, four times daily, led to a sustained resolution of the FK within 1 week. This case demonstrates the complexity of FK management and introduces autologous serum tears as a viable management option when conservative approaches to this condition fail.

Study in Mice Links Key Signaling Molecule to Underlying Cause of Osteogenesis Imperfecta

MedlinePlus

... by mutations in a gene that codes for collagen, an abundant structural component of bone. This type ... linked to defects in enzymes that help process collagen to its mature form. These types of OI ...

Crystal Structures of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

PubMed Central

Mileni, Mauro; Kamtekar, Satwik; Wood, David C.; Benson, Timothy E.; Cravatt, Benjamin F.; Stevens, Raymond C.

2010-01-01

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 Å resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network, and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH’s catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 Å) than previously reported. The higher resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggest a functional convergence between the amidase signature enzymes and serine proteases. PMID:20493882

Crystal structure of fatty acid amide hydrolase bound to the carbamate inhibitor URB597: discovery of a deacylating water molecule and insight into enzyme inactivation.

PubMed

Mileni, Mauro; Kamtekar, Satwik; Wood, David C; Benson, Timothy E; Cravatt, Benjamin F; Stevens, Raymond C

2010-07-23

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 A resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 A) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

RAYSAW: a log sawing simulator for 3D laser-scanned hardwood logs

Treesearch

R. Edward Thomas

2013-01-01

Laser scanning of hardwood logs provides detailed high-resolution imagery of log surfaces. Characteristics such as sweep, taper, and crook, as well as most surface defects, are visible to the eye in the scan data. In addition, models have been developed that predict interior knot size and position based on external defect information. Computerized processing of...

Defect-induced local variation of crystal phase transition temperature in metal-halide perovskites.

PubMed

Dobrovolsky, Alexander; Merdasa, Aboma; Unger, Eva L; Yartsev, Arkady; Scheblykin, Ivan G

2017-06-26

Solution-processed organometal halide perovskites are hybrid crystalline semiconductors highly interesting for low-cost and efficient optoelectronics. Their properties are dependent on the crystal structure. Literature shows a variety of crystal phase transition temperatures and often a spread of the transition over tens of degrees Kelvin. We explain this inconsistency by demonstrating that the temperature of the tetragonal-to-orthorhombic phase transition in methylammonium lead triiodide depends on the concentration and nature of local defects. Phase transition in individual nanowires was studied by photoluminescence microspectroscopy and super-resolution imaging. We propose that upon cooling from 160 to 140 K, domains of the crystal containing fewer defects stay in the tetragonal phase longer than highly defected domains that readily transform to the high bandgap orthorhombic phase at higher temperatures. The existence of relatively pure tetragonal domains during the phase transition leads to drastic photoluminescence enhancement, which is inhomogeneously distributed across perovskite microcrystals.Understanding crystal phase transition in materials is of fundamental importance. Using luminescence spectroscopy and super-resolution imaging, Dobrovolsky et al. study the transition from the tetragonal to orthorhombic crystal phase in methylammonium lead triiodide nanowires at low temperature.

High resolution structural characterisation of laser-induced defect clusters inside diamond

NASA Astrophysics Data System (ADS)

Salter, Patrick S.; Booth, Martin J.; Courvoisier, Arnaud; Moran, David A. J.; MacLaren, Donald A.

2017-08-01

Laser writing with ultrashort pulses provides a potential route for the manufacture of three-dimensional wires, waveguides, and defects within diamond. We present a transmission electron microscopy study of the intrinsic structure of the laser modifications and reveal a complex distribution of defects. Electron energy loss spectroscopy indicates that the majority of the irradiated region remains as sp3 bonded diamond. Electrically conductive paths are attributed to the formation of multiple nano-scale, sp2-bonded graphitic wires and a network of strain-relieving micro-cracks.

Plant photonics: application of optical coherence tomography to monitor defects and rots in onion

NASA Astrophysics Data System (ADS)

Meglinski, I. V.; Buranachai, C.; Terry, L. A.

2010-04-01

The incidence of physiological and/or pathological defects in many fresh produce types is still unacceptably high and accounts for a large proportion of waste. With increasing interest in food security their remains strong demand in developing reliable and cost effective technologies for non-destructive screening of internal defects and rots, these being deemed unacceptable by consumers. It is well recognized that the internal defects and structure of turbid scattering media can be effectively visualized by using optical coherence tomography (OCT). In the present study, the high spatial resolution and advantages of OCT have been demonstrated for imaging the skins and outer laminae (concentric tissue layers) of intact whole onion bulbs with a view to non-invasively visualizing potential incidence/severity of internal defects.

Stalking The Silent Killers.

ERIC Educational Resources Information Center

DeYoung, H. Garrett

1983-01-01

New technologies have been developed to diagnose such disorders as cancer, heart disease, and defects of the newborn in presymptomatic stages. Several technologies, including amniocentesis, radioimmunoassay, enzyme immunoassay, multichannel analysis and others are discussed. Development of these technologies and issues related to their use are…

Cariogenic properties of Streptococcus mutans clinical isolates with sortase defects.

PubMed

Lapirattanakul, Jinthana; Takashima, Yukiko; Tantivitayakul, Pornpen; Maudcheingka, Thaniya; Leelataweewud, Pattarawadee; Nakano, Kazuhiko; Matsumoto-Nakano, Michiyo

2017-09-01

In Streptococcus mutans, a Gram-positive pathogen of dental caries, several surface proteins are anchored by the activity of sortase enzyme. Although various reports have shown that constructed S. mutans mutants deficient of sortase as well as laboratory reference strains with a sortase gene mutation have low cariogenic potential, no known studies have investigated clinical isolates with sortase defects. Here, we examined the cariogenic properties of S. mutans clinical isolates with sortase defects as well as caries status in humans harboring such defective isolates. Sortase-defective clinical isolates were evaluated for biofilm formation, sucrose-dependent adhesion, stress-induced dextran-dependent aggregation, acid production, and acid tolerance. Additionally, caries indices of subjects possessing such defective isolates were determined. Our in vitro results indicated that biofilm with a lower quantity was formed by sortase-defective as compared to non-defective isolates. Moreover, impairments of sucrose-dependent adhesion and stress-induced dextran-dependent aggregation were found among the isolates with defects, whereas no alterations were seen in regard to acid production or tolerance. Furthermore, glucan-binding protein C, a surface protein anchored by sortase activity, was predominantly detected in culture supernatants of all sortase-defective S. mutans isolates. Although the sortase-defective isolates showed lower cariogenic potential because of a reduction in some cariogenic properties, deft/DMFT indices revealed that all subjects harboring those isolates had caries experience. Our findings suggest the impairment of cariogenic properties in S. mutans clinical isolates with sortase defects, though the detection of these defective isolates seemed not to imply low caries risk in the subjects harboring them. Copyright © 2017 Elsevier Ltd. All rights reserved.

Serine biosynthesis and transport defects.

PubMed

El-Hattab, Ayman W

2016-07-01

l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

HRTEM Analysis of Crystallographic Defects in CdZnTe Single Crystal

NASA Astrophysics Data System (ADS)

Yasar, Bengisu; Ergunt, Yasin; Kabukcuoglu, Merve Pinar; Parlak, Mehmet; Turan, Rasit; Kalay, Yunus Eren

2018-01-01

In recent years, CdZnTe has attracted much attention due to its superior electrical and structural properties for room-temperature operable gamma and x-ray detectors. However, CdZnTe (CZT) material has often suffered from crystallographic defects encountered during the growth and post-growth processes. The identification and structural characterization of these defects is crucial to synthesize defect-free CdZnTe single crystals. In this study, Cd0.95 Zn0.05 Te single crystals were grown using a three-zone vertical Bridgman system. The single crystallinity of the material was ensured by using x-ray diffraction measurements. High-resolution electron microscopy (HRTEM) was used to characterize the nano-scale defects on the CdZnTe matrix. The linear defects oriented along the ⟨211⟩ direction were examined by transmission electron microscopy (TEM) and the corresponding HRTEM image simulations were performed by using a quantitative scanning TEM simulation package.

Wafer defect detection by a polarization-insensitive external differential interference contrast module.

PubMed

Nativ, Amit; Feldman, Haim; Shaked, Natan T

2018-05-01

We present a system that is based on a new external, polarization-insensitive differential interference contrast (DIC) module specifically adapted for detecting defects in semiconductor wafers. We obtained defect signal enhancement relative to the surrounding wafer pattern when compared with bright-field imaging. The new DIC module proposed is based on a shearing interferometer that connects externally at the output port of an optical microscope and enables imaging thin samples, such as wafer defects. This module does not require polarization optics (such as Wollaston or Nomarski prisms) and is insensitive to polarization, unlike traditional DIC techniques. In addition, it provides full control of the DIC shear and orientation, which allows obtaining a differential phase image directly on the camera (with no further digital processing) while enhancing defect detection capabilities, even if the size of the defect is smaller than the resolution limit. Our technique has the potential of future integration into semiconductor production lines.

Actinic imaging and evaluation of phase structures on EUV lithography masks

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

Mochi, Iacopo; Goldberg, Kenneth; Huh, Sungmin

2010-09-28

The authors describe the implementation of a phase-retrieval algorithm to reconstruct phase and complex amplitude of structures on EUV lithography masks. Many native defects commonly found on EUV reticles are difficult to detect and review accurately because they have a strong phase component. Understanding the complex amplitude of mask features is essential for predictive modeling of defect printability and defect repair. Besides printing in a stepper, the most accurate way to characterize such defects is with actinic inspection, performed at the design, EUV wavelength. Phase defect and phase structures show a distinct through-focus behavior that enables qualitative evaluation of themore » object phase from two or more high-resolution intensity measurements. For the first time, phase of structures and defects on EUV masks were quantitatively reconstructed based on aerial image measurements, using a modified version of a phase-retrieval algorithm developed to test optical phase shifting reticles.« less

The Application of High-Resolution Electron Microscopy to Problems in Solid State Chemistry: The Exploits of a Peeping TEM.

ERIC Educational Resources Information Center

Eyring, LeRoy

1980-01-01

Describes methods for using the high-resolution electron microscope in conjunction with other tools to reveal the identity and environment of atoms. Problems discussed include the ultimate structure of real crystalline solids including defect structure and the mechanisms of chemical reactions. (CS)

Improved spatial resolution of luminescence images acquired with a silicon line scanning camera

NASA Astrophysics Data System (ADS)

Teal, Anthony; Mitchell, Bernhard; Juhl, Mattias K.

2018-04-01

Luminescence imaging is currently being used to provide spatially resolved defect in high volume silicon solar cell production. One option to obtain the high throughput required for on the fly detection is the use a silicon line scan cameras. However, when using a silicon based camera, the spatial resolution is reduced as a result of the weakly absorbed light scattering within the camera's chip. This paper address this issue by applying deconvolution from a measured point spread function. This paper extends the methods for determining the point spread function of a silicon area camera to a line scan camera with charge transfer. The improvement in resolution is quantified in the Fourier domain and in spatial domain on an image of a multicrystalline silicon brick. It is found that light spreading beyond the active sensor area is significant in line scan sensors, but can be corrected for through normalization of the point spread function. The application of this method improves the raw data, allowing effective detection of the spatial resolution of defects in manufacturing.

Optical signatures of deep level defects in Ga2O3

NASA Astrophysics Data System (ADS)

Gao, Hantian; Muralidharan, Shreyas; Pronin, Nicholas; Karim, Md Rezaul; White, Susan M.; Asel, Thaddeus; Foster, Geoffrey; Krishnamoorthy, Sriram; Rajan, Siddharth; Cao, Lei R.; Higashiwaki, Masataka; von Wenckstern, Holger; Grundmann, Marius; Zhao, Hongping; Look, David C.; Brillson, Leonard J.

2018-06-01

We used depth-resolved cathodoluminescence spectroscopy and surface photovoltage spectroscopy to measure the effects of near-surface plasma processing and neutron irradiation on native point defects in β-Ga2O3. The near-surface sensitivity and depth resolution of these optical techniques enabled us to identify spectral changes associated with removing or creating these defects, leading to identification of one oxygen vacancy-related and two gallium vacancy-related energy levels in the β-Ga2O3 bandgap. The combined near-surface detection and processing of Ga2O3 suggests an avenue for identifying the physical nature and reducing the density of native point defects in this and other semiconductors.

Hyperspectral range imaging for transportation systems evaluation

NASA Astrophysics Data System (ADS)

Bridgelall, Raj; Rafert, J. B.; Atwood, Don; Tolliver, Denver D.

2016-04-01

Transportation agencies expend significant resources to inspect critical infrastructure such as roadways, railways, and pipelines. Regular inspections identify important defects and generate data to forecast maintenance needs. However, cost and practical limitations prevent the scaling of current inspection methods beyond relatively small portions of the network. Consequently, existing approaches fail to discover many high-risk defect formations. Remote sensing techniques offer the potential for more rapid and extensive non-destructive evaluations of the multimodal transportation infrastructure. However, optical occlusions and limitations in the spatial resolution of typical airborne and space-borne platforms limit their applicability. This research proposes hyperspectral image classification to isolate transportation infrastructure targets for high-resolution photogrammetric analysis. A plenoptic swarm of unmanned aircraft systems will capture images with centimeter-scale spatial resolution, large swaths, and polarization diversity. The light field solution will incorporate structure-from-motion techniques to reconstruct three-dimensional details of the isolated targets from sequences of two-dimensional images. A comparative analysis of existing low-power wireless communications standards suggests an application dependent tradeoff in selecting the best-suited link to coordinate swarming operations. This study further produced a taxonomy of specific roadway and railway defects, distress symptoms, and other anomalies that the proposed plenoptic swarm sensing system would identify and characterize to estimate risk levels.

Structure of Ce2RhIn8: an example of complementary use of high-resolution neutron powder diffraction and reciprocal-space mapping to study complex materials.

PubMed

Moshopoulou, E G; Ibberson, R M; Sarrao, J L; Thompson, J D; Fisk, Z

2006-04-01

The room-temperature crystal structure of the heavy fermion antiferromagnet Ce2RhIn8, dicerium rhodium octaindide, has been studied by a combination of high-resolution synchrotron X-ray reciprocal-space mapping of single crystals and high-resolution time-of-flight neutron powder diffraction. The structure is disordered, exhibiting a complex interplay of non-periodic, partially correlated planar defects, coexistence and segregation of polytypic phases (induced by periodic planar ;defects'), mosaicity (i.e. domain misalignment) and non-uniform strain. These effects evolve as a function of temperature in a complicated way, but they remain down to low temperatures. The room-temperature diffraction data are best represented by a complex mixture of two polytypic phases, which are affected by non-periodic, partially correlated planar defects, differ slightly in their tetragonal structures, and exhibit different mosaicities and strain values. Therefore, Ce2RhIn8 approaches the paracrystalline state, rather than the classic crystalline state and thus several of the concepts of conventional single-crystal crystallography are inapplicable. The structural results are discussed in the context of the role of disorder in the heavy-fermion state and in the interplay between superconductivity and magnetism.

Mapping disease at an approximated individual level using aggregate data: a case study of mapping New Hampshire birth defects.

PubMed

Shi, Xun; Miller, Stephanie; Mwenda, Kevin; Onda, Akikazu; Reese, Judy; Onega, Tracy; Gui, Jiang; Karagas, Margret; Demidenko, Eugene; Moeschler, John

2013-09-06

Limited by data availability, most disease maps in the literature are for relatively large and subjectively-defined areal units, which are subject to problems associated with polygon maps. High resolution maps based on objective spatial units are needed to more precisely detect associations between disease and environmental factors. We propose to use a Restricted and Controlled Monte Carlo (RCMC) process to disaggregate polygon-level location data to achieve mapping aggregate data at an approximated individual level. RCMC assigns a random point location to a polygon-level location, in which the randomization is restricted by the polygon and controlled by the background (e.g., population at risk). RCMC allows analytical processes designed for individual data to be applied, and generates high-resolution raster maps. We applied RCMC to the town-level birth defect data for New Hampshire and generated raster maps at the resolution of 100 m. Besides the map of significance of birth defect risk represented by p-value, the output also includes a map of spatial uncertainty and a map of hot spots. RCMC is an effective method to disaggregate aggregate data. An RCMC-based disease mapping maximizes the use of available spatial information, and explicitly estimates the spatial uncertainty resulting from aggregation.

Computational imaging of defects in commercial substrates for electronic and photonic devices

NASA Astrophysics Data System (ADS)

Fukuzawa, Masayuki; Kashiwagi, Ryo; Yamada, Masayoshi

2012-03-01

Computational defect imaging has been performed in commercial substrates for electronic and photonic devices by combining the transmission profile acquired with an imaging type of linear polariscope and the computational algorithm to extract a small amount of birefringence. The computational images of phase retardation δ exhibited spatial inhomogeneity of defect-induced birefringence in GaP, LiNbO3, and SiC substrates, which were not detected by conventional 'visual inspection' based on simple optical refraction or transmission because of poor sensitivity. The typical imaging time was less than 30 seconds for 3-inch diameter substrate with the spatial resolution of 200 μm, while that by scanning polariscope was 2 hours to get the same spatial resolution. Since our proposed technique have been achieved high sensitivity, short imaging time, and wide coverage of substrate materials, which are practical advantages over the laboratory-scale apparatus such as X-ray topography and electron microscope, it is useful for nondestructive inspection of various commercial substrates in production of electronic and photonic devices.

A facile strategy for enzyme immobilization with highly stable hierarchically porous metal-organic frameworks.

PubMed

Liu, Xiao; Qi, Wei; Wang, Yuefei; Su, Rongxin; He, Zhimin

2017-11-16

Metal-organic frameworks (MOFs) have drawn extensive research interest as candidates for enzyme immobilization owing to their tunable porosity, high surface area, and excellent chemical/thermal stability. Herein, we report a facile and universal strategy for enzyme immobilization using highly stable hierarchically porous metal-organic frameworks (HP-MOFs). The HP-MOFs were stable over a wide pH range (pH = 2-11 for HP-DUT-5) and met the catalysis conditions of most enzymes. The as-prepared hierarchical micro/mesoporous MOFs with mesoporous defects showed a superior adsorption capacity towards enzymes. The maximum adsorption capacity of HP-DUT-5 for glucose oxidase (GOx) and uricase was 208 mg g -1 and 225 mg g -1 , respectively. Furthermore, we constructed two multi-enzyme biosensors for glucose and uric acid (UA) by immobilizing GOx and uricase with horseradish peroxidase (HRP) on HP-DUT-5, respectively. These sensors were efficiently applied in the colorimetric detection of glucose and UA and showed good sensitivity, selectivity, and recyclability.

Non-targeted analysis of electronics waste by comprehensive two-dimensional gas chromatography combined with high-resolution mass spectrometry: Using accurate mass information and mass defect analysis to explore the data.

PubMed

Ubukata, Masaaki; Jobst, Karl J; Reiner, Eric J; Reichenbach, Stephen E; Tao, Qingping; Hang, Jiliang; Wu, Zhanpin; Dane, A John; Cody, Robert B

2015-05-22

Comprehensive two-dimensional gas chromatography (GC×GC) and high-resolution mass spectrometry (HRMS) offer the best possible separation of their respective techniques. Recent commercialization of combined GC×GC-HRMS systems offers new possibilities for the analysis of complex mixtures. However, such experiments yield enormous data sets that require new informatics tools to facilitate the interpretation of the rich information content. This study reports on the analysis of dust obtained from an electronics recycling facility by using GC×GC in combination with a new high-resolution time-of-flight (TOF) mass spectrometer. New software tools for (non-traditional) Kendrick mass defect analysis were developed in this research and greatly aided in the identification of compounds containing chlorine and bromine, elements that feature in most persistent organic pollutants (POPs). In essence, the mass defect plot serves as a visual aid from which halogenated compounds are recognizable on the basis of their mass defect and isotope patterns. Mass chromatograms were generated based on specific ions identified in the plots as well as region of the plot predominantly occupied by halogenated contaminants. Tentative identification was aided by database searches, complementary electron-capture negative ionization experiments and elemental composition determinations from the exact mass data. These included known and emerging flame retardants, such as polybrominated diphenyl ethers (PBDEs), hexabromobenzene, tetrabromo bisphenol A and tris (1-chloro-2-propyl) phosphate (TCPP), as well as other legacy contaminants such as polychlorinated biphenyls (PCBs) and polychlorinated terphenyls (PCTs). Copyright © 2015 Elsevier B.V. All rights reserved.

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

Imamura, Kayo; Matsuura, Takanori; Ye, Zhengmao

Disproportionating enzyme from potato was crystallized and preliminarily analyzed using X-ray diffraction. Disproportionating enzyme (D-enzyme; EC 2.4.1.25) is a 59 kDa protein that belongs to the α-amylase family. D-enzyme catalyses intramolecular and intermolecular transglycosylation reactions of α-1,4 glucan. A crystal of the D-enzyme from potato was obtained by the hanging-drop vapour-diffusion method. Preliminary X-ray data showed that the crystal diffracts to 2.0 Å resolution and belongs to space group C222{sub 1}, with unit-cell parameters a = 69.7, b = 120.3, c = 174.2 Å.

Dissipative Dynamics of Enzymes

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

2014-11-01

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C ; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

Dissipative Dynamics of Enzymes

NASA Astrophysics Data System (ADS)

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni; Zocchi LabMolecular Biophysics Team

2015-03-01

We explore enzyme conformational dynamics at sub - Å resolution, specifically temperature effects. The ensemble averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor 2 as the temperature is raised from 10 C to 45 C; the elastic parameter K shows a similar decrease. Thus when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

Dissipative dynamics of enzymes.

PubMed

Ariyaratne, Amila; Wu, Chenhao; Tseng, Chiao-Yu; Zocchi, Giovanni

2014-11-07

We explore enzyme conformational dynamics at sub-Å resolution, specifically, temperature effects. The ensemble-averaged mechanical response of the folded enzyme is viscoelastic in the whole temperature range between the warm and cold denaturation transitions. The dissipation parameter γ of the viscoelastic description decreases by a factor of 2 as the temperature is raised from 10 to 45 °C; the elastic parameter K shows a similar decrease. Thus, when probed dynamically, the enzyme softens for increasing temperature. Equilibrium mechanical experiments with the DNA spring (and a different enzyme) also show, qualitatively, a small softening for increasing temperature.

The Use of Confocal Photoluminescence Microscopy for Determination of Defect Densities in Various 2-6 Semiconductors

DTIC Science & Technology

2014-03-11

optical configuration that significantly enhances both lateral and depth resolution and returns crisp PL images 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND...that significantly enhances both lateral and depth resolution and returns crisp PL images with high contrast. This technique revolutionized fluorescent...resolution and returns crisp PL images with high contrast. This technique revolutionized fluorescent imaging in biology, and has the potential to

Low surface energy polymeric release coating for improved contact print lithography

NASA Astrophysics Data System (ADS)

Mancini, David P.; Resnick, Douglas J.; Gehoski, Kathleen A.; Popovich, Laura L.; Chang, Daniel

2002-03-01

Contact printing has been used for decades in many various lithography applications in the microelectronic industry. While vacuum contact printing processes offer sub-micron resolution and high throughput, they often suffer from some important drawbacks. One of the most common problems is degradation in both resolution and defect density which occurs when the same mask si used for multiple exposures without frequent mask cleans. This is largely due to the relatively high surface energy of both quartz and chrome and the tendency of most photoresists to adhere to these surfaces. As a result, when a mask and wafer are pressed into intimate contact, resist will tend to stick to the mask creating a defect on the wafer, effectively propagating defects to subsequent wafers. In this study, DuPont Teflon AF 1601S is used as a photomask coating and evaluated for its ability to act as a release agent and reduce defects while maintaining resolution for multiple exposures. Teflon AF is an amorphous, transparent, low surface energy, polymeric material that can be spin coated into a thin conformal film. Tests have shown that when using an uncoated mask in vacuum contact, resolution of 0.75 micrometers dense lines is severely degraded after less than 10 consecutive exposures. However, when the mask is coated, 0.75 micrometers dense lines were successfully resolved using vacuum contact for over 200 exposures without cleaning. In addition, it has been demonstrated that Teflon AF coatings impart to a mask a self-cleaning capability, since particles tend to stick to the photoresist rather than the mask. A coated mask, which was purposefully contaminated with particulates, resolved 0.75 micrometers dense lines on all but the first wafer of a series of 25 consecutive exposures. The patented mask releases layer process has successfully been demonstrated with a positive novolak resist. Additional data which describes the system chemistry, dilution and coating process, and film morphology are also presented.

Probing defect states in polycrystalline GaN grown on Si(111) by sub-bandgap laser-excited scanning tunneling spectroscopy

NASA Astrophysics Data System (ADS)

Hsiao, F.-M.; Schnedler, M.; Portz, V.; Huang, Y.-C.; Huang, B.-C.; Shih, M.-C.; Chang, C.-W.; Tu, L.-W.; Eisele, H.; Dunin-Borkowski, R. E.; Ebert, Ph.; Chiu, Y.-P.

2017-01-01

We demonstrate the potential of sub-bandgap laser-excited cross-sectional scanning tunneling microscopy and spectroscopy to investigate the presence of defect states in semiconductors. The characterization method is illustrated on GaN layers grown on Si(111) substrates without intentional buffer layers. According to high-resolution transmission electron microscopy and cathodoluminescence spectroscopy, the GaN layers consist of nanoscale wurtzite and zincblende crystallites with varying crystal orientations and hence contain high defect state densities. In order to discriminate between band-to-band excitation and defect state excitations, we use sub-bandgap laser excitation. We probe a clear increase in the tunnel current at positive sample voltages during sub-bandgap laser illumination for the GaN layer with high defect density, but no effect is found for high quality GaN epitaxial layers. This demonstrates the excitation of free charge carriers at defect states. Thus, sub-bandgap laser-excited scanning tunneling spectroscopy is a powerful complimentary characterization tool for defect states.

Evaluation of anti-sticking layers performances for 200mm wafer scale Smart NILTM process through surface and defectivity characterizations

NASA Astrophysics Data System (ADS)

Delachat, F.; Phillipe, J.-C.; Larrey, V.; Fournel, F.; Bos, S.; Teyssèdre, H.; Chevalier, Xavier; Nicolet, Célia; Navarro, Christophe; Cayrefourcq, Ian

2018-03-01

In this work, an evaluation of various ASL processes for 200 mm wafer scale in the HERCULES® NIL equipment platform available at the CEA-Leti through the INSPIRE program is reported. The surface and adherence energies were correlated to the AFM and defectivity results in order to select the most promising ASL process for high resolution etch mask applications. The ASL performances of the selected process were evaluated by multiple working stamp fabrication using unpatterned and patterned masters though defectivity monitoring on optical based-inspection tools. Optical and SEM defect reviews were systematically performed. Multiple working stamps fabrication without degradation of the master defectivity was witnessed. This evaluation enabled to benchmark several ASL solutions based on the grafted technology develop by ARKEMA in order to reduce and optimize the soft stamp defectivity prior to its replication and therefore considerably reduce the final imprint defectivity for the Smart NIL process.

Platelet rich fibrin combined with decalcified freeze-dried bone allograft for the treatment of human intrabony periodontal defects: a randomized split mouth clinical trail.

PubMed

Agarwal, Ashish; Gupta, Narinder Dev; Jain, Avikal

2016-01-01

Polypeptide growth factors of platelet rich fibrin (PRF) have the potential to regenerate periodontal tissues. Osteoinductive property of demineralized freeze-dried bone allograft (DFDBA) has been successfully utilized in periodontal regeneration. The aim of the present randomized, split mouth, clinical trial was to determine the additive effects of PRF with a DFDBA in the treatment of human intrabony periodontal defects. Sixty interproximal infrabony defects in 30 healthy, non-smoker patients diagnosed with chronic periodontitis were randomly assigned to PRF/DFDBA group or the DFDBA/saline. Clinical [pocket depth (PD), clinical attachment level (CAL) and gingival recession (REC)] and radiographic (bone fill, defect resolution and alveolar crest resorption) measurements were made at baseline and at a 12-month evaluation. Compared with baseline, 12-month results indicated that both treatment modalities resulted in significant changes in all clinical and radiographic parameters. However, the PRP/DFDBA group exhibited statistically significantly greater changes compared with the DFDBA/saline group in PD (4.15 ± 0.84 vs 3.60 ± 0.51 mm), CAL (3.73 ± 0.74 vs 2.61 ± 0.68 mm), REC (0.47 ± 0.56 vs 1.00 ± 0.61 mm), bone fill (3.50 ± 0.67 vs 2.49 ± 0.64 mm) and defect resolution (3.73 ± 0.63 vs 2.75 ± 0.57 mm). Observations indicate that a combination of PRF and DFDBA is more effective than DFDBA with saline for the treatment of infrabony periodontal defects.

Age-Related Defects in Erythrocyte 2,3-Diphosphoglycerate Metabolism in Dementia

PubMed Central

Kaminsky, Yury G.; Reddy, V. Prakash; Ashraf, Ghulam Md; Ahmad, Ausaf; Benberin, Valery V.; Kosenko, Elena A.; Aliev, Gjumrakch

2013-01-01

Alzheimer disease (AD) is the most common dementing illness. Metabolic defects in the brain with aging contribute to the pathogenesis of AD. These changes can be found systematically and thus can be used as potential biomarkers. Erythrocytes (RBCs) are passive “reporter cells” that are not well studied in AD. In the present study, we analyzed an array of glycolytic and related enzymes and intermediates in RBCs from patients with AD and non-Alzheimer dementia (NA), age-matched controls (AC) and young adult controls (YC). AD is characterized by higher activities of hexokinase, phosphofructokinase, and bisphosphoglycerate mutase and bisphosphoglycerate phosphatase in RBCs. In our study, we observed that glycolytic and related enzymes displayed significantly lower activities in AC. However, similar or significantly higher activities were observed in AD and NA groups as compared to YC group. 2,3-diphosphoglycerate (2,3-DPG) levels were significantly decreased in AD and NA patients. The pattern of changes between groups in the above indices strongly correlates with each other. Collectively, our data suggested that AD and NA patients are associated with chronic disturbance of 2,3-DPG metabolism in RBCs. These defects may play a pivotal role in physiological processes, which predispose elderly subjects to AD and NA. PMID:24124630

NAD Deficiency, Congenital Malformations, and Niacin Supplementation.

PubMed

Shi, Hongjun; Enriquez, Annabelle; Rapadas, Melissa; Martin, Ella M M A; Wang, Roni; Moreau, Julie; Lim, Chai K; Szot, Justin O; Ip, Eddie; Hughes, James N; Sugimoto, Kotaro; Humphreys, David T; McInerney-Leo, Aideen M; Leo, Paul J; Maghzal, Ghassan J; Halliday, Jake; Smith, Janine; Colley, Alison; Mark, Paul R; Collins, Felicity; Sillence, David O; Winlaw, David S; Ho, Joshua W K; Guillemin, Gilles J; Brown, Matthew A; Kikuchi, Kazu; Thomas, Paul Q; Stocker, Roland; Giannoulatou, Eleni; Chapman, Gavin; Duncan, Emma L; Sparrow, Duncan B; Dunwoodie, Sally L

2017-08-10

Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. II. Isolation and characterization of phosphatidylserine auxotrophs

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

Kuge, O.; Nishijima, M.; Akamatsu, Y.

1986-05-05

Chinese hamster ovary (CHO) cell mutants that required exogenously added phosphatidylserine for cell growth were isolated by using the replica technique with polyester cloth, and three such mutants were characterized. Labeling experiments on intact cells with /sup 32/Pi and L-(U-/sup 14/C)serine revealed that a phosphatidylserine auxotroph, designated as PSA-3, was strikingly defective in phosphatidylserine biosynthesis. When cells were grown for 2 days without phosphatidylserine, the phosphatidylserine content of PSA-3 was about one-third of that of the parent. In extracts of the mutant, the enzymatic activity of the base-exchange reaction of phospholipids with serine producing phosphatidylserine was reduced to 33% ofmore » that in the parent; in addition, the activities of base-exchange reactions of phospholipids with choline and ethanolamine in the mutant were also reduced to 1 and 45% of those in the parent, respectively. Furthermore, it was demonstrated that the serine-exchange activity in the parent was inhibited approximately 60% when choline was added to the reaction mixture whereas that in the mutant was not significantly affected. From the results presented here, we conclude the following. There are at least two kinds of serine-exchange enzymes in CHO cells; one (serine-exchange enzyme I) can catalyze the base-exchange reactions of phospholipids with serine, choline, and ethanolamine while the other (serine-exchange enzyme II) does not use the choline as a substrate. Serine-exchange enzyme I, in which mutant PSA-3 is defective, plays a major role in phosphatidylserine biosynthesis in CHO cells. Serine-exchange enzyme I is essential for the growth of CHO cells.« less

Evaluation of Parallel and Fan-Beam Data Acquisition Geometries and Strategies for Myocardial SPECT Imaging

NASA Astrophysics Data System (ADS)

Qi, Yujin; Tsui, B. M. W.; Gilland, K. L.; Frey, E. C.; Gullberg, G. T.

2004-06-01

This study evaluates myocardial SPECT images obtained from parallel-hole (PH) and fan-beam (FB) collimator geometries using both circular-orbit (CO) and noncircular-orbit (NCO) acquisitions. A newly developed 4-D NURBS-based cardiac-torso (NCAT) phantom was used to simulate the /sup 99m/Tc-sestamibi uptakes in human torso with myocardial defects in the left ventricular (LV) wall. Two phantoms were generated to simulate patients with thick and thin body builds. Projection data including the effects of attenuation, collimator-detector response and scatter were generated using SIMSET Monte Carlo simulations. A large number of photon histories were generated such that the projection data were close to noise free. Poisson noise fluctuations were then added to simulate the count densities found in clinical data. Noise-free and noisy projection data were reconstructed using the iterative OS-EM reconstruction algorithm with attenuation compensation. The reconstructed images from noisy projection data show that the noise levels are lower for the FB as compared to the PH collimator due to increase in detected counts. The NCO acquisition method provides slightly better resolution and small improvement in defect contrast as compared to the CO acquisition method in noise-free reconstructed images. Despite lower projection counts the NCO shows the same noise level as the CO in the attenuation corrected reconstruction images. The results from the channelized Hotelling observer (CHO) study show that FB collimator is superior to PH collimator in myocardial defect detection, but the NCO shows no statistical significant difference from the CO for either PH or FB collimator. In conclusion, our results indicate that data acquisition using NCO makes a very small improvement in the resolution over CO for myocardial SPECT imaging. This small improvement does not make a significant difference on myocardial defect detection. However, an FB collimator provides better defect detection than a PH collimator with similar spatial resolution for myocardial SPECT imaging.

Phase measurements of EUV mask defects

DOE PAGES

Claus, Rene A.; Wang, Yow-Gwo; Wojdyla, Antoine; ...

2015-02-22

Extreme Ultraviolet (EUV) Lithography mask defects were examined on the actinic mask imaging system, SHARP, at Lawrence Berkeley National Laboratory. Also, a quantitative phase retrieval algorithm based on the Weak Object Transfer Function was applied to the measured through-focus aerial images to examine the amplitude and phase of the defects. The accuracy of the algorithm was demonstrated by comparing the results of measurements using a phase contrast zone plate and a standard zone plate. Using partially coherent illumination to measure frequencies that would otherwise fall outside the numerical aperture (NA), it was shown that some defects are smaller than themore » conventional resolution of the microscope. We found that the programmed defects of various sizes were measured and shown to have both an amplitude and a phase component that the algorithm is able to recover.« less

Anterior vaginal introitoplasty for an acquired sensation of wide vagina: a case report and new surgical intervention.

PubMed

Ostrzenski, Adam

2014-01-01

A vaginal introital defect case and its symptomatology have never been published before. The objective of this presentation was to describe symptoms associated with an acquired sensation of wide vagina and to present a new surgical treatment for anterior vaginal introital defects. A 42-year-old, Caucasian woman, G4P4013, presented with a sensation of wide vagina at the vaginal opening and a history of colpoperineoplasty, without mesh, for the same condition 4 years prior. Clinical evaluation documented anterior vaginal introital defects and the absence of vaginal site-specific defects. Reconstruction of a vaginal introital defect was completed without complications. Surgical resolution of symptoms and signs of this condition were noted. Anterior vaginal introitoplasty can assist in the management of an acquired sensation of wide vagina.

Defect of focus in two-line resolution with Hanning amplitude filters

NASA Astrophysics Data System (ADS)

Karunasagar, D.; Bhikshamaiah, G.; Keshavulu Goud, M.; Lacha Goud, S.

In the presence of defocusing the modified Sparrow limits of resolution for two-line objects have been investigated for a diffraction-limited coherent optical system apodized by generalized Hanning amplitude filters. These limits have been studied as a function of different parameters such as intensity ratio, the order of the filter for various amounts of apodization parameter. Results reveal that in some situations the defocusing is effective in enhancing the resolution of an optical system.

Novel mutants of Erwinia carotovora subsp. carotovora defective in the production of plant cell wall degrading enzymes generated by Mu transpososome-mediated insertion mutagenesis.

PubMed

Laasik, Eve; Ojarand, Merli; Pajunen, Maria; Savilahti, Harri; Mäe, Andres

2005-02-01

As in Erwinia carotovora subsp. carotovora the regulation details of the main virulence factors, encoding extracellular enzymes that degrade the plant cell wall, is only rudimentally understood, we performed a genetic screen to identify novel candidate genes involved in the process. Initially, we used Mu transpososome-mediated mutagenesis approach to generate a comprehensive transposon insertion mutant library of ca. 10000 clones and screened the clones for the loss of extracellular enzyme production. Extracellular enzymes production was abolished by mutations in the chromosomal helEcc, trkAEcc yheLEcc, glsEcc, igaAEcc and cysQEcc genes. The findings reported here demonstrate that we have isolated six new representatives that belong to the pool of genes modulating the production of virulence factors in E. carotovora.

Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency.

PubMed

Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C; Wallace, John C; Polyak, Steven W

2008-01-01

The attachment of biotin onto the biotin-dependent enzymes is catalysed by biotin protein ligase (BPL), also known as holocarboxylase synthase HCS in mammals. Mammals contain five biotin-enzymes that participate in a number of important metabolic pathways such as fatty acid biogenesis, gluconeogenesis and amino acid catabolism. All mammalian biotin-enzymes are post-translationally biotinylated, and therefore activated, through the action of a single HCS. Substrate recognition by BPLs occurs through conserved structural cues that govern the specificity of biotinylation. Defects in biotin metabolism, including HCS, give rise to multiple carboxylase deficiency (MCD). Here we review the literature on this important enzyme. In particular, we focus on the new information that has been learned about BPL's from a number of recently published protein structures. Through molecular modelling studies insights into the structural basis of HCS deficiency in MCD are discussed.

Chevron Defect at the Intersection of Grain Boundaries with Free Surfaces in Au

NASA Astrophysics Data System (ADS)

Radetic, T.; Lançon, F.; Dahmen, U.

2002-08-01

We have identified a new defect at the intersection between grain boundaries and surfaces in Au using atomic resolution transmission electron microscopy. At the junction line of 90° <110> tilt grain boundaries of (110)-(001) orientation with the free surface, a small segment of the grain boundary, about 1nm in length, dissociates into a triangular region with a chevronlike stacking disorder and a distorted hcp structure. The structure and stability of these defects are confirmed by atomistic simulations, and we point out the relationship with the one-dimensional incommensurate structure of the grain boundary.

Evaluation of Dentin Defect Formation during Retreatment with Hand and Rotary Instruments: A Micro-CT Study.

PubMed

Yilmaz, Ayca; Helvacioglu-Yigit, Dilek; Gur, Cansu; Ersev, Handan; Kiziltas Sendur, Gullu; Avcu, Egemen; Baydemir, Canan; Abbott, Paul Vincent

2017-01-01

The purpose of this study was to compare the incidence and longitudinal propagation of dentin defects after gutta-percha removal with hand and rotary instruments using microcomputed tomography. Twenty mandibular incisors were prepared using the balanced-force technique and scanned in a 19.9  μ m resolution. Following filling with the lateral compaction technique, gutta-percha was removed with ProTaper Universal Retreatment (PTUR) or hand instruments. After rescanning, a total of 24,120 cross-sectional images were analyzed. The numbers, types, and longitudinal length changes of defects were recorded. Defects were observed in 36.90% of the cross sections. A total of 73 defects were comprised of 87.67% craze lines, 2.73% partial cracks, and 9.58% fractures. No significant difference in terms of new defect formation was detected between the retreatment groups. The apical and middle portions of the roots had more dentin defects than the coronal portions. Defects in three roots of the PTUR instrument group increased in length. Under the conditions of this in vitro study, gutta-percha removal seemed to not increase the incidence of dentin defect formation, but the longitudinal defect propagation finding suggests possible cumulative dentinal damage due to additional endodontic procedures. Hand and rotary instrumentation techniques caused similar dentin defect formation during root canal retreatment.

Synaptic Polarity Depends on Phosphatidylinositol Signaling Regulated by myo-Inositol Monophosphatase in Caenorhabditis elegans

PubMed Central

Kimata, Tsubasa; Tanizawa, Yoshinori; Can, Yoko; Ikeda, Shingo; Kuhara, Atsushi; Mori, Ikue

2012-01-01

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP2). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP2 on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP2 synthesis through inhibition of IMPase. We propose that the PIP2 signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity. PMID:22446320

Enhanced resolution imaging of ultrathin ZnO layers on Ag(111) by multiple hydrogen molecules in a scanning tunneling microscope junction

NASA Astrophysics Data System (ADS)

Liu, Shuyi; Shiotari, Akitoshi; Baugh, Delroy; Wolf, Martin; Kumagai, Takashi

2018-05-01

Molecular hydrogen in a scanning tunneling microscope (STM) junction has been found to enhance the lateral spatial resolution of the STM imaging, referred to as scanning tunneling hydrogen microscopy (STHM). Here we report atomic resolution imaging of 2- and 3-monolayer (ML) thick ZnO layers epitaxially grown on Ag(111) using STHM. The enhanced resolution can be obtained at a relatively large tip to surface distance and resolves a more defective structure exhibiting dislocation defects for 3-ML-thick ZnO than for 2 ML. In order to elucidate the enhanced imaging mechanism, the electric and mechanical properties of the hydrogen molecular junction (HMJ) are investigated by a combination of STM and atomic force microscopy. It is found that the HMJ shows multiple kinklike features in the tip to surface distance dependence of the conductance and frequency shift curves, which are absent in a hydrogen-free junction. Based on a simple modeling, we propose that the junction contains several hydrogen molecules and sequential squeezing of the molecules out of the junction results in the kinklike features in the conductance and frequency shift curves. The model also qualitatively reproduces the enhanced resolution image of the ZnO films.

Caenorhabditis elegans glutamylating enzymes function redundantly in male mating.

PubMed

Chawla, Daniel G; Shah, Ruchi V; Barth, Zachary K; Lee, Jessica D; Badecker, Katherine E; Naik, Anar; Brewster, Megan M; Salmon, Timothy P; Peel, Nina

2016-09-15

Microtubule glutamylation is an important modulator of microtubule function and has been implicated in the regulation of centriole stability, neuronal outgrowth and cilia motility. Glutamylation of the microtubules is catalyzed by a family of tubulin tyrosine ligase-like (TTLL) enzymes. Analysis of individual TTLL enzymes has led to an understanding of their specific functions, but how activities of the TTLL enzymes are coordinated to spatially and temporally regulate glutamylation remains relatively unexplored. We have undertaken an analysis of the glutamylating TTLL enzymes in C. elegans We find that although all five TTLL enzymes are expressed in the embryo and adult worm, loss of individual enzymes does not perturb microtubule function in embryonic cell divisions. Moreover, normal dye-filling, osmotic avoidance and male mating behavior indicate the presence of functional amphid cilia and male-specific neurons. A ttll-4(tm3310); ttll-11(tm4059); ttll-5(tm3360) triple mutant, however, shows reduced male mating efficiency due to a defect in the response step, suggesting that these three enzymes function redundantly, and that glutamylation is required for proper function of the male-specific neurons. © 2016. Published by The Company of Biologists Ltd.

Modeling defect cluster evolution in irradiated structural materials: Focus on comparing to high-resolution experimental characterization studies

DOE PAGES

Wirth, Brian D.; Hu, Xunxiang; Kohnert, Aaron; ...

2015-03-02

Exposure of metallic structural materials to irradiation environments results in significant microstructural evolution, property changes, and performance degradation, which limits the extended operation of current generation light water reactors and restricts the design of advanced fission and fusion reactors. Further, it is well recognized that these irradiation effects are a classic example of inherently multiscale phenomena and that the mix of radiation-induced features formed and the corresponding property degradation depend on a wide range of material and irradiation variables. This inherently multiscale evolution emphasizes the importance of closely integrating models with high-resolution experimental characterization of the evolving radiation-damaged microstructure. Lastly,more » this article provides a review of recent models of the defect microstructure evolution in irradiated body-centered cubic materials, which provide good agreement with experimental measurements, and presents some outstanding challenges, which will require coordinated high-resolution characterization and modeling to resolve.« less

The hydration structure at yttria-stabilized cubic zirconia (110)-water interface with sub-Ångström resolution

DOE PAGES

Hou, Binyang; Kim, Seunghyun; Kim, Taeho; ...

2016-06-15

The interfacial hydration structure of yttria-stabilized cubic zirconia (110) surface in contact with water was determined with ~0.5 Å resolution by high-resolution X-ray reflectivity measurement. The terminal layer shows a reduced electron density compared to the following substrate lattice layers, which indicates there are additional defects generated by metal depletion as well as intrinsic oxygen vacancies, both of which are apparently filled by water species. Above this top surface layer, two additional adsorbed layers are observed forming a characteristic interfacial hydration structure. The first adsorbed layer shows abnormally high density as pure water and likely includes metal species, whereas themore » second layer consists of pure water. The observed interfacial hydration structure seems responsible for local equilibration of the defective surface in water and eventually regulating the long-term degradation processes. As a result, the multitude of water interactions with the zirconia surface results in the complex but highly ordered interfacial structure constituting the reaction front.« less

High-volume manufacturing compatible dry development rinse process (DDRP): patterning and defectivity performance for EUVL

NASA Astrophysics Data System (ADS)

Sayan, Safak; Vanelderen, Pieter; Hetel, Iulian; Chan, BT; Raghavan, Praveen; Blanco, Victor; Foubert, Philippe; D'urzo, Lucia; De Simone, Danilo; Vandenberghe, Geert

2017-04-01

There are many knobs available that change the chemical and physical properties of the photoresists to "break" the RLS (Resolution, Sensitivity, Line edge/width roughness) trade-off, however those are not enough today to realize a material to satisfy all requirements at once for 7nm technology and beyond. DDRP improves the ultimate achievable resolution via pattern collapse mitigation, hence the priority of requirements for the EUV photoresist development may be changed with more focus on Sensitivity and LWR. This may potentially provide a new conceptual approach towards EUV PR development for DDRP applications. We have previously demonstrated pattern collapse (PC) mitigation via DDRP on different EUVL photoresists (including different resist platforms), achieving ultimate resolution and exposure latitude improvements [1,2]. In this contribution, we report patterning and material defect performance of HVM compatible (all aqueous) dry development rinse material. We will also report on process window improvement on 2-dimensional metal structures towards standard cell size reduction with elimination of mask layer(s) using single EUV exposure.

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

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

Azim, N.; Deery, E.; Warren, M. J.

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. Two near-atomic resolution structures of PBGD from B. megaterium are reported that demonstrate the time-dependent accumulation of partially oxidized forms of the cofactor, including one that possesses a tetrahedral C atom in the terminal pyrrole ring. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form amore » linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.« less

Restoring balance to B cells in ADA deficiency.

PubMed

Luning Prak, Eline T

2012-06-01

It is paradoxical that immunodeficiency disorders are associated with autoimmunity. Adenosine deaminase (ADA) deficiency, a cause of X-linked severe combined immunodeficiency (SCID), is a case in point. In this issue of the JCI, Sauer and colleagues investigate the B cell defects in ADA-deficient patients. They demonstrate that ADA patients receiving enzyme replacement therapy had B cell tolerance checkpoint defects. Remarkably, gene therapy with a retrovirus that expresses ADA resulted in the apparent correction of these defects, with normalization of peripheral B cell autoantibody frequencies. In vitro, agents that either block ADA or overexpress adenosine resulted in altered B cell receptor and TLR signaling. Collectively, these data implicate a B cell-intrinsic mechanism for alterations in B cell tolerance in the setting of partial ADA deficiency that is corrected by gene therapy.

ADA Deficiency: Evaluation of the Clinical and Laboratory Features and the Outcome.

PubMed

Cagdas, Deniz; Gur Cetinkaya, Pınar; Karaatmaca, Betül; Esenboga, Saliha; Tan, Cagman; Yılmaz, Togay; Gümüş, Ersin; Barış, Safa; Kuşkonmaz, Barış; Ozgur, Tuba Turul; Bali, Pawan; Santisteban, Ines; Orhan, Diclehan; Yüce, Aysel; Cetinkaya, Duygu; Boztug, Kaan; Hershfield, Michael; Sanal, Ozden; Tezcan, İlhan

2018-05-09

Adenosine deaminase (ADA) deficiency is an autosomal recessive primary immunodeficiency. It results in the intracellular accumulation of toxic metabolites which have effects particularly on lymphocytes and the brain. The aim of this study was to evaluate the outcome of 13 ADA-deficient patients. We planned to evaluate their clinical and laboratory findings before and after enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (aHSCT), and hematopoietic stem cell gene therapy (HSCGT). Measurement of ADA enzyme activity and metabolites and sequencing of the ADA gene were performed in most of the patients with ADA deficiency. One of the patients with late-onset ADA deficiency was diagnosed by the help of primary immunodeficiency panel screening. Ten out of 13 patients were diagnosed as SCID, while 3 out of 13 were diagnosed as delayed-/late-onset ADA deficiency. Late-onset ADA deficiency patients had clinical and laboratory findings of combined immunodeficiency (CID). Eight patients with ADA-SCID were found to have higher levels of ADA metabolite (dAXP%) (62.1% (34.6-71.9)) than 3 patients with delayed-/late-onset ADA deficiency (6.9% (2.1-8.9). All but one patient with SCID had T-B-NK- phenotype, one had T-B-NK+ phenotype. Genetic defect was documented in 11 patients. Four out of 11 patients had compound heterozygous defects. Three out of 4 patients with compound heterozygous defects had delayed-onset/late-onset ADA deficiency. Seven out of 11 patients with SCID had homozygous defects. Five out of 7 had the same homozygous indel frameshift mutation (c.955-959delGAAGA) showing a founder effect. There were two novel splice site defects: one (IVS10+2T>C) was heterozygous in a patient with late-onset ADA deficiency, and the other was homozygous (IVS2delT+2) in a SCID patient. Other defects were missense defects. Nine out of 13 patients were put on pegylated ADA ERT. Four out of six patients were transplanted without using a conditioning regimen. HSCGT was performed to one of the patients. The genetic diagnosis of SCID is utmost important. There is a chance to give ERT before the definitive therapy if the patient with SCID/CID has ADA deficiency. Although ERT was insufficient to restore a normal immune function in ADA-SCID patients, it was useful to improve and stabilize the clinical status before curative therapy (aHSCT/HSCGT). Enzyme replacement therapy was successful in patients with late-/delayed-onset ADA deficiency who presented with the features of combined immunodeficiency. Gastrointestinal polyposis in a patient with late-onset ADA deficiency may be an association or a coincidental finding. Intermittent neurodevelopmental evaluation especially for hearing impairment should be performed in most of the ADA-deficient patients. This may alleviate the speech delay and cognitive abnormalities which may be observed in the follow-up.

HOMOCYSTEINE RE-METHYLATION ENZYME POLYMORPHISMS AND INCREASED RISKS FOR NEURAL TUBE DEFECTS (NTDS). (R828292)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Enhanced truncated-correlation photothermal coherence tomography with application to deep subsurface defect imaging and 3-dimensional reconstructions

NASA Astrophysics Data System (ADS)

Tavakolian, Pantea; Sivagurunathan, Koneswaran; Mandelis, Andreas

2017-07-01

Photothermal diffusion-wave imaging is a promising technique for non-destructive evaluation and medical applications. Several diffusion-wave techniques have been developed to produce depth-resolved planar images of solids and to overcome imaging depth and image blurring limitations imposed by the physics of parabolic diffusion waves. Truncated-Correlation Photothermal Coherence Tomography (TC-PCT) is the most successful class of these methodologies to-date providing 3-D subsurface visualization with maximum depth penetration and high axial and lateral resolution. To extend the depth range and axial and lateral resolution, an in-depth analysis of TC-PCT, a novel imaging system with improved instrumentation, and an optimized reconstruction algorithm over the original TC-PCT technique is developed. Thermal waves produced by a laser chirped pulsed heat source in a finite thickness solid and the image reconstruction algorithm are investigated from the theoretical point of view. 3-D visualization of subsurface defects utilizing the new TC-PCT system is reported. The results demonstrate that this method is able to detect subsurface defects at the depth range of ˜4 mm in a steel sample, which exhibits dynamic range improvement by a factor of 2.6 compared to the original TC-PCT. This depth does not represent the upper limit of the enhanced TC-PCT. Lateral resolution in the steel sample was measured to be ˜31 μm.

X-ray Structure of Imidazolonepropionase from Agrobacterium tumefaciens at 1.87 angstrom Resolution

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

Tyagi,R.; Kumaran, D.; Burley, S.

2007-01-01

Histidine degradation in agrobacterium tumefaciens involves four enzymes, including histidase, urocanase, imidazolonepropionase, and N-formylglutamate amido hydrolase. The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-t-glutamate.

Imaging active topological defects in carbon nanotubes

NASA Astrophysics Data System (ADS)

Suenaga, Kazu; Wakabayashi, Hideaki; Koshino, Masanori; Sato, Yuta; Urita, Koki; Iijima, Sumio

2007-06-01

A single-walled carbon nanotube (SWNT) is a wrapped single graphene layer, and its plastic deformation should require active topological defects-non-hexagonal carbon rings that can migrate along the nanotube wall. Although in situ transmission electron microscopy (TEM) has been used to examine the deformation of SWNTs, these studies deal only with diameter changes and no atomistic mechanism has been elucidated experimentally. Theory predicts that some topological defects can form through the Stone-Wales transformation in SWNTs under tension at 2,000 K, and could act as a dislocation core. We demonstrate here, by means of high-resolution (HR)-TEM with atomic sensitivity, the first direct imaging of pentagon-heptagon pair defects found in an SWNT that was heated at 2,273 K. Moreover, our in situ HR-TEM observation reveals an accumulation of topological defects near the kink of a deformed nanotube. This result suggests that dislocation motions or active topological defects are indeed responsible for the plastic deformation of SWNTs.

Admittance spectroscopy or deep level transient spectroscopy: A contrasting juxtaposition

NASA Astrophysics Data System (ADS)

Bollmann, Joachim; Venter, Andre

2018-04-01

A comprehensive understanding of defects in semiconductors remains of primary importance. In this paper the effectiveness of two of the most commonly used semiconductor defect spectroscopy techniques, viz. deep level transient spectroscopy (DLTS) and admittance spectroscopy (AS) are reviewed. The analysis of defects present in commercially available SiC diodes shows that admittance spectroscopy allows the identification of deep traps with reduced measurement effort compared to deep Level Transient Spectroscopy (DLTS). Besides the N-donor, well-studied intrinsic defects were detected in these diodes. Determination of their activation energy and defect density, using the two techniques, confirm that the sensitivity of AS is comparable to that of DLTS while, due to its well defined peak shape, the spectroscopic resolution is superior. Additionally, admittance spectroscopy can analyze faster emission processes which make the study of shallow defects more practical and even that of shallow dopant levels, possible. A comparative summary for the relevant spectroscopic features of the two capacitance methods are presented.

Structure and function of enzymes in heme biosynthesis.

PubMed

Layer, Gunhild; Reichelt, Joachim; Jahn, Dieter; Heinz, Dirk W

2010-06-01

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V-shaped glutamyl-tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the "Radical SAM enzyme" coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure-function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.

Glucose-6-phosphate dehydrogenase deficiency: correlation between the genotype, biochemistry and phenotype.

PubMed

Chan, Daisy K L

2008-12-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic enzyme defect present in many people from African, Middle Eastern, Mediterranean and Asian countries. Individuals with the enzyme deficiency may remain asymptomatic, develop an acute haemolytic crises to infections or Fava beans, neonatal jaundice or chronic non-spherocytic haemolytic anaemia. Electrophoretic mobility may be fast, slow or normal. Over 160 mutations have been described, mostly due to single amino acid substitution. Although correlation of the genotype and biochemistry with the clinical phenotype of G6PD deficient individuals remains somewhat variable, there is better correlation among individuals presenting with chronic non-spherocytic haemolytic anaemia, which is related to the NADP structure of the enzyme.

Magnetic resonance imaging at a high field strength of ventricular septal defects in infants.

PubMed

Baker, E J; Ayton, V; Smith, M A; Parsons, J M; Ladusans, E J; Anderson, R H; Maisey, M N; Tynan, M; Fagg, N L; Deverall, P B

1989-10-01

Magnetic resonance imaging at a high field strength has potential benefits for the study of the heart in infants, which is when most congenital heart disease presents. Seventeen infants with various anatomical types of ventricular septal defect were studied by this technique. Good quality, high resolution, images were obtained in every case. There were no major practical problems. The morphology of the defects in all 17 hearts was displayed in great detail. In some instances, the interpretation of the images resembled that of equivalent images from cross sectional echocardiography. But this new technique allowed imaging in planes that cannot be obtained by echocardiography. One particularly valuable plane gave a face on view of the inlet and trabecular components of the septum. This allowed very precise localisation of defects in these areas. The relation between the defects and the atrioventricular and arterial valves was exceptionally well shown in various different imaging planes. One patient in the series had multiple trabecular defects that were clearly shown. Magnetic resonance imaging gives detailed morphological information about ventricular septal defects.

Signature of dislocations and stacking faults of face-centred cubic nanocrystals in coherent X-ray diffraction patterns: a numerical study.

PubMed

Dupraz, Maxime; Beutier, Guillaume; Rodney, David; Mordehai, Dan; Verdier, Marc

2015-06-01

Crystal defects induce strong distortions in diffraction patterns. A single defect alone can yield strong and fine features that are observed in high-resolution diffraction experiments such as coherent X-ray diffraction. The case of face-centred cubic nanocrystals is studied numerically and the signatures of typical defects close to Bragg positions are identified. Crystals of a few tens of nanometres are modelled with realistic atomic potentials and 'relaxed' after introduction of well defined defects such as pure screw or edge dislocations, or Frank or prismatic loops. Diffraction patterns calculated in the kinematic approximation reveal various signatures of the defects depending on the Miller indices. They are strongly modified by the dissociation of the dislocations. Selection rules on the Miller indices are provided, to observe the maximum effect of given crystal defects in the initial and relaxed configurations. The effect of several physical and geometrical parameters such as stacking fault energy, crystal shape and defect position are discussed. The method is illustrated on a complex structure resulting from the simulated nanoindentation of a gold nanocrystal.

Signature of dislocations and stacking faults of face-centred cubic nanocrystals in coherent X-ray diffraction patterns: a numerical study1

PubMed Central

Dupraz, Maxime; Beutier, Guillaume; Rodney, David; Mordehai, Dan; Verdier, Marc

2015-01-01

Crystal defects induce strong distortions in diffraction patterns. A single defect alone can yield strong and fine features that are observed in high-resolution diffraction experiments such as coherent X-ray diffraction. The case of face-centred cubic nanocrystals is studied numerically and the signatures of typical defects close to Bragg positions are identified. Crystals of a few tens of nanometres are modelled with realistic atomic potentials and ‘relaxed’ after introduction of well defined defects such as pure screw or edge dislocations, or Frank or prismatic loops. Diffraction patterns calculated in the kinematic approximation reveal various signatures of the defects depending on the Miller indices. They are strongly modified by the dissociation of the dislocations. Selection rules on the Miller indices are provided, to observe the maximum effect of given crystal defects in the initial and relaxed configurations. The effect of several physical and geometrical parameters such as stacking fault energy, crystal shape and defect position are discussed. The method is illustrated on a complex structure resulting from the simulated nanoindentation of a gold nanocrystal. PMID:26089755

Bio-photonic detection method for morphological analysis of anthracnose disease and physiological disorders of Diospyros kaki

NASA Astrophysics Data System (ADS)

Wijesinghe, Ruchire Eranga; Lee, Seung-Yeol; Ravichandran, Naresh Kumar; Shirazi, Muhammad Faizan; Moon, Byungin; Jung, Hee-Young; Jeon, Mansik; Kim, Jeehyun

2017-04-01

The pathological and physiological defects in various types of fruits lead to large amounts of economical waste. It is well recognized that internal fruit defects due to pathological infections and physiological disorders can be effectively visualized at an initial stage of the disease using a well-known bio-photonic detection method called optical coherence tomography (OCT). This work investigates the use of OCT for identifying the morphological variations of anthracnose (bitter rot) disease infected and physiologically disordered Diospyros kaki (Asian Persimmon) fruits. An experiment was conducted using fruit samples that were carefully selected from persimmon orchards. Depth-resolved images with a high axial resolution were acquired using 850-nm-based spectral-domain OCT (SD-OCT) system. The obtained exemplary high-resolution two-dimensional and volumetric three-dimensional images revealed complementary morphological differences between healthy and defected samples. Moreover, the obtained depth-profile analysis results confirmed the disappearance of the healthy cell layers among the healthy-infected boundary regions. Thus, the proposed method has the potential to increase the diagnostic accuracy of the OCT technique used in agricultural plantations.

Simulation of electron energy loss spectra of nanomaterials with linear-scaling density functional theory

DOE PAGES

Tait, E. W.; Ratcliff, L. E.; Payne, M. C.; ...

2016-04-20

Experimental techniques for electron energy loss spectroscopy (EELS) combine high energy resolution with high spatial resolution. They are therefore powerful tools for investigating the local electronic structure of complex systems such as nanostructures, interfaces and even individual defects. Interpretation of experimental electron energy loss spectra is often challenging and can require theoretical modelling of candidate structures, which themselves may be large and complex, beyond the capabilities of traditional cubic-scaling density functional theory. In this work, we present functionality to compute electron energy loss spectra within the onetep linear-scaling density functional theory code. We first demonstrate that simulated spectra agree withmore » those computed using conventional plane wave pseudopotential methods to a high degree of precision. The ability of onetep to tackle large problems is then exploited to investigate convergence of spectra with respect to supercell size. As a result, we apply the novel functionality to a study of the electron energy loss spectra of defects on the (1 0 1) surface of an anatase slab and determine concentrations of defects which might be experimentally detectable.« less

Imaging review of cerebrospinal fluid leaks

PubMed Central

Vemuri, Naga V; Karanam, Lakshmi S P; Manchikanti, Venkatesh; Dandamudi, Srinivas; Puvvada, Sampath K; Vemuri, Vineet K

2017-01-01

Cerebrospinal fluid (CSF) leak occurs due to a defect in the dura and skull base. Trauma remains the most common cause of CSF leak; however, a significant number of cases are iatrogenic, and result from a complication of functional endoscopic sinus surgery (FESS). Early diagnosis of CSF leak is of paramount importance to prevent life-threatening complications such as brain abscess and meningitis. Imaging plays a crucial role in the detection and characterization of CSF leaks. Three-dimensional, isotropic, high resolution computed tomography (HRCT) accurately detects the site and size of the bony defect. CT cisternography, though invasive, helps accurately identify the site of CSF leak, especially in the presence of multiple bony defects. Magnetic resonance imaging (MRI) accurately detects CSF leaks and associated complications such as the encephaloceles and meningoceles. In this review, we emphasize the importance and usefulness of 3D T2 DRIVE MR cisternography in localizing CSF leaks. This sequence has the advantages of effective bone and fat suppression, decreased artefacts, faster acquisition times, three-dimensional capability, y and high spatial resolution in addition to providing very bright signal from the CSF. PMID:29379240

Imaging review of cerebrospinal fluid leaks.

PubMed

Vemuri, Naga V; Karanam, Lakshmi S P; Manchikanti, Venkatesh; Dandamudi, Srinivas; Puvvada, Sampath K; Vemuri, Vineet K

2017-01-01

Cerebrospinal fluid (CSF) leak occurs due to a defect in the dura and skull base. Trauma remains the most common cause of CSF leak; however, a significant number of cases are iatrogenic, and result from a complication of functional endoscopic sinus surgery (FESS). Early diagnosis of CSF leak is of paramount importance to prevent life-threatening complications such as brain abscess and meningitis. Imaging plays a crucial role in the detection and characterization of CSF leaks. Three-dimensional, isotropic, high resolution computed tomography (HRCT) accurately detects the site and size of the bony defect. CT cisternography, though invasive, helps accurately identify the site of CSF leak, especially in the presence of multiple bony defects. Magnetic resonance imaging (MRI) accurately detects CSF leaks and associated complications such as the encephaloceles and meningoceles. In this review, we emphasize the importance and usefulness of 3D T2 DRIVE MR cisternography in localizing CSF leaks. This sequence has the advantages of effective bone and fat suppression, decreased artefacts, faster acquisition times, three-dimensional capability, y and high spatial resolution in addition to providing very bright signal from the CSF.

Resolution of Hydronephrosis in a Patient With Mucopolysaccharidosis Type II With Enzyme Replacement Therapy.

PubMed

Nishiyama, Kei; Imai, Takashi; Ohkubo, Kazuhiro; Sanefuji, Masafumi; Takada, Hidetoshi

2017-03-01

Mucopolysaccharidosis type II (MPS II) is caused by deficiency of lysosomal enzyme iduronate-2-sulfatase. Insufficient activity of the enzyme results in accumulation of glycosaminoglycans leading to progressive multisystem pathologies. MPS II is less likely to be complicated by kidney and urinary tract problems. We report a boy with MPS II, who developed left hydronephrosis. His hydronephrosis improved after starting enzyme replacement therapy. It was suggested that MPS II was closely associated with the pathogenesis of hydronephrosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Probing the Active Site of Candida Glabrata Dihydrofolate Reductase with High Resolution Crystal Structures and the Synthesis of New Inhibitors

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

Liu, J.; Bolstad, D; Smith, A

2009-01-01

Candida glabrata, a fungal strain resistant to many commonly administered antifungal agents, has become an emerging threat to human health. In previous work, we validated that the essential enzyme, dihydrofolate reductase, is a drug target in C. glabrata. Using a crystal structure of dihydrofolate reductase from C. glabrata bound to an initial lead compound, we designed a class of biphenyl antifolates that potently and selectively inhibit both the enzyme and the growth of the fungal culture. In this work, we explore the structure-activity relationships of this class of antifolates with four new high resolution crystal structures of enzyme:inhibitor complexes andmore » the synthesis of four new inhibitors. The designed inhibitors are intended to probe key hydrophobic pockets visible in the crystal structure. The crystal structures and an evaluation of the new compounds reveal that methyl groups at the meta and para positions of the distal phenyl ring achieve the greatest number of interactions with the pathogenic enzyme and the greatest degree of selectivity over the human enzyme. Additionally, antifungal activity can be tuned with substitution patterns at the propargyl and para-phenyl positions.« less

Biocatalytic induction of supramolecular order

NASA Astrophysics Data System (ADS)

Hirst, Andrew R.; Roy, Sangita; Arora, Meenakshi; Das, Apurba K.; Hodson, Nigel; Murray, Paul; Marshall, Stephen; Javid, Nadeem; Sefcik, Jan; Boekhoven, Job; van Esch, Jan H.; Santabarbara, Stefano; Hunt, Neil T.; Ulijn, Rein V.

2010-12-01

Supramolecular gels, which demonstrate tunable functionalities, have attracted much interest in a range of areas, including healthcare, environmental protection and energy-related technologies. Preparing these materials in a reliable manner is challenging, with an increased level of kinetic defects observed at higher self-assembly rates. Here, by combining biocatalysis and molecular self-assembly, we have shown the ability to more quickly access higher-ordered structures. By simply increasing enzyme concentration, supramolecular order expressed at molecular, nano- and micro-levels is dramatically enhanced, and, importantly, the gelator concentrations remain identical. Amphiphile molecules were prepared by attaching an aromatic moiety to a dipeptide backbone capped with a methyl ester. Their self-assembly was induced by an enzyme that hydrolysed the ester. Different enzyme concentrations altered the catalytic activity and size of the enzyme clusters, affecting their mobility. This allowed structurally diverse materials that represent local minima in the free energy landscape to be accessed based on a single gelator structure.

Impacts | Hydrogen and Fuel Cells | NREL

Science.gov Websites

Impacts Impacts Read about NREL's impacts on innovations in hydrogen and fuel cell research and -Splitting Electrodes NREL Shows How Cyanobacteria Build Hydrogen-Producing Enzyme Fuel Cell Systems R&D -Speed Scanner to Monitor Fuel Cell Material Defects Making Fuel Cells Cleaner, Better, and Cheaper GM

Modeling of Powder Bed Manufacturing Defects

NASA Astrophysics Data System (ADS)

Mindt, H.-W.; Desmaison, O.; Megahed, M.; Peralta, A.; Neumann, J.

2018-01-01

Powder bed additive manufacturing offers unmatched capabilities. The deposition resolution achieved is extremely high enabling the production of innovative functional products and materials. Achieving the desired final quality is, however, hampered by many potential defects that have to be managed in due course of the manufacturing process. Defects observed in products manufactured via powder bed fusion have been studied experimentally. In this effort we have relied on experiments reported in the literature and—when experimental data were not sufficient—we have performed additional experiments providing an extended foundation for defect analysis. There is large interest in reducing the effort and cost of additive manufacturing process qualification and certification using integrated computational material engineering. A prerequisite is, however, that numerical methods can indeed capture defects. A multiscale multiphysics platform is developed and applied to predict and explain the origin of several defects that have been observed experimentally during laser-based powder bed fusion processes. The models utilized are briefly introduced. The ability of the models to capture the observed defects is verified. The root cause of the defects is explained by analyzing the numerical results thus confirming the ability of numerical methods to provide a foundation for rapid process qualification.

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

Claus, Rene A.; Wang, Yow-Gwo; Wojdyla, Antoine

Extreme Ultraviolet (EUV) Lithography mask defects were examined on the actinic mask imaging system, SHARP, at Lawrence Berkeley National Laboratory. Also, a quantitative phase retrieval algorithm based on the Weak Object Transfer Function was applied to the measured through-focus aerial images to examine the amplitude and phase of the defects. The accuracy of the algorithm was demonstrated by comparing the results of measurements using a phase contrast zone plate and a standard zone plate. Using partially coherent illumination to measure frequencies that would otherwise fall outside the numerical aperture (NA), it was shown that some defects are smaller than themore » conventional resolution of the microscope. We found that the programmed defects of various sizes were measured and shown to have both an amplitude and a phase component that the algorithm is able to recover.« less

Metastability of the midgap level EL 2 in GaAs - Relationship with the As antisite defect

NASA Technical Reports Server (NTRS)

Skowronski, M.; Lagowski, J.; Gatos, H. C.

1985-01-01

It is found that the rate of the photoinduced transition of the GaAs midgap level EL 2 to its metastable state increases as its occupation increases. High-resolution optical spectra of this transition exhibit a sharp peak very similar to the no-phonon line of the intracenter absorption of the As antisite defect. These findings show that the transition to the metastable state is initiated from the ground state 1A1, and it is finalized via the excited state 1T2 of the neutral As antisite defect. They thus provide a new basis for the critical assessment of the EL 2 metastability models and further confirmation of the association of EL 2 with the isolated As antisite defect.

Biomarkers in Lysosomal Storage Diseases

PubMed Central

Bobillo Lobato, Joaquin; Jiménez Hidalgo, Maria; Jiménez Jiménez, Luis M.

2016-01-01

A biomarker is generally an analyte that indicates the presence and/or extent of a biological process, which is in itself usually directly linked to the clinical manifestations and outcome of a particular disease. The biomarkers in the field of lysosomal storage diseases (LSDs) have particular relevance where spectacular therapeutic initiatives have been achieved, most notably with the introduction of enzyme replacement therapy (ERT). There are two main types of biomarkers. The first group is comprised of those molecules whose accumulation is directly enhanced as a result of defective lysosomal function. These molecules represent the storage of the principal macro-molecular substrate(s) of a specific enzyme or protein, whose function is deficient in the given disease. In the second group of biomarkers, the relationship between the lysosomal defect and the biomarker is indirect. In this group, the biomarker reflects the effects of the primary lysosomal defect on cell, tissue, or organ functions. There is no “gold standard” among biomarkers used to diagnosis and/or monitor LSDs, but there are a number that exist that can be used to reasonably assess and monitor the state of certain organs or functions. A number of biomarkers have been proposed for the analysis of the most important LSDs. In this review, we will summarize the most promising biomarkers in major LSDs and discuss why these are the most promising candidates for screening systems. PMID:28933418

The Epidermis of Grhl3-Null Mice Displays Altered Lipid Processing and Cellular Hyperproliferation

PubMed Central

Ting, Stephen B; Caddy, Jacinta; Wilanowski, Tomasz; Auden, Alana; Cunningham, John M; Elias, Peter M; Holleran, Walter M

2005-01-01

The presence of an impermeable surface barrier is an essential homeostatic mechanism in almost all living organisms. We have recently described a novel gene that is critical for the developmental instruction and repair of the integument in mammals. This gene, Grainy head-like 3 (Grhl3) is a member of a large family of transcription factors that are homologs of the Drosophila developmental gene grainy head (grh). Mice lacking Grhl3 fail to form an adequate skin barrier, and die at birth due to dehydration. These animals are also unable to repair the epidermis, exhibiting failed wound healing in both fetal and adult stages of development. These defects are due, in part, to diminished expression of a Grhl3 target gene, Transglutaminase 1 (TGase 1), which encodes a key enzyme involved in cross-linking of epidermal structural proteins and lipids into the cornified envelope (CE). Remarkably, the Drosophila grh gene plays an analogous role, regulating enzymes involved in the generation of quinones, which are essential for cross-linking structural components of the fly epidermis. In an extension of our initial analyses, we focus this report on additional defects observed in the Grhl3-null epidermis, namely defective extra-cellular lipid processing, altered lamellar lipid architecture and cellular hyperproliferation. These abnormalities suggest that Grhl3 plays diverse mechanistic roles in maintaining homeostasis in the skin. PMID:19521564

The epidermis of grhl3-null mice displays altered lipid processing and cellular hyperproliferation.

PubMed

Ting, Stephen B; Caddy, Jacinta; Wilanowski, Tomasz; Auden, Alana; Cunningham, John M; Elias, Peter M; Holleran, Walter M; Jane, Stephen M

2005-04-01

The presence of an impermeable surface barrier is an essential homeostatic mechanism in almost all living organisms. We have recently described a novel gene that is critical for the developmental instruction and repair of the integument in mammals. This gene, Grainy head-like 3 (Grhl3) is a member of a large family of transcription factors that are homologs of the Drosophila developmental gene grainy head (grh). Mice lacking Grhl3 fail to form an adequate skin barrier, and die at birth due to dehydration. These animals are also unable to repair the epidermis, exhibiting failed wound healing in both fetal and adult stages of development. These defects are due, in part, to diminished expression of a Grhl3 target gene, Transglutaminase 1 (TGase 1), which encodes a key enzyme involved in cross-linking of epidermal structural proteins and lipids into the cornified envelope (CE). Remarkably, the Drosophila grh gene plays an analogous role, regulating enzymes involved in the generation of quinones, which are essential for cross-linking structural components of the fly epidermis. In an extension of our initial analyses, we focus this report on additional defects observed in the Grhl3-null epidermis, namely defective extra-cellular lipid processing, altered lamellar lipid architecture and cellular hyperproliferation. These abnormalities suggest that Grhl3 plays diverse mechanistic roles in maintaining homeostasis in the skin.

High resolution in-operando microimaging of solar cells with pulsed electrically-detected magnetic resonance

NASA Astrophysics Data System (ADS)

Katz, Itai; Fehr, Matthias; Schnegg, Alexander; Lips, Klaus; Blank, Aharon

2015-02-01

The in-operando detection and high resolution spatial imaging of paramagnetic defects, impurities, and states becomes increasingly important for understanding loss mechanisms in solid-state electronic devices. Electron spin resonance (ESR), commonly employed for observing these species, cannot meet this challenge since it suffers from limited sensitivity and spatial resolution. An alternative and much more sensitive method, called electrically-detected magnetic resonance (EDMR), detects the species through their magnetic fingerprint, which can be traced in the device's electrical current. However, until now it could not obtain high resolution images in operating electronic devices. In this work, the first spatially-resolved electrically-detected magnetic resonance images (EDMRI) of paramagnetic states in an operating real-world electronic device are provided. The presented method is based on a novel microwave pulse sequence allowing for the coherent electrical detection of spin echoes in combination with powerful pulsed magnetic-field gradients. The applicability of the method is demonstrated on a device-grade 1-μm-thick amorphous silicon (a-Si:H) solar cell and an identical device that was degraded locally by an electron beam. The degraded areas with increased concentrations of paramagnetic defects lead to a local increase in recombination that is mapped by EDMRI with ∼20-μm-scale pixel resolution. The novel approach presented here can be widely used in the nondestructive in-operando three-dimensional characterization of solid-state electronic devices with a resolution potential of less than 100 nm.

A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin

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

Budayova-Spano, Monika, E-mail: spano@embl-grenoble.fr; Institut Laue-Langevin, 6 Rue Jules Horowitz, BP 156, 38042 Grenoble; Bonneté, Françoise

2006-03-01

Neutron diffraction data of hydrogenated recombinant urate oxidase enzyme (Rasburicase), complexed with a purine-type inhibitor 8-azaxanthin, was collected to 2.1 Å resolution from a crystal grown in D{sub 2}O by careful control and optimization of crystallization conditions via knowledge of the phase diagram. Deuterium atoms were clearly seen in the neutron-scattering density map. Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 Å resolution using the LADI instrument from a crystal (grownmore » in D{sub 2}O) with volume 1.8 mm{sup 3}. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 Å) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.« less

The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles

PubMed Central

Alvarez, Diego E.

2016-01-01

Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207–238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207–238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. PMID:27068088

The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

PubMed

Alvarez, Diego E; Agaisse, Hervé

2016-06-01

Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Treatment of patients with neurotrophic keratitis stages 2 and 3 with plasma rich in growth factors (PRGF-Endoret) eye-drops.

PubMed

Sanchez-Avila, Ronald Mauricio; Merayo-Lloves, Jesus; Riestra, Ana Cristina; Fernandez-Vega Cueto, Luis; Anitua, Eduardo; Begoña, Leire; Muruzabal, Francisco; Orive, Gorka

2018-06-01

To provide preliminary data about efficacy and safety of plasma rich in growth factors (PRGF) eye-drops in neurotrophic keratitis (NK) and to analyze the possible influence of certain variables on treatment outcomes. This retrospective study included patients with stages 2-3 of NK treated with PRGF eye-drops. Primary endpoint was the resolution time of corneal ulcer defect. Outcome measures including percentage of ulcer closure at 4 weeks, Ocular Surface Disease Index (OSDI), Best-Corrected Visual Acuity (BCVA) and Visual Analogue Scale (VAS) were also evaluated before and after treatment with PRGF. The influence of some patients' clinical variables on results was assessed. Safety assessment was also performed reporting all adverse events. Thirty-eight treated eyes in a total of thirty-one patients were evaluated, of which five cases had no prior response to autologous serum treatment. Most cases (97.4%) achieved the complete resolution of corneal defect/ulcer. Mean resolution time was 11.4 weeks (SD = 13.7). A statistical significant (p < 0.05) reduction in OSDI (60.9%), VAS frequency (59.9%), VAS severity (57.0%) and improvement in BCVA (52.8%) was observed. The results were stratified according to the pathology stage and to the identified potential effect modifiers variables. Only one adverse event was reported in one patient (2.6%). PRGF eye-drops could be a safe and effective therapeutic option for patients with stages 2-3 of NK, showing high rates of corneal defect/ulcer resolution in short times, either in reducing signs and symptoms of NK, and therefore preventing the progression of NK to greater ocular complications.

Use of high-granularity CdZnTe pixelated detectors to correct response non-uniformities caused by defects in crystals

DOE PAGES

Bolotnikov, A. E.; Camarda, G. S.; Cui, Y.; ...

2015-09-06

Following our successful demonstration of the position-sensitive virtual Frisch-grid detectors, we investigated the feasibility of using high-granularity position sensing to correct response non-uniformities caused by the crystal defects in CdZnTe (CZT) pixelated detectors. The development of high-granularity detectors able to correct response non-uniformities on a scale comparable to the size of electron clouds opens the opportunity of using unselected off-the-shelf CZT material, whilst still assuring high spectral resolution for the majority of the detectors fabricated from an ingot. Here, we present the results from testing 3D position-sensitive 15×15×10 mm 3 pixelated detectors, fabricated with conventional pixel patterns with progressively smallermore » pixel sizes: 1.4, 0.8, and 0.5 mm. We employed the readout system based on the H3D front-end multi-channel ASIC developed by BNL's Instrumentation Division in collaboration with the University of Michigan. We use the sharing of electron clouds among several adjacent pixels to measure locations of interaction points with sub-pixel resolution. By using the detectors with small-pixel sizes and a high probability of the charge-sharing events, we were able to improve their spectral resolutions in comparison to the baseline levels, measured for the 1.4-mm pixel size detectors with small fractions of charge-sharing events. These results demonstrate that further enhancement of the performance of CZT pixelated detectors and reduction of costs are possible by using high spatial-resolution position information of interaction points to correct the small-scale response non-uniformities caused by crystal defects present in most devices.« less

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse.

PubMed

van den Brink, Joost; Maitan-Alfenas, Gabriela Piccolo; Zou, Gen; Wang, Chengshu; Zhou, Zhihua; Guimarães, Valéria Monteze; de Vries, Ronald P

2014-10-01

Plant-degrading enzymes can be produced by fungi on abundantly available low-cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two "second generation" substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi-)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non-washed SCB. The sensitivity to non-washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spatial distribution of enzyme activities along the root and in the rhizosphere of different plants

NASA Astrophysics Data System (ADS)

Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes:β-glucosidase, leucine amino peptidase and phosphatase, using fluorogenically labelled substrates. Spatial resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify spatial distribution of enzyme activities in the rhizosphere hotspots. References Spohn, M., Kuzyakov, Y., 2013. Phosphorus mineralization can be driven by microbial need for carbon. Soil Biology & Biochemistry 61: 69-75

X-Ray Diffraction and Imaging Study of Imperfections of Crystallized Lysozyme with Coherent X-Rays

NASA Technical Reports Server (NTRS)

Hu, Zheng-Wei; Chu, Y. S.; Lai, B.; Cai, Z.; Thomas, B. R.; Chernov, A. A.

2003-01-01

Phase-sensitive x-ray diffraction imaging and high angular-resolution diffraction combined with phase contrast radiographic imaging are employed to characterize defects and perfection of a uniformly grown tetragonal lysozyme crystal in symmetric Laue case. The fill width at half-maximum (FWHM) of a 4 4 0 rocking curve measured from the original crystal is approximately 16.7 arcseconds, and defects, which include point defects, line defects, and microscopic domains, have been clearly observed in the diffraction images of the crystal. The observed line defects carry distinct dislocation features running approximately along the <110> growth front, and they have been found to originate mostly at a central growth area and occasionally at outer growth regions. Individual point defects trapped at a crystal nucleus are resolved in the images of high sensitivity to defects. Slow dehydration has led to the broadening of the 4 4 0 rocking curve by a factor of approximately 2.4. A significant change of the defect structure and configuration with drying has been revealed, which suggests the dehydration induced migration and evolution of dislocations and lattice rearrangements to reduce overall strain energy. The sufficient details of the observed defects shed light upon perfection, nucleation and growth, and properties of protein crystals.

Virus scaffolds as enzyme nano-carriers.

PubMed

Cardinale, Daniela; Carette, Noëlle; Michon, Thierry

2012-07-01

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Flavivirus RNA cap methyltransferase: structure, function, and inhibition.

PubMed

Liu, Lihui; Dong, Hongping; Chen, Hui; Zhang, Jing; Ling, Hua; Li, Zhong; Shi, Pei-Yong; Li, Hongmin

2010-08-01

Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5' terminal cap 1 structure (m(7)GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m(7)GpppA → m(7)GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2'-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2'-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.

Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

PubMed

Harlalka, Gaurav V; Lehman, Anna; Chioza, Barry; Baple, Emma L; Maroofian, Reza; Cross, Harold; Sreekantan-Nair, Ajith; Priestman, David A; Al-Turki, Saeed; McEntagart, Meriel E; Proukakis, Christos; Royle, Louise; Kozak, Radoslaw P; Bastaki, Laila; Patton, Michael; Wagner, Karin; Coblentz, Roselyn; Price, Joy; Mezei, Michelle; Schlade-Bartusiak, Kamilla; Platt, Frances M; Hurles, Matthew E; Crosby, Andrew H

2013-12-01

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A resolution.

PubMed

Varrot, A; Hastrup, S; Schülein, M; Davies, G J

1999-01-15

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.

An efficient way of layout processing based on calibre DRC and pattern matching for defects inspection application

NASA Astrophysics Data System (ADS)

Li, Helen; Lee, Robben; Lee, Tyzy; Xue, Teddy; Liu, Hermes; Wu, Hall; Wan, Qijian; Du, Chunshan; Hu, Xinyi; Liu, Zhengfang

2018-03-01

As technology advances, escalating layout design complexity and chip size make defect inspection becomes more challenging than ever before. The YE (Yield Enhancement) engineers are seeking for an efficient strategy to ensure accuracy without suffering running time. A smart way is to set different resolutions for different pattern structures, for examples, logic pattern areas have a higher scan resolution while the dummy areas have a lower resolution, SRAM area may have another different resolution. This can significantly reduce the scan processing time meanwhile the accuracy does not suffer. Due to the limitation of the inspection equipment, the layout must be processed in order to output the Care Area marker in line with the requirement of the equipment, for instance, the marker shapes must be rectangle and the number of the rectangle shapes should be as small as possible. The challenge is how to select the different Care Areas by pattern structures, merge the areas efficiently and then partition them into pieces of rectangle shapes. This paper presents a solution based on Calibre DRC and Pattern Matching. Calibre equation-based DRC is a powerful layout processing engine and Calibre Pattern Matching's automated visual capture capability enables designers to define these geometries as layout patterns and store them in libraries which can be re-used in multiple design layouts. Pattern Matching simplifies the description of very complex relationships between pattern shapes efficiently and accurately. Pattern matching's true power is on display when it is integrated with normal DRC deck. In this application of defects inspection, we first run Calibre DRC to get rule based Care Area then use Calibre Pattern Matching's automated pattern capture capability to capture Care Area shapes which need a higher scan resolution with a tune able pattern halo. In the pattern matching step, when the patterns are matched, a bounding box marker will be output to identify the high resolution area. The equation-based DRC and Pattern Matching effectively work together for different scan phases.

Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension.

PubMed

Johnson, Jennifer A; Hemnes, Anna R; Perrien, Daniel S; Schuster, Manfred; Robinson, Linda J; Gladson, Santhi; Loibner, Hans; Bai, Susan; Blackwell, Tom R; Tada, Yuji; Harral, Julie W; Talati, Megha; Lane, Kirk B; Fagan, Karen A; West, James

2012-03-01

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.

The possibility of identifying the spatial location of single dislocations by topo-tomography on laboratory setups

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

Zolotov, D. A., E-mail: zolotovden@crys.ras.ru; Buzmakov, A. V.; Elfimov, D. A.

2017-01-15

The spatial arrangement of single linear defects in a Si single crystal (input surface (111)) has been investigated by X-ray topo-tomography using laboratory X-ray sources. The experimental technique and the procedure of reconstructing a 3D image of dislocation half-loops near the Si crystal surface are described. The sizes of observed linear defects with a spatial resolution of about 10 μm are estimated.

Microstructural study of Mg-doped p-type GaN: Correlation between high-resolution electron microscopy and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Tsen, S.-C. Y.; Smith, David J.; Tsen, K. T.; Kim, W.; Morkoç, H.

1997-12-01

A series of Mg-doped GaN films (˜1-1.3 μm) grown by reactive molecular beam epitaxy at substrate temperatures of 750 and 800 °C has been studied by high-resolution electron microscopy (HREM) and Raman spectroscopy. Stacking defects parallel to the substrate surface were observed in samples grown on sapphire substrates at 750 °C with AlN buffer layers (60-70 nm) at low Mg concentration. A transition region with mixed zinc-blende cubic (c) and wurtzite hexagonal (h) phases having the relative orientations of (111)c//(00.1)h and (11¯0)c//(10.0)h was observed for increased Mg concentration. The top surfaces of highly doped samples were rough and assumed a completely zinc-blende phase with some inclined stacking faults. Samples grown with a Mg cell temperature of 350 °C and high doping levels were highly disordered with many small crystals having inclined stacking faults, microtwins, and defective wurtzite and zinc-blende phases. Correlation between HREM and Raman scattering results points towards the presence of compressive lattice distortion along the growth direction which might be attributable to structural defects. The films grown at 800 °C had better quality with less observable defects and less yellow luminescence than samples grown at 750 °C.

Band gap modulation in magnetically doped low-defect thin films of (Bi1-xSbx)2 Te3 with minimized bulk carrier concentration

NASA Astrophysics Data System (ADS)

Maximenko, Yulia; Scipioni, Kane; Wang, Zhenyu; Katmis, Ferhat; Steiner, Charles; Weis, Adam; van Harlingen, Dale; Madhavan, Vidya

Topological insulators Bi2Te3 and Sb2Te3 are promising materials for electronics, but both are naturally prone to vacancies and anti-site defects that move the Fermi energy onto the bulk bands. Fabricating (Bi1-xSbx)2 Te3 (BST) with the tuned x minimizes point defects and unmasks topological surface states by reducing bulk carriers. BST thin films have shown topological surface states and quantum anomalous Hall effect. However, different studies reported variable Sb:Bi ratios used to grow an undoped BST film. Here, we develop a reliable way to grow defect-free subnanometer-flat BST thin films having the Fermi energy tuned to the Dirac point. High-resolution scanning tunneling microscopy (STM) and Landau level spectroscopy prove the importance of crystallinity and surface roughness-not only Sb:Bi ratio-for the final bulk carrier concentration. The BST thin films were doped with Cr and studied with STM with atomic resolution. Counterintuitively, Cr density is anticorrelated with the local band gap due to Cr's antiferromagnetic order. We analyze the correlations and report the relevant band gap values. Predictably, high external magnetic field compromises antiferromagnetic order, and the local band gap increases. US DOE DE-SC0014335; Moore Found. GBMF4860; F. Seitz MRL.

Azasugar inhibitors as pharmacological chaperones for Krabbe disease

DOE PAGES

Hill, Chris H.; Viuff, Agnete H.; Spratley, Samantha J.; ...

2015-03-23

Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe amore » new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.« less

Golgi Traffic and Integrity Depend on N-Myristoyl Transferase-1 in Arabidopsis[W

PubMed Central

Renna, Luciana; Stefano, Giovanni; Majeran, Wojciech; Micalella, Chiara; Meinnel, Thierry; Giglione, Carmela; Brandizzi, Federica

2013-01-01

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes. PMID:23673980

Update on the Genetic Polymorphisms of Drug-Metabolizing Enzymes in Antiepileptic Drug Therapy

PubMed Central

Saruwatari, Junji; Ishitsu, Takateru; Nakagawa, Kazuko

2010-01-01

Genetic polymorphisms in the genes that encode drug-metabolizing enzymes are implicated in the inter-individual variability in the pharmacokinetics and pharmaco-dynamics of antiepileptic drugs (AEDs). However, the clinical impact of these polymorphisms on AED therapy still remains controversial. The defective alleles of cytochrome P450 (CYP) 2C9 and/or CYP2C19 could affect not only the pharmacokinetics, but also the pharmacodynamics of phenytoin therapy. CYP2C19 deficient genotypes were associated with the higher serum concentration of an active metabolite of clobazam, N-desmethylclobazam, and with the higher clinical efficacy of clobazam therapy than the other CYP2C19 genotypes. The defective alleles of CYP2C9 and/or CYP2C19 were also found to have clinically significant effects on the inter-individual variabilities in the population pharmacokinetics of phenobarbital, valproic acid and zonisamide. EPHX1 polymorphisms may be associated with the pharmacokinetics of carbamazepine and the risk of phenytoin-induced congenital malformations. Similarly, the UDP-glucuronosyltransferase 2B7 genotype may affect the pharmacokinetics of lamotrigine. Gluthatione S-transferase null genotypes are implicated in an increased risk of hepatotoxicity caused by carbamazepine and valproic acid. This article summarizes the state of research on the effects of mutations of drug-metabolizing enzymes on the pharmacokinetics and pharmacodynamics of AED therapies. Future directions for the dose-adjustment of AED are discussed. PMID:27713373

MULTIPLE ENZYME RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS FOR HIGH RESOLUTION DISTINCTION OF PSEUDOMONAS (SENSU STRICTO) 16S RRNA GENES

EPA Science Inventory

Pseudomonas specific 16S rDNA PCR amplification and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis using a single digestion mixture of Alu I, Hinf I, Rsa I, and Tru 9I distinguished 150 published sequences and reference strains of authentic Pseudomonas...

Enzyme-directed assembly of nanoparticles in tumors monitored by in vivo whole animal imaging and ex vivo super-resolution fluorescence imaging.

PubMed

Chien, Miao-Ping; Carlini, Andrea S; Hu, Dehong; Barback, Christopher V; Rush, Anthony M; Hall, David J; Orr, Galya; Gianneschi, Nathan C

2013-12-18

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Single molecule super-resolution imaging of proteins in living Salmonella enterica using self-labelling enzymes

PubMed Central

Barlag, Britta; Beutel, Oliver; Janning, Dennis; Czarniak, Frederik; Richter, Christian P.; Kommnick, Carina; Göser, Vera; Kurre, Rainer; Fabiani, Florian; Erhardt, Marc; Piehler, Jacob; Hensel, Michael

2016-01-01

The investigation of the subcellular localization, dynamics and interaction of proteins and protein complexes in prokaryotes is complicated by the small size of the cells. Super-resolution microscopy (SRM) comprise various new techniques that allow light microscopy with a resolution that can be up to ten-fold higher than conventional light microscopy. Application of SRM techniques to living prokaryotes demands the introduction of suitable fluorescent probes, usually by fusion of proteins of interest to fluorescent proteins with properties compatible to SRM. Here we describe an approach that is based on the genetically encoded self-labelling enzymes HaloTag and SNAP-tag. Proteins of interest are fused to HaloTag or SNAP-tag and cell permeable substrates can be labelled with various SRM-compatible fluorochromes. Fusions of the enzyme tags to subunits of a type I secretion system (T1SS), a T3SS, the flagellar rotor and a transcription factor were generated and analysed in living Salmonella enterica. The new approach is versatile in tagging proteins of interest in bacterial cells and allows to determine the number, relative subcellular localization and dynamics of protein complexes in living cells. PMID:27534893

TopoIIα prevents telomere fragility and formation of ultra thin DNA bridges during mitosis through TRF1-dependent binding to telomeres.

PubMed

d'Alcontres, Martina Stagno; Palacios, Jose Alejandro; Mejias, Diego; Blasco, Maria A

2014-01-01

Telomeres are repetitive nucleoprotein structures at the ends of chromosomes. Like most genomic regions consisting of repetitive DNA, telomeres are fragile sites prone to replication fork stalling and generation of chromosomal instability. In particular, abrogation of the TRF1 telomere binding protein leads to stalled replication forks and aberrant telomere structures known as "multitelomeric signals". Here, we report that TRF1 deficiency also leads to the formation of "ultra-fine bridges" (UFB) during mitosis, and to an increased time to complete mitosis mediated by the spindle assembly checkpoint proteins (SAC). We find that topoisomerase IIα (TopoIIα), an enzyme essential for resolution of DNA replication intermediates, binds telomeres in a TRF1-mediated manner. Indeed, similar to TRF1 abrogation, TopoIIα downregulation leads to telomere fragility and UFB, suggesting that these phenotypes are due to decreased TopoIIα at telomeres. We find that SAC proteins bind telomeres in vivo, and that this is disrupted upon TRF1 deletion. These findings suggest that TRF1 links TopoIIα and SAC proteins in a pathway that ensures correct telomere replication and mitotic segregation, unveiling how TRF1 protects from telomere fragility and mitotic defects.

Recombination protein Tid1p controls resolution of cohesin-dependent linkages in meiosis in Saccharomyces cerevisiae

PubMed Central

Kateneva, Anna V.; Konovchenko, Anton A.; Guacci, Vincent; Dresser, Michael E.

2005-01-01

Sister chromatid cohesion and interhomologue recombination are coordinated to promote the segregation of homologous chromosomes instead of sister chromatids at the first meiotic division. During meiotic prophase in Saccharomyces cerevisiae, the meiosis-specific cohesin Rec8p localizes along chromosome axes and mediates most of the cohesion. The mitotic cohesin Mcd1p/Scc1p localizes to discrete spots along chromosome arms, and its function is not clear. In cells lacking Tid1p, which is a member of the SWI2/SNF2 family of helicase-like proteins that are involved in chromatin remodeling, Mcd1p and Rec8p persist abnormally through both meiotic divisions, and chromosome segregation fails in the majority of cells. Genetic results indicate that the primary defect in these cells is a failure to resolve Mcd1p-mediated connections. Tid1p interacts with recombination enzymes Dmc1p and Rad51p and has an established role in recombination repair. We propose that Tid1p remodels Mcd1p-mediated cohesion early in meiotic prophase to facilitate interhomologue recombination and the subsequent segregation of homologous chromosomes. PMID:16230461

High resolution Talbot self-imaging applied to structural characterization of self-assembled monolayers of microspheres.

PubMed

Garcia-Sucerquia, J; Alvarez-Palacio, D C; Kreuzer, H J

2008-09-10

We report the observation of the Talbot self-imaging effect in high resolution digital in-line holographic microscopy (DIHM) and its application to structural characterization of periodic samples. Holograms of self-assembled monolayers of micron-sized polystyrene spheres are reconstructed at different image planes. The point-source method of DIHM and the consequent high lateral resolution allows the true image (object) plane to be identified. The Talbot effect is then exploited to improve the evaluation of the pitch of the assembly and to examine defects in its periodicity.

Effect of Te inclusions in CdZnTe crystals at different temperatures

NASA Astrophysics Data System (ADS)

Hossain, A.; Bolotnikov, A. E.; Camarda, G. S.; Gul, R.; Kim, K.-H.; Cui, Y.; Yang, G.; Xu, L.; James, R. B.

2011-02-01

CdZnTe crystals often exhibit nonuniformities due to the presence of Te inclusions and dislocations. High concentrations of such defects in these crystals generally entail severe charge-trapping, a major problem in ensuring the device's satisfactory performance. In this study, we employed a high-intensity, high-spatial-resolution synchrotron x-ray beam as the ideal tool to generate charges by focusing it over the large Te inclusions, and then observing the carrier's response at room- and at low-temperatures. A high spatial 5-μm resolution raster scan revealed the fine details of the presence of extended defects, like Te inclusions and dislocations in the CdZnTe crystals. A noticeable change was observed in the efficiency of electron charge collection at low temperature (1 °C), but it was hardly altered at room-temperature.

Restoring defect structures in 3C-SiC/Si (001) from spherical aberration-corrected high-resolution transmission electron microscope images by means of deconvolution processing.

PubMed

Wen, C; Wan, W; Li, F H; Tang, D

2015-04-01

The [110] cross-sectional samples of 3C-SiC/Si (001) were observed with a spherical aberration-corrected 300 kV high-resolution transmission electron microscope. Two images taken not close to the Scherzer focus condition and not representing the projected structures intuitively were utilized for performing the deconvolution. The principle and procedure of image deconvolution and atomic sort recognition are summarized. The defect structure restoration together with the recognition of Si and C atoms from the experimental images has been illustrated. The structure maps of an intrinsic stacking fault in the area of SiC, and of Lomer and 60° shuffle dislocations at the interface have been obtained at atomic level. Copyright © 2015 Elsevier Ltd. All rights reserved.

X-ray-induced acoustic computed tomography of concrete infrastructure

NASA Astrophysics Data System (ADS)

Tang, Shanshan; Ramseyer, Chris; Samant, Pratik; Xiang, Liangzhong

2018-02-01

X-ray-induced Acoustic Computed Tomography (XACT) takes advantage of both X-ray absorption contrast and high ultrasonic resolution in a single imaging modality by making use of the thermoacoustic effect. In XACT, X-ray absorption by defects and other structures in concrete create thermally induced pressure jumps that launch ultrasonic waves, which are then received by acoustic detectors to form images. In this research, XACT imaging was used to non-destructively test and identify defects in concrete. For concrete structures, we conclude that XACT imaging allows multiscale imaging at depths ranging from centimeters to meters, with spatial resolutions from sub-millimeter to centimeters. XACT imaging also holds promise for single-side testing of concrete infrastructure and provides an optimal solution for nondestructive inspection of existing bridges, pavement, nuclear power plants, and other concrete infrastructure.

Resonant tunneling through electronic trapping states in thin MgO magnetic junctions.

PubMed

Teixeira, J M; Ventura, J; Araujo, J P; Sousa, J B; Wisniowski, P; Cardoso, S; Freitas, P P

2011-05-13

We report an inelastic electron tunneling spectroscopy study on MgO magnetic junctions with thin barriers (0.85-1.35 nm). Inelastic electron tunneling spectroscopy reveals resonant electronic trapping within the barrier for voltages V>0.15  V. These trapping features are associated with defects in the barrier crystalline structure, as confirmed by high-resolution transmission electron microscopy. Such defects are responsible for resonant tunneling due to energy levels that are formed in the barrier. A model was applied to determine the average location and energy level of the traps, indicating that they are mostly located in the middle of the MgO barrier, in accordance with the high-resolution transmission electron microscopy data and trap-assisted tunneling conductance theory. Evidence of the influence of trapping on the voltage dependence of tunnel magnetoresistance is shown.

Laplace deep level transient spectroscopy: Embodiment and evolution

NASA Astrophysics Data System (ADS)

Peaker, A. R.; Markevich, V. P.; Hawkins, I. D.; Hamilton, B.; Bonde Nielsen, K.; Gościński, K.

2012-08-01

This paper is to commemorate the work of Leszek Dobaczewski who devoted much of his life to the development and application of high resolution DLTS. Under good experimental conditions Laplace DLTS provides an order of magnitude higher energy resolution than conventional DLTS techniques. This has had a profound effect on electrical defect spectroscopy enabling the effect of external probes, such as uniaxial stress, and internal perturbations, such as the proximity of atoms isovalent with the host, to be quantified in terms of electronic behaviour. Laplace DLTS provides a synergy with other techniques that was difficult or impossible to achieve previously. In this paper we present an overview of the development of LDLTS and illustrate some of its uses by describing its application in a number of key areas of defect research. Leszek Dobaczewski was born on 25th December 1954. He received his education in Warsaw taking his PhD in 1986 with Jerzy Langer at the Institute of Physics on “Recombination Processes at defects with the large lattice relaxation”. He held a research position at the institute in Warsaw until he came to Manchester in 1990 and thereafter alternated between Manchester and Warsaw. He worked primarily on the development and application of high resolution DLTS. He was awarded the degree of DSc in 1994 for his work on DX centres and held an appointment as full professor in Warsaw with Visiting Professor posts at Manchester and Aarhus. Professor Leszek Dobaczewski died in April 2010.

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

PubMed

Newby, A C; Chrambach, A

1979-02-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins.

Mechanisms of Mitochondrial Defects in Gulf War Syndrome

DTIC Science & Technology

2014-10-01

parameters: uncoupling ratio, net routine flux control ratio, respiratory control ratio, leak flux control ratio, phosphorylation respiratory... oxidative phosphorylation subunit) Quantitative analysis of individual mitochondrial proteins. The technique has been established and validated for muscle...Blue Native and Clear Native Analyses (non-denatured analysis of supercomplex formation and monomeric oxidative phosphorylation enzyme assembly

[Congenital hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency].

PubMed

Mura, M; Saidi, R; Wolf, A; Moalic, J L; Oliver, M

2009-12-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzyme defect with a wide range of clinical manifestations that can be severe. A variety of factors including many medications can induce hemolytic episodes. Screening for G6PD deficiency is required before use of some drugs especially primaquine or dapsone.

Glucose-6-phosphate dehydrogenase deficiency: not exclusively in males.

PubMed

van den Broek, Leonie; Heylen, Evelien; van den Akker, Machiel

2016-12-01

Glucose-6-phosphate (G6PD) deficiency is the most common human enzyme defect, often presenting with neonatal jaundice and/or acute hemolytic anemia, triggered by oxidizing agents. G6PD deficiency is an X-linked, hereditary disease, mainly affecting men, but should also be considered in females with an oxidative hemolysis.

Seventeen Alpha-Hydroxylase Deficiency Associated with Absent Gonads and Myelolipoma: A Case Report and Review of Literature.

PubMed

Soveid, Mahmood; Rais-Jalali, Ghanbar Ali

2016-11-01

Congenital adrenal hyperplasia comprises a group of disorders resulting from defects in enzymes required for the synthesis of cortisol. The clinical presentation depends on the specific enzyme defect. We report a rare case of congenital adrenal hyperplasia due to 17 alpha-hydroxylase deficiency. A 26-year-old female patient referred with hypertension and hypokalemia. She also had primary amenorrhea and lack of sexual development. The karyotype was 46, XY. Hormonal evaluation showed low serum levels of all steroid hormones, requiring alpha-hydroxylation, which included cortisol, 17 alpha-hydroxy progesterone, dehydroepiandrosterone sulfate, estradiol, and testosterone. The levels of adrenocorticotropic, follicle-stimulating, and luteinizing hormones were high. Radiological and surgical investigations failed to show a gonad. She also had a large myelolipoma. Treatment was commenced with low-dose dexamethasone and conjugated estrogen. Her hypertension and hypokalemia were resolved. The myelolipoma was removed by laparoscopy due to pain and sensation of heaviness. Our review of literature revealed that a combination of this disorder with either agonadism or myelolipoma is very rare and that only 2 previous cases have been reported for each entity.

Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene.

PubMed Central

Shields, M S; Montgomery, S O; Cuskey, S M; Chapman, P J; Pritchard, P H

1991-01-01

Pseudomonas cepacia G4 possesses a novel pathway of toluene catabolism that is shown to be responsible for the degradation of trichloroethylene (TCE). This pathway involves conversion of toluene via o-cresol to 3-methylcatechol. In order to determine the enzyme of toluene degradation that is responsible for TCE degradation, chemically induced mutants, blocked in the toluene ortho-monooxygenase (TOM) pathway of G4, were examined. Mutants of the phenotypic class designated TOM A- were all defective in their ability to oxidize toluene, o-cresol, m-cresol, and phenol, suggesting that a single enzyme is responsible for conversion of these compounds to their hydroxylated products (3-methylcatechol from toluene, o-cresol, and m-cresol and catechol from phenol) in the wild type. Mutants of this class did not degrade TCE. Two other mutant classes which were blocked in toluene catabolism, TOM B-, which lacked catechol-2,3-dioxygenase, and TOM C-, which lacked 2-hydroxy-6-oxoheptadienoic acid hydrolase activity, were fully capable of TCE degradation. Therefore, TCE degradation is directly associated with the monooxygenation capability responsible for toluene, cresol, and phenol hydroxylation. PMID:1892384

An essential role for LPA signalling in telencephalon development.

PubMed

Geach, Timothy J; Faas, Laura; Devader, Christelle; Gonzalez-Cordero, Anai; Tabler, Jacqueline M; Brunsdon, Hannah; Isaacs, Harry V; Dale, Leslie

2014-02-01

Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.

Atomic Defects and Doping of Monolayer NbSe2.

PubMed

Nguyen, Lan; Komsa, Hannu-Pekka; Khestanova, Ekaterina; Kashtiban, Reza J; Peters, Jonathan J P; Lawlor, Sean; Sanchez, Ana M; Sloan, Jeremy; Gorbachev, Roman V; Grigorieva, Irina V; Krasheninnikov, Arkady V; Haigh, Sarah J

2017-03-28

We have investigated the structure of atomic defects within monolayer NbSe 2 encapsulated in graphene by combining atomic resolution transmission electron microscope imaging, density functional theory (DFT) calculations, and strain mapping using geometric phase analysis. We demonstrate the presence of stable Nb and Se monovacancies in monolayer material and reveal that Se monovacancies are the most frequently observed defects, consistent with DFT calculations of their formation energy. We reveal that adventitious impurities of C, N, and O can substitute into the NbSe 2 lattice stabilizing Se divacancies. We further observe evidence of Pt substitution into both Se and Nb vacancy sites. This knowledge of the character and relative frequency of different atomic defects provides the potential to better understand and control the unusual electronic and magnetic properties of this exciting two-dimensional material.

Local and transient nanoscale strain mapping during in situ deformation

DOE PAGES

Gammer, C.; Kacher, J.; Czarnik, C.; ...

2016-08-26

The mobility of defects such as dislocations controls the mechanical properties of metals. This mobility is determined both by the characteristics of the defect and the material, as well as the local stress and strain applied to the defect. Therefore, the knowledge of the stress and strain during deformation at the scale of defects is important for understanding fundamental deformation mechanisms. In this paper, we demonstrate a method of measuring local stresses and strains during continuous in situ deformation with a resolution of a few nanometers using nanodiffraction strain mapping. Finally, our results demonstrate how large multidimensional data sets capturedmore » with high speed electron detectors can be analyzed in multiple ways after an in situ TEM experiment, opening the door for true multimodal analysis from a single electron scattering experiment.« less

Manufacturability improvements in EUV resist processing toward NXE:3300 processing

NASA Astrophysics Data System (ADS)

Kuwahara, Yuhei; Matsunaga, Koichi; Shimoaoki, Takeshi; Kawakami, Shinichiro; Nafus, Kathleen; Foubert, Philippe; Goethals, Anne-Marie; Shimura, Satoru

2014-03-01

As the design rule of semiconductor process gets finer, extreme ultraviolet lithography (EUVL) technology is aggressively studied as a process for 22nm half pitch and beyond. At present, the studies for EUV focus on manufacturability. It requires fine resolution, uniform, smooth patterns and low defectivity, not only after lithography but also after the etch process. In the first half of 2013, a CLEAN TRACKTM LITHIUS ProTMZ-EUV was installed at imec for POR development in preparation of the ASML NXE:3300. This next generation coating/developing system is equipped with state of the art defect reduction technology. This tool with advanced functions can achieve low defect levels. This paper reports on the progress towards manufacturing defectivity levels and latest optimizations towards the NXE:3300 POR for both lines/spaces and contact holes at imec.

Lipase-Mediated Kinetic Resolution: An Introductory Approach to Practical Biocatalysis

ERIC Educational Resources Information Center

Bandeira, Pamela T.; Thomas, Juliana C.; de Oliveira, Alfredo R. M.; Piovan, Leandro

2017-01-01

An experimental protocol that provides an excellent way to discuss concepts at the crossroads of organic chemistry and biochemistry employing biocatalysis is presented. By evaluating several reaction parameters (enzyme source, organic solvent, and acyl donor), it was possible to conduct an enzymatic kinetic resolution experiment using…

Microscopic resolution broadband dielectric spectroscopy

NASA Astrophysics Data System (ADS)

Mukherjee, S.; Watson, P.; Prance, R. J.

2011-08-01

Results are presented for a non-contact measurement system capable of micron level spatial resolution. It utilises the novel electric potential sensor (EPS) technology, invented at Sussex, to image the electric field above a simple composite dielectric material. EP sensors may be regarded as analogous to a magnetometer and require no adjustments or offsets during either setup or use. The sample consists of a standard glass/epoxy FR4 circuit board, with linear defects machined into the surface by a PCB milling machine. The sample is excited with an a.c. signal over a range of frequencies from 10 kHz to 10 MHz, from the reverse side, by placing it on a conducting sheet connected to the source. The single sensor is raster scanned over the surface at a constant working distance, consistent with the spatial resolution, in order to build up an image of the electric field, with respect to the reference potential. The results demonstrate that both the surface defects and the internal dielectric variations within the composite may be imaged in this way, with good contrast being observed between the glass mat and the epoxy resin.

Folate and epigenetic mechanisms in neural tube development and defects.

PubMed

Meethal, Sivan Vadakkadath; Hogan, Kirk J; Mayanil, Chandra S; Iskandar, Bermans J

2013-09-01

Multiple genetic and epigenetic factors involved in central nervous system (CNS) development influence the incidence of neural tube defects (NTDs). The beneficial effect of periconceptional folic acid on NTD prevention denotes a vital role for the single-carbon biochemical pathway in NTD genesis. Indeed, NTDs are associated with polymorphisms in a diversity of genes that encode folate pathway enzymes. Recent evidence suggests that CNS development and function, and consequently NTDs, are also associated with epigenetic mechanisms, many of which participate in the folate cycle and its input and output pathways. We provide an overview with select examples drawn from the authors' research.

Probing localized strain in solution-derived YB a2C u3O7 -δ nanocomposite thin films

NASA Astrophysics Data System (ADS)

Guzman, Roger; Gazquez, Jaume; Mundet, Bernat; Coll, Mariona; Obradors, Xavier; Puig, Teresa

2017-07-01

Enhanced pinning due to nanoscale strain is unique to the high-Tc cuprates, where pairing may be modified with lattice distortion. Therefore a comprehensive understanding of the defect landscape is required for a broad range of applications. However, determining the type and distribution of defects and their associated strain constitutes a critical task, and for this aim, real-space techniques for atomic resolution characterization are necessary. Here, we use scanning transmission electron microscopy (STEM) to study the atomic structure of individual defects of solution-derived YB a2C u3O7 (YBCO) nanocomposites, where the inclusion of incoherent secondary phase nanoparticles within the YBCO matrix dramatically increases the density of Y1B a2C u4O8 (Y124) intergrowths, the commonest defect in YBCO thin films. The formation of the Y124 is found to trigger a concatenation of strain-derived interactions with other defects and the concomitant nucleation of intrinsic defects, which weave a web of randomly distributed nanostrained regions that profoundly transform the vortex-pinning landscape of the YBCO nanocomposite thin films.

Topological defects in liquid crystals and molecular self-assembly (Conference Presentation)

NASA Astrophysics Data System (ADS)

Abbott, Nicholas L.

2017-02-01

Topological defects in liquid crystals (LCs) have been widely used to organize colloidal dispersions and template polymerizations, leading to a range of elastomers and gels with complex mechanical and optical properties. However, little is understood about molecular-level assembly processes within defects. This presentation will describe an experimental study that reveals that nanoscopic environments defined by LC topological defects can selectively trigger processes of molecular self-assembly. By using fluorescence microscopy, cryogenic transmission electron microscopy and super-resolution optical microscopy, key signatures of molecular self-assembly of amphiphilic molecules in topological defects are observed - including cooperativity, reversibility, and controlled growth of the molecular assemblies. By using polymerizable amphiphiles, we also demonstrate preservation of molecular assemblies templated by defects, including nanoscopic "o-rings" synthesized from "Saturn-ring" disclinations. Our results reveal that topological defects in LCs are a versatile class of three-dimensional, dynamic and reconfigurable templates that can direct processes of molecular self-assembly in a manner that is strongly analogous to other classes of macromolecular templates (e.g., polymer—surfactant complexes). Opportunities for the design of exquisitely responsive soft materials will be discussed using bacterial endotoxin as an example.

Dual-mode lensless imaging device for digital enzyme linked immunosorbent assay

NASA Astrophysics Data System (ADS)

Sasagawa, Kiyotaka; Kim, Soo Heyon; Miyazawa, Kazuya; Takehara, Hironari; Noda, Toshihiko; Tokuda, Takashi; Iino, Ryota; Noji, Hiroyuki; Ohta, Jun

2014-03-01

Digital enzyme linked immunosorbent assay (ELISA) is an ultra-sensitive technology for detecting biomarkers and viruses etc. As a conventional ELISA technique, a target molecule is bonded to an antibody with an enzyme by antigen-antibody reaction. In this technology, a femto-liter droplet chamber array is used as reaction chambers. Due to its small volume, the concentration of fluorescent product by single enzyme can be sufficient for detection by a fluorescent microscopy. In this work, we demonstrate a miniaturized lensless imaging device for digital ELISA by using a custom image sensor. The pixel array of the sensor is coated with a 20 μm-thick yellow filter to eliminate excitation light at 470 nm and covered by a fiber optic plate (FOP) to protect the sensor without resolution degradation. The droplet chamber array formed on a 50μm-thick glass plate is directly placed on the FOP. In the digital ELISA, microbeads coated with antibody are loaded into the droplet chamber array, and the ratio of the fluorescent to the non-fluorescent chambers with the microbeads are observed. In the fluorescence imaging, the spatial resolution is degraded by the spreading through the glass plate because the fluorescence is irradiated omnidirectionally. This degradation is compensated by image processing and the resolution of ~35 μm was achieved. In the bright field imaging, the projected images of the beads with collimated illumination are observed. By varying the incident angle and image composition, microbeads were successfully imaged.

Hypogammaglobulinaemia secondary to cow-milk allergy in children under 2 years of age

PubMed Central

Bezrodnik, Liliana; Raccio, Andrea C Gómez; Canil, Laura M; Rey, Maria Amanda; Carabajal, Patricia C; Fossati, Carlos A; Docena, Guillermo H

2007-01-01

Symptomatic hypogammaglobulinaemia in children younger than 2 years of age was studied to rule out a primary immunodeficiency. Thirty-four patients were referred to the Immunology Service to study the hypogammaglobulinaemia-associated clinical picture. Food allergy was documented in 10 patients by personal and familial history, presence of specific immunoglobulin E (IgE) and elevated total serum IgE levels. Coeliac disease and human immunodeficiency virus infection were also ruled out. Protein loss through stools was assessed by clearance of α1-antitrypsin (AAT). Serum immunoglobulin levels were determined by nephelometry and functional antibodies were studied by enzyme-linked immunosorbent assay. The cellular immune response was assessed by in vitro lymphocyte proliferation in response to mitogens and cell subsets were analysed by flow cytometry. In five patients of the 10 patients we suspected a protein loss through the mucosa. Four of these five patients showed an increased AAT and the other showed an extensive cutaneous lesion. Immunological studies revealed normal antibody function, in vitro lymphoproliferative responses and cell numbers in four of the 5 patients. One patient showed abnormally low numbers of CD4+ T cells as well as a defective proliferative response to mitogens. After diagnosis of cow milk allergy, milk was replaced with infant milk formula containing hydrolysed proteins. Recovery of immunoglobulin values and clinical resolution were achieved. Hypogammaglobulinaemia during early childhood in some children may be secondary to cow milk allergy, and immunoglobulins and cells may leak through the inflamed mucosa. Resolution of symptoms as well as normalization of immunoglobulin values may be easily achieved by avoidance of the offending allergen. PMID:17498216

Infrared-thermographic screening of the activity and enantioselectivity of enzymes.

PubMed

Reetz, M T; Hermes, M; Becker, M H

2001-05-01

The infrared radiation caused by the heat of reaction of an enantioselective enzyme-catalyzed transformation can be detected by modern photovoltaic infrared (IR)-thermographic cameras equipped with focal-plane array detectors. Specifically, in the lipase-catalyzed enantioselective acylation of racemic 1-phenylethanol, the (R)- and (S)-substrates are allowed to react separately in the wells of microtiter plates, the (R)-alcohol showing hot spots in the IR-thermographic images. Thus, highly enantioselective enzymes can be identified at kinetic resolution.

Phonon Scattering in Silicon by Multiple Morphological Defects: A Multiscale Analysis

NASA Astrophysics Data System (ADS)

Lorenzi, Bruno; Dettori, Riccardo; Dunham, Marc T.; Melis, Claudio; Tonini, Rita; Colombo, Luciano; Sood, Aditya; Goodson, Kenneth E.; Narducci, Dario

2018-05-01

Ideal thermoelectric materials should possess low thermal conductivity κ along with high electrical conductivity σ . Thus, strategies are needed to impede the propagation of phonons mostly responsible for thermal conduction while only marginally affecting charge carrier diffusion. Defect engineering may provide tools to fulfill this aim, provided that one can achieve an adequate understanding of the role played by multiple morphological defects in scattering thermal energy carriers. In this paper, we study how various morphological defects such as grain boundaries and dispersed nanovoids reduce the thermal conductivity of silicon. A blended approach has been adopted, using data from both simulations and experiments in order to cover a wide range of defect densities. We show that the co-presence of morphological defects with different characteristic scattering length scales is effective in reducing the thermal conductivity. We also point out that non-gray models (i.e. models with spectral resolution) are required to improve the accuracy of predictive models explaining the dependence of κ on the density of morphological defects. Finally, the application of spectral models to Matthiessen's rule is critically addressed with the aim of arriving at a compact model of phonon scattering in highly defective materials showing that non-local descriptors would be needed to account for lattice distortion due to nanometric voids.

Pre-clinical and Clinical Evaluation of High Resolution, Mobile Gamma Camera and Positron Imaging Devices

DTIC Science & Technology

2009-10-01

Field-of-View, Mobile PET/SPECT System for Bedside Environments: A Dynamic Cardiac Phantom Study using 99mTc and 18F- FDG . Presented at the American...using Tc-99m tracers and viability imaging using F- 18 tracers [3]-[7]. For cardiac F-18 imaging in a bedside environment, the 511 keV SPECT approach...SPECT system may have difficulty imaging subtle myocardial defects with F-18 tracers , but it may effectively image moderate to severe defects. The

Quantum metrology with a single spin-3/2 defect in silicon carbide

NASA Astrophysics Data System (ADS)

Soykal, Oney O.; Reinecke, Thomas L.

We show that implementations for quantum sensing with exceptional sensitivity and spatial resolution can be made using the novel features of semiconductor high half-spin multiplet defects with easy-to-implement optical detection protocols. To achieve this, we use the spin- 3 / 2 silicon monovacancy deep center in hexagonal silicon carbide based on our rigorous derivation of this defect's ground state and of its electronic and optical properties. For a single VSi- defect, we obtain magnetic field sensitivities capable of detecting individual nuclear magnetic moments. We also show that its zero-field splitting has an exceptional strain and temperature sensitivity within the technologically desirable near-infrared window of biological systems. Other point defects, i.e. 3d transition metal or rare-earth impurities in semiconductors, may also provide similar opportunities in quantum sensing due to their similar high spin (S >= 3 / 2) configurations. This work was supported in part by ONR and by the Office of Secretary of Defense, Quantum Science and Engineering Program.

EUV patterning improvement toward high-volume manufacturing

NASA Astrophysics Data System (ADS)

Kuwahara, Yuhei; Matsunaga, Koichi; Kawakami, Shinichiro; Nafus, Kathleen; Foubert, Philippe; Goethals, Anne-Marie

2015-03-01

Extreme ultraviolet lithography (EUVL) technology is a promising candidate for a semiconductor process for 18nm half pitch and beyond. So far, the studies of EUV for manufacturability have been focused on particular aspects. It still requires fine resolution, uniform and smooth patterns, and low defectivity, not only after lithography but also after the etch process. Tokyo Electron Limited and imec are continuously collaborating to improve manufacturing quality of the process of record (POR) on a CLEAN TRACKTM LITHIUS ProTMZ-EUV. This next generation coating/developing system has been upgraded with defectivity reduction enhancements which are applied along with TELTM best known methods. We have evaluated process defectivity post lithography and post etch. Apart from defectivity, FIRMTM rinse material and application compatibility with sub 18nm patterning is improved to prevent line pattern collapse and increase process window on next generation resist materials. This paper reports on the progress of defectivity and patterning performance optimization towards the NXE:3300 POR.

Electrical detection and analysis of surface acoustic wave in line-defect two-dimensional piezoelectric phononic crystals

NASA Astrophysics Data System (ADS)

Cai, Feida; Li, Honglang; Tian, Yahui; Ke, Yabing; Cheng, Lina; Lou, Wei; He, Shitang

2018-03-01

Line-defect piezoelectric phononic crystals (PCs) show good potential applications in surface acoustic wave (SAW) MEMS devices for RF communication systems. To analyze the SAW characteristics in line-defect two-dimensional (2D) piezoelectric PCs, optical methods are commonly used. However, the optical instruments are complex and expensive, whereas conventional electrical methods can only measure SAW transmission of the whole device and lack spatial resolution. In this paper, we propose a new electrical experimental method with multiple receiving interdigital transducers (IDTs) to detect the SAW field distribution, in which an array of receiving IDTs of equal aperture was used to receive the SAW. For this new method, SAW delay lines with perfect and line-defect 2D Al/128°YXLiNbO3 piezoelectric PCs on the transmitting path were designed and fabricated. The experimental results showed that the SAW distributed mainly in the line-defect region, which agrees with the theoretical results.

Abnormality in catalase import into peroxisomes leads to severe neurological disorder

PubMed Central

Sheikh, Faruk G.; Pahan, Kalipada; Khan, Mushfiquddin; Barbosa, Ernest; Singh, Inderjit

1998-01-01

Peroxisomal disorders are lethal inherited diseases caused by either defects in peroxisome assembly or dysfunction of single or multiple enzymatic function(s). The peroxisomal matrix proteins are targeted to peroxisomes via the interaction of peroxisomal targeting signal sequences 1 and 2 (PTS1 or PTS2) with their respective cytosolic receptors. We have studied human skin fibroblast cell lines that have multiple peroxisomal dysfunctions with normal packaging of PTS1 and PTS2 signal-containing proteins but lack catalase in peroxisomes. To understand the defect in targeting of catalase to peroxisomes and the loss of multiple enzyme activities, we transfected the mutant cells with normal catalase modified to contain either PTS1 or PTS2 signal sequence. We demonstrate the integrity of these pathways by targeting catalase into peroxisomes via PTS1 or PTS2 pathways. Furthermore, restoration of peroxisomal functions by targeting catalase-SKL protein (a catalase fused to the PTS1 sequence) to peroxisomes indicates that loss of multiple functions may be due to their inactivation by H2O2 or other oxygen species in these catalase-negative peroxisomes. In addition to enzyme activities, targeting of catalase-SKL chimera to peroxisomes also corrected the in situ levels of fatty acids and plasmalogens in these mutant cell lines. In normal fibroblasts treated with aminotriazole to inhibit catalase, we found that peroxisomal functions were inhibited to the level found in mutant cells, an observation that supports the conclusion that multiple peroxisomal enzyme defects in these patients are caused by H2O2 toxicity in catalase-negative peroxisomes. Moreover, targeting of catalase to peroxisomes via PTS1 and PTS2 pathways in these mutant cell lines suggests that there is another pathway for catalase import into peroxisomes and that an abnormality in this pathway manifests as a peroxisomal disease. PMID:9501198

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases.

PubMed

Oh, Doo-Byoung

2015-08-01

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy.

Lamb waves increase sensitivity in nondestructive testing

NASA Technical Reports Server (NTRS)

Di Novi, R.

1967-01-01

Lamb waves improve sensitivity and resolution in the detection of small defects in thin plates and small diameter, thin-walled tubing. This improvement over shear waves applies to both longitudinal and transverse flaws in the specimens.

Characterization of Human Aspartoacylase: the brain enzyme responsible for Canavan disease†

PubMed Central

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E.

2008-01-01

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate, and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity, but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some posttranslational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc results in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme. PMID:16669630

Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease.

PubMed

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E

2006-05-09

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.

EBIC investigation of hydrogenation of crystal defects in EFG solar silicon ribbons

NASA Technical Reports Server (NTRS)

Sullivan, T.; Ast, D. G.

1983-01-01

Changes in the contrast and resolution of defect structures in 205 Ohm-cm EFG polysilicon ribbon subjected to annealing and hydrogenation treatments were observed in a JEOL 733 Superprobe scanning electron microscope, using electron beam induced current (EBIC) collected at an A1 Schottky barrier. The Schottky barrier was formed by evaporation of A1 onto the cleaned and polished surface of the ribbon material. Measurement of beam energy, beam current, and the current induced in the Schottky diode enabled observations to be quantified. Exposure to hydrogen plasma increased charge collection efficiency. However, no simple causal relationship between the hydrogenation and charge collection efficiency could be inferred, because the collection efficiency also displayed an unexpected thermal dependence. Good quality intermediate-magnification (1000X-5400X) EBIC micrographs of several specific defect structures were obtained. Comparison of grown-in and stress-induced dislocations after annealing in vacuum at 500 C revealed that stress-induced dislocations are hydrogenated to a much greater degree than grown-in dislocations. The theoretical approximations used to predict EBIC contrast and resolution may not be entirely adequate to describe them under high beam energy and low beam current conditions.

Mitochondrial Translation and Beyond: Processes Implicated in Combined Oxidative Phosphorylation Deficiencies

PubMed Central

Smits, Paulien; Smeitink, Jan; van den Heuvel, Lambert

2010-01-01

Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multisubunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders. PMID:20396601

Hydroxycinnamic acid decarboxylase activity of Brettanomyces bruxellensis involved in volatile phenol production: relationship with cell viability.

PubMed

Laforgue, R; Lonvaud-Funel, A

2012-12-01

Brettanomyces bruxellensis populations have been correlated with an increase in phenolic off-flavors in wine. The volatile phenols causing the olfactory defect result from the successive decarboxylation and reduction of hydroxycinnamic acids that are normal components of red wines. The growth of B. bruxellensis is preventable by adding sulfur dioxide (SO(2)), with variable effectiveness. Moreover, it was hypothesized that SO(2) was responsible for the entry of B. bruxellensis into a viable but non-culturable (VBNC) state. The aim of this project was to investigate the effects of SO(2) on the remaining enzyme activities of B. bruxellensis populations according to their viability and cultivability, focusing on the hydroxycinnamate decarboxylase enzyme, the first enzyme needed, rather than the metabolites produced. Enzyme activity was determined both in cell-free extracts and resting cells after various SO(2) treatments in synthetic media. After slight sulfiting (around 50 mg/L total SO(2)), the yeasts had lost part of their enzyme activity but not their cultivability. At higher doses (at least 75 mg/L total SO(2)) the majority of yeasts had lost their cultivability but still retained part of their enzyme activity. These results suggested that non culturable cells retained some enzyme activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Poorly expressed endogenous ecotropic provirus of DBA/2 mice encodes a mutant Pr65gag protein that is not myristylated.

PubMed Central

Copeland, N G; Jenkins, N A; Nexø, B; Schultz, A M; Rein, A; Mikkelsen, T; Jørgensen, P

1988-01-01

DBA/2 mice carry a single endogenous ecotropic murine leukemia provirus designated Emv-3. Although this provirus appears to be nondefective by genomic restriction enzyme mapping, weanling mice do not produce virus and only about one-third of adult mice ever express virus. 5-Iododeoxyuridine and 5-azacytidine, two potent inducers of ecotropic virus expression, are relatively ineffective at inducing Emv-3 expression. However, the chemical carcinogen 7,12-dimethylbenz(a)anthracene can induce ecotropic virus expression in approximately 95% of treated DBA/2 mice. Previous experiments involving DNA transfection and marker rescue analysis of molecularly cloned Emv-3 DNA suggested that Emv-3 carries a small defect(s) in the gag gene, not detectable by restriction enzyme mapping, that inhibits virus expression in vivo and in vitro. Using a combination of approaches, including DNA sequencing, peptide mapping, and metabolic labeling of cells with [3H]myristate, we have demonstrated that the defect in Emv-3 most likely results from a single nucleotide substitution within the gene for p15gag that inhibits myristylation of the Pr65gag N terminus. Myristylation of Pr65gag is thought to be required for this protein to associate with the plasma membrane and is essential for virus particle formation. These results provide a conceptual framework for understanding how Emv-3 expression is regulated during development and after chemical induction. Images PMID:2826810

Randomized clinical study assessing two membranes for guided bone regeneration of peri-implant bone defects: clinical and histological outcomes at 6 months.

PubMed

Naenni, Nadja; Schneider, David; Jung, Ronald E; Hüsler, Jürg; Hämmerle, Christoph H F; Thoma, Daniel S

2017-10-01

To test whether or not one of two membranes is superior for peri-implant-guided bone regeneration in terms of clinical and histologic outcomes. In 27 patients, 27 two-piece dental implants were placed in single-tooth gaps in the esthetic area. Buccal dehiscence and/or fenestration-type defects were regenerated using demineralized bovine bone mineral and randomly covered with either a resorbable membrane (RES) or a titanium-reinforced non-resorbable membrane (N-RES). Clinical measurements included vertical defect resolution and the horizontal thickness of regenerated bone at implant placement and at 6 months. Statistics were performed by means of nonparametric testing. The remaining mean vertical defect measured 4 mm (±2.07) (RES) and 2.36 mm (±2.09) (N-RES) (P = 0.044) at baseline and 0.77 mm (±0.85) (RES) and 0.21 mm (±0.80) (N-RES) (P = 0.021) at re-entry. This translated into a defect resolution of 85% (RES) and 90.7% (N-RES) (P = 0.10). The horizontal thickness after augmentation measured 3.46 mm (±0.52) (RES) and 2.82 mm (±0.50) (N-RES) (P = 0.004). The mean loss in horizontal thickness from baseline to re-entry measured 2.23 mm (SD ±1.21) (RES) and 0.14 mm (±0.79) (N-RES) (P = 0.017). The horizontal changes in thickness at the implant shoulder level were statistically significant between the groups (P = 0.0001). Both treatment modalities were clinically effective in regenerating bone as demonstrated by a similar horizontal thickness and vertical defect fill at 6 months. The N-RES group exhibited significantly less horizontal bone thickness reduction from baseline to follow-up. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Impact of defective pixels in AMLCDs on the perception of medical images

NASA Astrophysics Data System (ADS)

Kimpe, Tom; Sneyders, Yuri

2006-03-01

With LCD displays, each pixel has its own individual transistor that controls the transmittance of that pixel. Occasionally, these individual transistors will short or alternatively malfunction, resulting in a defective pixel that always shows the same brightness. With ever increasing resolution of displays the number of defect pixels per display increases accordingly. State of the art processes are capable of producing displays with no more than one faulty transistor out of 3 million. A five Mega Pixel medical LCD panel contains 15 million individual sub pixels (3 sub pixels per pixel), each having an individual transistor. This means that a five Mega Pixel display on average will have 5 failing pixels. This paper investigates the visibility of defective pixels and analyzes the possible impact of defective pixels on the perception of medical images. JND simulations were done to study the effect of defective pixels on medical images. Our results indicate that defective LCD pixels can mask subtle features in medical images in an unexpectedly broad area around the defect and therefore may reduce the quality of diagnosis for specific high-demanding areas such as mammography. As a second contribution an innovative solution is proposed. A specialized image processing algorithm can make defective pixels completely invisible and moreover can also recover the information of the defect so that the radiologist perceives the medical image correctly. This correction algorithm has been validated with both JND simulations and psycho visual tests.

Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution.

PubMed

Eibinger, Manuel; Zahel, Thomas; Ganner, Thomas; Plank, Harald; Nidetzky, Bernd

2016-01-01

Enzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field. We have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through "random-walk" lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate. Salient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application.

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

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

Abramchik, Yu. A.; Timofeev, V. I., E-mail: tostars@mail.ru; Muravieva, T. I.

2017-01-15

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus (T. th HB27) has high thermal stability and shows maximum activity at 75°Ð¡, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample,more » which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P2{sub 1} and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.« less

Crystallization and preliminary X-ray diffraction analysis of recombinant phosphoribosylpyrophosphate synthetase from the Thermophilic thermus thermophilus strain HB27

NASA Astrophysics Data System (ADS)

Abramchik, Yu. A.; Timofeev, V. I.; Muravieva, T. I.; Sinitsyna, E. V.; Esipov, R. S.; Kuranova, I. P.

2017-01-01

Phosphoribosylpyrophosphate synthetases (PRPP synthetases) are among the key enzymes essential for vital functions of organisms and are involved in the biosynthesis of purine and pyrimidine nucleotides, coenzymes, and the amino acids histidine and tryptophan. These enzymes are used in biotechnology for the combined chemoenzymatic synthesis of natural nucleotide analogs. Recombinant phosphoribosylpyrophosphate synthetase I from the thermophilic strain HB27 of the bacterium Thermus thermophilus ( T. th HB27) has high thermal stability and shows maximum activity at 75°C, due to which this enzyme holds promise for biotechnological applications. In order to grow crystals and study them by X-ray crystallography, an enzyme sample, which was produced using a highly efficient producer strain, was purified by affinity and gel-filtration chromatography. The screening of crystallization conditions was performed by the vapor-diffusion technique. The crystals of the enzyme suitable for X-ray diffraction were grown by the counter-diffusion method through a gel layer. These crystals were used to collect the X-ray diffraction data set at the SPring-8 synchrotron radiation facility (Japan) to 3-Å resolution. The crystals belong to sp. gr. P21 and have the following unitcell parameters: a = 107.7 Å, b = 112.6 Å, c = 110.2 Å, α = γ = 90°, β = 116.6°. The X-ray diffraction data set is suitable for determining the three-dimensional structure of the enzyme at 3.0-Å resolution.

Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

PubMed

Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

2010-08-10

Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

Kinetically controlled fabrication of single-crystalline TiO 2 nanobrush architectures with high energy {001} facets

DOE PAGES

Fan, Lisha; Gao, Xiang; Lee, Dongkyu; ...

2017-03-01

Here, this study demonstrates that precise control of nonequilibrium growth conditions during pulsed laser deposition (PLD) can be exploited to produce single-crystalline anatase TiO 2 nanobrush architectures with large surface areas terminated with high energy {001} facets. The data indicate that the key to nanobrush formation is controlling the atomic surface transport processes to balance defect aggregation and surface-smoothing processes. High-resolution scanning transmission electron microscopy data reveal that defect-mediated aggregation is the key to TiO 2 nanobrush formation. The large concentration of defects present at the intersection of domain boundaries promotes aggregation of PLD growth species, resulting in the growthmore » of the single-crystalline nanobrush architecture. This study proposes a model for the relationship between defect creation and growth mode in nonequilibrium environments, which enables application of this growth method to novel nanostructure design in a broad range of materials.« less

Measuring Te inclusion uniformity over large areas for CdTe/CZT imaging and spectrometry sensors

NASA Astrophysics Data System (ADS)

Bolke, Joe; O'Brien, Kathryn; Wall, Peter; Spicer, Mike; Gélinas, Guillaume; Beaudry, Jean-Nicolas; Alexander, W. Brock

2017-09-01

CdTe and CZT materials are technologies for gamma and x-ray imaging for applications in industry, homeland security, defense, space, medical, and astrophysics. There remain challenges in uniformity over large detector areas (50 75 mm) due to a combination of material purity, handling, growth process, grown in defects, doping/compensation, and metal contacts/surface states. The influence of these various factors has yet to be explored at the large substrate level required for devices with higher resolution both spatially and spectroscopically. In this study, we looked at how the crystal growth processes affect the size and density distributions of microscopic Te inclusion defects. We were able to grow single crystals as large as 75 mm in diameter and spatially characterize three-dimensional defects and map the uniformity using IR microscopy. We report on the pattern of observed defects within wafers and its relation to instabilities at the crystal growth interface.

Adult onset urea cycle disorder in a patient with presumed hepatic encephalopathy.

PubMed

Atiq, Muslim; Holt, Andrew F; Safdar, Kamran; Weber, Frederick; Ravinuthala, Ravi; Jonas, Mark E; Neff, Guy W

2008-02-01

Deficiency of any of the 5 enzymes in the urea cycle results in the accumulation of ammonia, leading to encephalopathy; which if untreated, can be lethal and produce devastating neurologic sequelae in long-term survivors. We hereby present an interesting case that presented with hyperammonemia and encephalopathy; later found to have an urea cycle defect.

Endocrine Therapy of Breast Cancer

DTIC Science & Technology

2006-06-01

Becker muscular dystrophy (BMD, hypomorphic for dys- trophin, n = 5), Dysferlin deficiency (putative vesicle traf- fic defect, n = 9), and Calpain III...this study. (1) Limb-girdle muscular dystrophy (LGMD, provided by Children National Medical Center, Center for Genetic Medicine): 4 diagnostic...groups, Fukutin related protein de- ficiency (FKRP) (homozygous missense for glycosylation enzyme, limb-girdle muscular dystrophy sub-type, n = 7

[CONE BEAM COMPUTED TOMOGRAPHY IN DIAGNOSTICS OF ODONTOGENIC MAXILLARY SINUSITIS (CASE REPORTS)].

PubMed

Demidova, E; Khurdzidze, G

2017-06-01

Diagnostic studies performed by cone beam computed tomography Morita 3D made possible to obtain high resolution images of hard tissues of upper jawbone and maxillary sinus, to detect bony tissue defects, such as odontogenic cysts, cystogranulomas and granulomas. High-resolution and three dimensional tomographic image reconstructions allowed for optimal and prompt determination of the scope of surgical treatment and planning of effective conservative treatment regimen. Interactive diagnostics helped to estimate cosmetic and functional results of surgical treatment, to prevent the occurrence of surgical complications, and to evaluate the efficacy of conservative treatment. The obtained data contributed to determination of particular applications of cone beam computed tomography in the diagnosis of odontogenic maxillary sinusitis, detection of specific defects with cone beam tomography as the most informative method of diagnosis; as well as to determination of weak and strong sides, and helped to offer mechanisms of x-ray diagnostics to dental surgeons and ENT specialists.

Effect of Ce doping on structural, optical and photocatalytic properties of ZnO nano-structures.

PubMed

Selvam, N Clament Sagaya; Vijaya, J Judith; Kennedy, L John

2014-03-01

A novel self-assembled pure and Ce doped ZnO nano-particles (NPs) were successfully synthesized by a simple low temperature co-precipitation method. The prepared photocatalysts were characterized by X-ray diffraction (XRD), High resolution scanning electron microscopy (HR-SEM), High resolution transmission electron microscopy (HR-TEM), diffuse reflectance spectroscopy (DRS) and Photoluminescence (PL) spectroscopy. The results indicated that the prepared photocatalysts shows a novel morphology, high crystallinity, uniform size distribution, and more defects. Photocatalytic degradation (PCD) of nonylphenol, a potent endocrine disrupting chemical in aqueous medium was investigated. Higher amount of oxygen defects exhibits enhanced PCD of nonylphenol. In addition, the influence of the Ce contents on the structure, morphology, absorption, emission and photocatalytic activity of ZnO nanoparticles (NPs) were investigated systematically. The relative PCD efficiency of pure ZnO, Ce-doped ZnO NPs and commercial TiO2 (Degussa P-25) have also been discussed.

Inhibitors of amino acids biosynthesis as antifungal agents.

PubMed

Jastrzębowska, Kamila; Gabriel, Iwona

2015-02-01

Fungal microorganisms, including the human pathogenic yeast and filamentous fungi, are able to synthesize all proteinogenic amino acids, including nine that are essential for humans. A number of enzymes catalyzing particular steps of human-essential amino acid biosynthesis are fungi specific. Numerous studies have shown that auxotrophic mutants of human pathogenic fungi impaired in biosynthesis of particular amino acids exhibit growth defect or at least reduced virulence under in vivo conditions. Several chemical compounds inhibiting activity of one of these enzymes exhibit good antifungal in vitro activity in minimal growth media, which is not always confirmed under in vivo conditions. This article provides a comprehensive overview of the present knowledge on pathways of amino acids biosynthesis in fungi, with a special emphasis put on enzymes catalyzing particular steps of these pathways as potential targets for antifungal chemotherapy.

Evaluation of myocardial defect detection between parallel-hole and fan-beam SPECT using the Hotelling trace

NASA Astrophysics Data System (ADS)

Wollenweber, S. D.; Tsui, B. M. W.; Lalush, D. S.; Frey, E. C.; Gullberg, G. T.

1998-08-01

The objective of this study was to implement the Hotelling trace (HT) to evaluate the potential increase in defect detection in myocardial SPECT using high-resolution fan-beam (HRF) versus parallel-hole (HRP) collimation and compare results to a previously reported human observer study (G.K. Gregoriou et al., ibid., vol. 42, p. 1267-75, 1995). Projection data from the 3D MCAT torso phantom were simulated including the effects of attenuation, collimator-detector response blurring and scatter. Poisson noise fluctuations were then simulated. The HRP and HRF collimators had the same spatial resolution at 20 cm. The total counts in the projection data sets were proportional to the detection efficiencies of the collimators and on the order of that found in clinical Tc-99m studies. In six left-ventricular defect locations, the HT found for HRF was superior to that for HRP collimation. For HRF collimation, the HT was calculated for reconstructed images using 64/spl times/64, 128/spl times/128 and 192/spl times/192 grid sizes. The results demonstrate substantial improvement in myocardial defect detection when the grid size was increased from 64/spl times/64 to 128/spl times/128 and slight improvement from 128/spl times/128 to 192/spl times/192. Also, the performance of the Hotelling observer in terms of the HT at the different grid sizes correlates at better than 0.95 to that found in human observers in a previously reported observer experiment and ROC study.

Low-voltage cross-sectional EBIC for characterisation of GaN-based light emitting devices.

PubMed

Moldovan, Grigore; Kazemian, Payam; Edwards, Paul R; Ong, Vincent K S; Kurniawan, Oka; Humphreys, Colin J

2007-01-01

Electron beam induced current (EBIC) characterisation can provide detailed information on the influence of crystalline defects on the diffusion and recombination of minority carriers in semiconductors. New developments are required for GaN light emitting devices, which need a cross-sectional approach to provide access to their complex multi-layered structures. A sample preparation approach based on low-voltage Ar ion milling is proposed here and shown to produce a flat cross-section with very limited surface recombination, which enables low-voltage high resolution EBIC characterisation. Dark defects are observed in EBIC images and correlation with cathodoluminescence images identify them as threading dislocations. Emphasis is placed on one-dimensional quantification which is used to show that junction delineation with very good spatial resolution can be achieved, revealing significant roughening of this GaN p-n junction. Furthermore, longer minority carrier diffusion lengths along the c-axis are found at dislocation sites, in both p-GaN and the multi-quantum well (MQW) region. This is attributed to gettering of point defects at threading dislocations in p-GaN and higher escape rate from quantum wells at dislocation sites in the MQW region, respectively. These developments show considerable promise for the use of low-voltage cross-sectional EBIC in the characterisation of point and extended defects in GaN-based devices and it is suggested that this technique will be particularly useful for degradation analysis.

Defect Engineering by Codoping in KCaI3 :Eu2 + Single-Crystalline Scintillators

NASA Astrophysics Data System (ADS)

Wu, Yuntao; Li, Qi; Jones, Steven; Dun, Chaochao; Hu, Sheng; Zhuravleva, Mariya; Lindsey, Adam C.; Stand, Luis; Loyd, Matthew; Koschan, Merry; Auxier, John; Hall, Howard L.; Melcher, Charles L.

2017-09-01

Eu2 + -doped alkali or alkali earth iodide scintillators with energy resolutions ≤3 % at 662 keV promise the excellent discrimination ability for radioactive isotopes required for homeland-security and nuclear-nonproliferation applications. To extend their applications to x-ray imaging, such as computed tomography scans, the intense afterglow which delays the response time of such materials is an obstacle that needs to be overcome. However, a clear understanding of the origin of the afterglow and feasible solutions is still lacking. In this work, we present a combined experimental and theoretical investigation of the physical insights of codoping-based defect engineering which can reduce the afterglow effectively in KCaI3:Eu2 + single-crystal scintillators. We illustrate that Sc3 + codoping greatly suppresses the afterglow, whereas Y3 + , Gd3 + , or La3 + codoping enhances the afterglow. Meanwhile, a light yield of 57 000 photons / MeV and an energy resolution of 3.4% at 662 keV can be maintained with the appropriate concentration of Sc3 + codoping, which makes the material promising for medical-imaging applications. Through our thermoluminescence techniques and density-functional-theory calculations, we are able to identify the defect structures and understand the mechanism by which codoping affects the scintillation performance of KCaI3:Eu2 + crystals. The proposed defect-engineering strategy is further validated by achieving afterglow suppression in Mg2 + codoped KCaI3:Eu2 + single crystals.

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase

PubMed Central

Borgnia, Mario J.; Banerjee, Soojay; Merk, Alan; Matthies, Doreen; Bartesaghi, Alberto; Rao, Prashant; Pierson, Jason; Earl, Lesley A.; Falconieri, Veronica

2016-01-01

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. PMID:27036132

Analyzing genetic diversity in conifers...isozyme resolution by starch gel electrophoresis

Treesearch

M. Thompson Conkle

1972-01-01

Enzymes in forest tree materials can be resolved by starch gel electrophoresis. A gel slab is prepared in a mold assembled from glass and plastic. Wicks containing an aqueous extract of macerated plant material are inserted in the gel and processed. The gel is sliced, stained, examined, and photographed. Isozyme bands produced by differential migration of enzymes...

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by In Vivo Whole Animal and Ex Vivo Super-Resolution Fluorescence Imaging

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

Chien, Miao-Ping; Carlini, Andrea S.; Hu, Dehong

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block, and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo, and in ex vivo organ analysis following intratumoral injectionmore » into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micron-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.« less

Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx.

PubMed

Romano, Christine A; Zhou, Mowei; Song, Yang; Wysocki, Vicki H; Dohnalkova, Alice C; Kovarik, Libor; Paša-Tolić, Ljiljana; Tebo, Bradley M

2017-09-29

Bacteria that produce Mn oxides are extraordinarily skilled engineers of nanomaterials that contribute significantly to global biogeochemical cycles. Their enzyme-based reaction mechanisms may be genetically tailored for environmental remediation applications or bioenergy production. However, significant challenges exist for structural characterization of the enzymes responsible for biomineralization. The active Mn oxidase in Bacillus sp. PL-12, Mnx, is a complex composed of a multicopper oxidase (MCO), MnxG, and two accessory proteins, MnxE and MnxF. MnxG shares sequence similarity with other, structurally characterized MCOs. MnxE and MnxF have no similarity to any characterized proteins. The ~200 kDa complex has been recalcitrant to crystallization, so its structure is unknown. Here, we show that native mass spectrometry defines the subunit topology and copper binding of Mnx, while high-resolution electron microscopy visualizes the protein and nascent Mn oxide minerals. These data provide critical structural information for understanding Mn biomineralization by such unexplored enzymes.Significant challenges exist for structural characterization of enzymes responsible for biomineralization. Here the authors show that native mass spectrometry and high resolution electron microscopy can define the subunit topology and copper binding of a manganese oxidizing complex, and describe early stage formation of its mineral products.

Combining Zebrafish and Mouse Models to Test the Function of Deubiquitinating Enzyme (Dubs) Genes in Development: Role of USP45 in the Retina.

PubMed

Toulis, Vasileios; Garanto, Alejandro; Marfany, Gemma

2016-01-01

Ubiquitination is a dynamic and reversible posttranslational modification. Much effort has been devoted to characterize the function of ubiquitin pathway genes in the cell context, but much less is known on their functional role in the development and maintenance of organs and tissues in the organism. In fact, several ubiquitin ligases and deubiquitinating enzymes (DUBs) are implicated in human pathological disorders, from cancer to neurodegeneration. The aim of our work is to explore the relevance of DUBs in retinal function in health and disease, particularly since some genes related to the ubiquitin or SUMO pathways cause retinal dystrophies, a group of rare diseases that affect 1:3000 individuals worldwide. We propose zebrafish as an extremely useful and informative genetic model to characterize the function of any particular gene in the retina, and thus complement the expression data from mouse. A preliminary characterization of gene expression in mouse retinas (RT-PCR and in situ hybridization) was performed to select particularly interesting genes, and we later replicated the experiments in zebrafish. As a proof of concept, we selected ups45 to be knocked down by morpholino injection in zebrafish embryos. Morphant phenotypic analysis showed moderate to severe eye morphological defects, with a defective formation of the retinal structures, therefore supporting the relevance of DUBs in the formation and differentiation of the vertebrate retina, and suggesting that genes encoding ubiquitin pathway enzymes are good candidates for causing hereditary retinal dystrophies.

Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

PubMed Central

Matuschewski, Kai; Haussig, Joana M.

2016-01-01

Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

Coater/developer based techniques to improve high-resolution EUV patterning defectivity

NASA Astrophysics Data System (ADS)

Hontake, Koichi; Huli, Lior; Lemley, Corey; Hetzer, Dave; Liu, Eric; Ko, Akiteru; Kawakami, Shinichiro; Shimoaoki, Takeshi; Hashimoto, Yusaku; Tanaka, Koichiro; Petrillo, Karen; Meli, Luciana; De Silva, Anuja; Xu, Yongan; Felix, Nelson; Johnson, Richard; Murray, Cody; Hubbard, Alex

2017-10-01

Extreme ultraviolet lithography (EUVL) technology is one of the leading candidates under consideration for enabling the next generation of devices, for 7nm node and beyond. As the focus shifts to driving down the 'effective' k1 factor and enabling the full scaling entitlement of EUV patterning, new techniques and methods must be developed to reduce the overall defectivity, mitigate pattern collapse, and eliminate film-related defects. In addition, CD uniformity and LWR/LER must be improved in terms of patterning performance. Tokyo Electron Limited (TEL™) and IBM Corporation are continuously developing manufacturing quality processes for EUV. In this paper, we review the ongoing progress in coater/developer based processes (coating, developing, baking) that are required to enable EUV patterning.

Optimizing defect inspection strategy through the use of design-aware database control layers

NASA Astrophysics Data System (ADS)

Stoler, Dvori; Ruch, Wayne; Ma, Weimin; Chakravarty, Swapnajit; Liu, Steven; Morgan, Ray; Valadez, John; Moore, Bill; Burns, John

2007-10-01

Resolution limitations in the mask making process can cause differences between the features that appear in a database and those printed to a reticle. These differences may result from intentional or unintentional features in the database exceeding the resolution limit of the mask making process such as small gaps or lines in the data, line end shortening on small sub-resolution assist features etc creating challenges to both mask writing and mask inspection. Areas with high variance from design to mask, often referred to as high MEEF areas (mask error enhancement factor), become highly problematic and can directly impact mask and device yield, mask manufacturing cycle time and ultimately mask costs. Specific to mask inspection it may be desirable to inspect certain non-critical or non-relevant features at reduced sensitivity so as not to detect real, but less significant process defects. In contrast there may also be times where increased sensitivity is required for critical mask features or areas. Until recently, this process was extremely manual, creating added time and cost to the mask inspection cycle. Shifting to more intelligent and automated inspection flows is the key focus of this paper. A novel approach to importing design data directly into the mask inspection to include both MDP generated MRC errors files and LRC generated MEEF files. The results of recently developed inspection and review capability based upon controlling defect inspection using design aware data base control layers on a pixel basis are discussed. Typical mask shop applications and implementations will be shown.

Kinetic Resolution of Secondary Alcohols Using Amidine-Based Catalysts

PubMed Central

Li, Ximin; Jiang, Hui; Uffman, Eric W.; Guo, Lei; Zhang, Yuhua; Yang, Xing; Birman, Vladimir B.

2012-01-01

Kinetic resolution of racemic alcohols has been traditionally achieved via enzymatic enantioselective esterification and ester hydrolysis. However, there has long been considerable interest in devising nonenzymatic alternative methods for this transformation. Amidine-Based Catalysts (ABCs), a new class of enantioselective acyl transfer catalysts developed in our group, have demonstrated, inter alia, high efficacy in the kinetic resolution of benzylic, allylic and propargylic secondary alcohols and 2-substituted cycloalkanols, and thus provide a viable alternative to enzymes. PMID:22283696

Isolation of a Mutant of Salmonella typhimurium Dependent on D-Arabinose-5-phosphate for Growth and Synthesis of 3-Deoxy-D-mannoctulosonate (Ketodeoxyoctonate)

PubMed Central

Rick, P. D.; Osborn, M. J.

1972-01-01

A new type of auxotrophic mutant of Salmonella typhimurium has been isolated that is defective in the synthesis of the 3-deoxy-D-mannooctulosonate (ketodeoxyoctonate) region of the lipopolysaccharide and requires D-arabinose-5-phosphate for growth. Genetic and biochemical evidence indicated that the mutant defect was due to an altered ketodeoxyoctonate-8-phosphate synthetase with an apparent Km for D-arabinose-5-phosphate 35-fold higher than that of the parental enzyme. As a result of this enzymatic lesion, the mutant strain was dependent on exogenous D-arabinose-5-phosphate both for growth and for synthesis of a complete lipopolysaccharide. PMID:4566459

ATP Synthase Diseases of Mitochondrial Genetic Origin

PubMed Central

Dautant, Alain; Meier, Thomas; Hahn, Alexander; Tribouillard-Tanvier, Déborah; di Rago, Jean-Paul; Kucharczyk, Roza

2018-01-01

Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae, we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits 8 and a of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures. PMID:29670542

The Aminoacyl-tRNA Synthetase Complex.

PubMed

Mirande, Marc

2017-01-01

Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.

Atomic resolution crystal structures and quantum chemistry meet to reveal subtleties of hydroxynitrile lyase catalysis.

PubMed

Schmidt, Andrea; Gruber, Karl; Kratky, Christoph; Lamzin, Victor S

2008-08-01

Hydroxynitrile lyases are versatile enzymes that enantiospecifically cope with cyanohydrins, important intermediates in the production of various agrochemicals or pharmaceuticals. We determined four atomic resolution crystal structures of hydroxynitrile lyase from Hevea brasiliensis: one native and three complexes with acetone, isopropyl alcohol, and thiocyanate. We observed distinct distance changes among the active site residues related to proton shifts upon substrate binding. The combined use of crystallography and ab initio quantum chemical calculations allowed the determination of the protonation states in the enzyme active site. We show that His(235) of the catalytic triad must be protonated in order for catalysis to proceed, and we could reproduce the cyanohydrin synthesis in ab initio calculations. We also found evidence for the considerable pK(a) shifts that had been hypothesized earlier. We envision that this knowledge can be used to enhance the catalytic properties and the stability of the enzyme for industrial production of enantiomerically pure cyanohydrins.

Three-dimensional structure of E. Coli purine nucleoside phosphorylase at 0.99 Å resolution

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

Timofeev, V. I., E-mail: tostars@mail.ru; Abramchik, Yu. A., E-mail: ugama@yandex.ru; Zhukhlistova, N. E., E-mail: inna@ns.crys.ras.ru

2016-03-15

Purine nucleoside phosphorylases (PNPs) catalyze the reversible phosphorolysis of nucleosides and are key enzymes involved in nucleotide metabolism. They are essential for normal cell function and can catalyze the transglycosylation. Crystals of E. coli PNP were grown in microgravity by the capillary counterdiffusion method through a gel layer. The three-dimensional structure of the enzyme was determined by the molecular-replacement method at 0.99 Å resolution. The structural features are considered, and the structure of E. coli PNP is compared with the structures of the free enzyme and its complexes with purine base derivatives established earlier. A comparison of the environment ofmore » the purine base in the complex of PNP with formycin A and of the pyrimidine base in the complex of uridine phosphorylase with thymidine revealed the main structural features of the base-binding sites. Coordinates of the atomic model determined with high accuracy were deposited in the Protein Data Bank (PDB-ID: 4RJ2).« less

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

Xu, Ting; Ooi, Amy; Lee, Hooi Chen

An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pHmore » 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.« less

Multiple focused EMAT designs for improved surface breaking defect characterization

NASA Astrophysics Data System (ADS)

Thring, C. B.; Fan, Y.; Edwards, R. S.

2017-02-01

Ultrasonic Rayleigh waves can be employed for the detection of surface breaking defects such as rolling contact fatigue and stress corrosion cracking. Electromagnetic Acoustic Transducers (EMATs) are well suited to this technique as they can directly generate Rayleigh waves within the sample without the requirement for wedges, and they are robust and inexpensive compared to laser ultrasonics. Three different EMAT coil types have been developed, and these are compared to assess their ability to detect and characterize small (down to 0.5 mm depth, 1 mm diameter) surface breaking defects in aluminium. These designs are: a pair of linear meander coils used in a pseudo-pulse-echo mode, a pair of focused meander coils also used in pseudo-pulse-echo mode, and a pair of focused racetrack coils used in pitch-catch mode. The linear meander coils are able to detect most of the defects tested, but have a much lower signal to noise ratio and give limited sizing information. The focused meander coils and the focused racetrack coils can detect all defects tested, but have the advantage that they can also characterize the defect sizes on the sample surface, and have a stronger sensitivity at their focal point. Measurements using all three EMAT designs are presented and compared for high resolution imaging of surface-breaking defects.

Microwave-Assisted Resolution of α-Lipoic Acid Catalyzed by an Ionic Liquid Co-Lyophilized Lipase.

PubMed

Liu, Ning; Wang, Lei; Wang, Zhi; Jiang, Liyan; Wu, Zhuofu; Yue, Hong; Xie, Xiaona

2015-05-29

The combination of the ionic liquid co-lyophilized lipase and microwave irradiation was used to improve enzyme performance in enantioselective esterification of α-lipoic acid. Effects of various reaction conditions on enzyme activity and enantioselectivity were investigated. Under optimal condition, the highest enantioselectivity (E = 41.2) was observed with a high enzyme activity (178.1 μmol/h/mg) when using the ionic liquid co-lyophilized lipase with microwave assistance. Furthermore, the ionic liquid co-lyophilized lipase exhibited excellent reusability under low power microwave.

Resolution Quality and Atom Positions in Sub-Angstrom Electron Microscopy

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

O'Keefe, Michael A.; Allard, Lawrence F.; Blom, Douglas A.

2005-02-15

Ability to determine whether an image peak represents one single atom or several depends on resolution of the HR-(S)TEM. Rayleigh's resolution criterion, an accepted standard in optics, was derived as a means for judging when two image intensity peaks from two sources of light (stars) are distinguishable from a single source. Atom spacings closer than the Rayleigh limit have been resolved in HR-TEM, suggesting that it may be useful to consider other limits, such as the Sparrow resolution criterion. From the viewpoint of the materials scientist, it is important to be able to use the image to determine whether anmore » image feature represents one or more atoms (resolution), and where the atoms (or atom columns) are positioned relative to one another (resolution quality). When atoms and the corresponding image peaks are separated by more than the Rayleigh limit of the HR-(S)TEM, it is possible to adjust imaging parameters so that relative peak positions in the image correspond to relative atom positions in the specimen. When atoms are closer than the Rayleigh limit, we must find the relationship of the peak position to the atom position by peak fitting or, if we have a suitable model, by image simulation. Our Rayleigh-Sparrow parameter QRS reveals the ''resolution quality'' of a microscope image. QRS values greater than 1 indicate a clearly resolved twin peak, while values between 1 and 0 mean a lower-quality resolution and an image with peaks displaced from the relative atom positions. The depth of the twin-peak minimum can be used to determine the value of QRS and the true separation of the atom peaks that sum to produce the twin peak in the image. The Rayleigh-Sparrow parameter can be used to refine relative atom positions in defect images where atoms are closer than the Rayleigh limit of the HR-(S)TEM, reducing the necessity for full image simulations from large defect models.« less

Preface of 16th International conference on Defects, Recognition, Imaging and Physics in Semiconductors

NASA Astrophysics Data System (ADS)

Yang, Deren; Xu, Ke

2016-11-01

The 16th International conference on Defects-Recognition, Imaging and Physics in Semiconductors (DRIP-XVI) was held at the Worldhotel Grand Dushulake in Suzhou, China from 6th to 10th September 2015, around the 30th anniversary of the first DRIP conference. It was hosted by the Suzhou Institute of Nano-tech and Nano-bionics (SINANO), Chinese Academy of Sciences. On this occasion, about one hundred participants from nineteen countries attended the event. And a wide range of subjects were addressed during the conference: physics of point and extended defects in semiconductors: origin, electrical, optical and magnetic properties of defects; diagnostics techniques of crystal growth and processing of semiconductor materials (in-situ and process control); device imaging and mapping to evaluate performance and reliability; defect analysis in degraded optoelectronic and electronic devices; imaging techniques and instruments (proximity probe, x-ray, electron beam, non-contact electrical, optical and thermal imaging techniques, etc.); new frontiers of atomic-scale-defect assessment (STM, AFM, SNOM, ballistic electron energy microscopy, TEM, etc.); new approaches for multi-physic-parameter characterization with Nano-scale space resolution. Within these subjects, there were 58 talks, of which 18 invited, and 50 posters.

In situ defect annealing of swift heavy ion irradiated CeO 2 and ThO 2 using synchrotron X-ray diffraction and a hydrothermal diamond anvil cell

DOE PAGES

Palomares, Raul I.; Tracy, Cameron L.; Zhang, Fuxiang; ...

2015-04-16

Hydrothermal diamond anvil cells (HDACs) provide facile means for coupling synchrotron Xray techniques with pressure up to 10 GPa and temperature up to 1300 K. This manuscript reports on an application of the HDAC as an ambient-pressure sample environment for performing in situ defect annealing and thermal expansion studies of swift heavy ion irradiated CeO 2 and ThO 2 using synchrotron X-ray diffraction. The advantages of the in situ HDAC technique over conventional annealing methods include: rapid temperature ramping and quench times, high-resolution measurement capability, simultaneous annealing of multiple samples, and prolonged temperature- and apparatus stability at high temperatures. Isochronalmore » annealing between 300 K and 1100 K revealed 2-stage and 1-stage defect recovery processes for irradiated CeO 2 and ThO 2, respectively; indicating that the morphology of the defects produced by swift heavy ion irradiation of these two materials differs significantly. These results suggest that electronic configuration plays a major role in both the radiation-induced defect production and high temperature defect recovery mechanisms of CeO 2 and ThO 2.« less

Mapping crystal defects with a digital scanning ultramicroscope

NASA Astrophysics Data System (ADS)

Springer, John M., Jr.; Silberman, Enrique; Kroes, Roger L.; Reiss, Don

1991-12-01

A computer controlled scanning ultramicroscope has been built to assist in the characterization of transparent crystals. The device measures the scattering of a focused He-Ne laser beam by crystalline defects. As an XYZ translation table moves the crystal under the ultramicroscope, the scattered light is measured by a photodetector whose output is digitized and recorded. From this data, contour maps or 3-D perspective plots of the scattering regions of the crystal can be generated to assist in finding patterns of defects which might be correlated with perturbations in the growth process. The verified resolution of the present instrument is about 1 micrometers , which is limited by the minimum step of the stepper-motor driven translation stages, optical diffraction effects, and the sensitivity of the detector at the laser light frequency. The instrument was used to build a database of defects patterns in commercial laboratory grown triglycine sulphate (TGS) crystals, and to map defects in a TGS crystal grown from aqueous solution during the flight of Spacelab 3. This crystal shows indications of a reduction both in the generation of defects at the seed-new growth interface and in their propagation into the new crystal.

Mapping crystal defects with a digital scanning ultramicroscope

NASA Astrophysics Data System (ADS)

Springer, J. M.; Silberman, E.; Kroes, Roger; Reiss, D.

A computer controlled scanning ultramicroscope has been built to assist in the characterization of transparent crystals. The device measures the scattering of a focused He-Ne laser beam by crystalline defects. As an XYZ translation table moves the crystal under the ultramicroscope, the scattered light is measured by a photodetector whose output is digitized and recorded. From this data, contour maps or three dimensional perspective plots of the scattering regions of the crystal can be generated to assist in finding patterns of defects which might be correlated with perturbations in the growth process. The verified resolution of the present instrument is about 1 micrometer, which is limited by the minimum step of the stepper-motor driven translation stages, optical diffraction effects and the sensitivity of the detector at the laser light frequency. The instrument was used to build a database of defects patterns in commercial laboratory grown triglycine sulphate (TGS) crystals, and to map defects in a TGS crystal grown from aqueous solution during the flight of Spacelab 3. This crystal shows indications of a reduction both in the generation of defects at the seed-new growth interface and in their propagation into the new crystal.

Enhanced Mass Defect Filtering To Simplify and Classify Complex Mixtures of Lignin Degradation Products.

PubMed

Dier, Tobias K F; Egele, Kerstin; Fossog, Verlaine; Hempelmann, Rolf; Volmer, Dietrich A

2016-01-19

High resolution mass spectrometry was utilized to study the highly complex product mixtures resulting from electrochemical breakdown of lignin. As most of the chemical structures of the degradation products were unknown, enhanced mass defect filtering techniques were implemented to simplify the characterization of the mixtures. It was shown that the implemented ionization techniques had a major impact on the range of detectable breakdown products, with atmospheric pressure photoionization in negative ionization mode providing the widest coverage in our experiments. Different modified Kendrick mass plots were used as a basis for mass defect filtering, where Kendrick mass defect and the mass defect of the lignin-specific guaiacol (C7H7O2) monomeric unit were utilized, readily allowing class assignments independent of the oligomeric state of the product. The enhanced mass defect filtering strategy therefore provided rapid characterization of the sample composition. In addition, the structural similarities between the compounds within a degradation sequence were determined by comparison to a tentatively identified product of this compound series. In general, our analyses revealed that primarily breakdown products with low oxygen content were formed under electrochemical conditions using protic ionic liquids as solvent for lignin.

Mapping crystal defects with a digital scanning ultramicroscope

NASA Technical Reports Server (NTRS)

Springer, J. M.; Silberman, E.; Kroes, Roger; Reiss, D.

1991-01-01

A computer controlled scanning ultramicroscope has been built to assist in the characterization of transparent crystals. The device measures the scattering of a focused He-Ne laser beam by crystalline defects. As an XYZ translation table moves the crystal under the ultramicroscope, the scattered light is measured by a photodetector whose output is digitized and recorded. From this data, contour maps or three dimensional perspective plots of the scattering regions of the crystal can be generated to assist in finding patterns of defects which might be correlated with perturbations in the growth process. The verified resolution of the present instrument is about 1 micrometer, which is limited by the minimum step of the stepper-motor driven translation stages, optical diffraction effects and the sensitivity of the detector at the laser light frequency. The instrument was used to build a database of defects patterns in commercial laboratory grown triglycine sulphate (TGS) crystals, and to map defects in a TGS crystal grown from aqueous solution during the flight of Spacelab 3. This crystal shows indications of a reduction both in the generation of defects at the seed-new growth interface and in their propagation into the new crystal.

Detection of orbicularis oris muscle defects in first degree relatives of cleft lip children using ultrasound.

PubMed

Mittal, M; Maheshwari, N; Ahlawat, K; Sharma, V; Sultan, A; Chopra, R

2012-01-01

The severity of cleft lip (CL) varies considerably from complete bilateral CL and palate at one end of the spectrum to a minimal CL at the other. In some cases of microform clefting, there may be no visible manifestation of the defect on the lip surface (i.e., the defect is occult) and no residual functional deficit. This study used high resolution ultrasonography to detect subclinical anomalies of orbicularis oris muscle (OOM) in first degree relatives of CL +- cleft palate children and compared it with controls. Thirty relatives of 25 children with non-syndromic CL or CL+ CP were identified for the study. Thirty subjects having negative family history of CL/P in three generations and absence of any minimal cleft features were taken as controls. Ultrasound scans of OOM of all the controls and relatives were taken. Statistical analysis was performed using standard χ2 tests with Yates correction. Defects were seen in 13.3% of relatives and no defects were seen in controls, this was not statistically significant. The data support the hypothesis that subclinical CL cases with subepithelial OOM defects do exist and Orbicularis oris discontinuities represent the mildest form of CL.

The role of folate metabolism in orofacial development and clefting

PubMed Central

Wahl, Stacey E.; Kennedy, Allyson E.; Wyatt, Brent H.; Moore, Alexander D.; Pridgen, Deborah E.; Cherry, Amanda M.; Mavila, Catherine B.; Dickinson, Amanda J.G.

2015-01-01

Folate deficiency has been associated with numerous diseases and birth defects including orofacial defects. However, whether folate has a role in the face during early orofacial development has been unclear. The present study reveals that pharmacological and antisense oligonucleotide mediated inhibition of DHFR, an integral enzyme in the folate pathway, results in specific changes in the size and shape of the midface and embryonic mouth. Such defects are accompanied by a severe reduction in the muscle and cartilage jaw elements without significant change in neural crest pattern or global levels of methylation. We propose that the orofacial defects associated with DHFR deficient function are the result of decreased cell proliferation and increased cell death via DNA damage. In particular, localized apoptosis may also be depleting the cells of the face that express crucial genes for the differentiation of the jaw structures. Folate supplementation is widely known to reduce human risk for orofacial clefts. In the present study, we show that activating folate metabolism can reduce median oral clefts in the primary palate by increasing cell survival. Moreover, we demonstrate that a minor decrease in DHFR function exacerbates median facial clefts caused by RAR inhibition. This work suggests that folate deficiencies could be a major contributing factor to multifactorial orofacial defects. PMID:26144049

Unraveling the mystery of natural rubber biosythesis part I: investigation of the composition and growth of in vitro natural rubber using high resolution size exclusion chromatography

USDA-ARS?s Scientific Manuscript database

Monitoring the growth of in vitro natural rubberwas accomplished by high resolution size exclusion chromatography, SEC.Washed rubber particles isolated from H. brasiliensis latex, containing the rubber transferase enzyme, were used to catalyze the polymerization of synthetic isopentenyl pyrophosphat...

Inductive Non-Contact Position Sensor

NASA Technical Reports Server (NTRS)

Youngquist, Robert; Garcia, Alyssa; Simmons, Stephen

2010-01-01

Optical hardware has been developed to measure the depth of defects in the Space Shuttle Orbiter's windows. In this hardware, a mirror is translated such that its position corresponds to the defect's depth, so the depth measurement problem is transferred to a mirror-position measurement problem. This is preferable because the mirror is internal to the optical system and thus accessible. Based on requirements supplied by the window inspectors, the depth of the defects needs to be measured over a range of 200 microns with a resolution of about 100 nm and an accuracy of about 400 nm. These same requirements then apply to measuring the position of the mirror, and in addition, since this is a scanning system, a response time of about 10 ms is needed. A market search was conducted and no sensor that met these requirements that also fit into the available housing volume (less than one cubic inch) was found, so a novel sensor configuration was constructed to meet the requirements. This new sensor generates a nearly linearly varying magnetic field over a small region of space, which can easily be sampled, resulting in a voltage proportional to position. Experiments were done with a range of inductor values, drive voltages, drive frequencies, and inductor shapes. A rough mathematical model was developed for the device that, in most aspects, describes how it operates and what electrical parameters should be chosen for best performance. The final configuration met all the requirements, yielding a small rugged sensor that was easy to use and had nanometer resolution over more than the 200-micron range required. The inductive position sensor is a compact device (potentially as small as 2 cubic centimeters), which offers nanometer-position resolution over a demonstrated range of nearly 1 mm. One of its advantages is the simplicity of its electrical design. Also, the sensor resolution is nearly uniform across its operational range, which is in contrast to eddy current and capacitive sensors whose sensitivity is dependent upon position.

Ion channeling study of defects in compound crystals using Monte Carlo simulations

NASA Astrophysics Data System (ADS)

Turos, A.; Jozwik, P.; Nowicki, L.; Sathish, N.

2014-08-01

Ion channeling is a well-established technique for determination of structural properties of crystalline materials. Defect depth profiles have been usually determined basing on the two-beam model developed by Bøgh (1968) [1]. As long as the main research interest was focused on single element crystals it was considered as sufficiently accurate. New challenge emerged with growing technological importance of compound single crystals and epitaxial heterostructures. Overlap of partial spectra due to different sublattices and formation of complicated defect structures makes the two beam method hardly applicable. The solution is provided by Monte Carlo computer simulations. Our paper reviews principal aspects of this approach and the recent developments in the McChasy simulation code. The latter made it possible to distinguish between randomly displaced atoms (RDA) and extended defects (dislocations, loops, etc.). Hence, complex defect structures can be characterized by the relative content of these two components. The next refinement of the code consists of detailed parameterization of dislocations and dislocation loops. Defect profiles for variety of compound crystals (GaN, ZnO, SrTiO3) have been measured and evaluated using the McChasy code. Damage accumulation curves for RDA and extended defects revealed non monotonous defect buildup with some characteristic steps. Transition to each stage is governed by the different driving force. As shown by the complementary high resolution XRD measurements lattice strain plays here the crucial role and can be correlated with the concentration of extended defects.

A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition.

PubMed

Ilari, Andrea; Baiocco, Paola; Messori, Luigi; Fiorillo, Annarita; Boffi, Alberto; Gramiccia, Marina; Di Muccio, Trentina; Colotti, Gianni

2012-02-01

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin-trypanothione reductase-NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.

Topologically Allowed Nonsixfold Vortices in a Sixfold Multiferroic Material: Observation and Classification

DOE PAGES

Cheng, Shaobo; Li, Jun; Han, Myung-Geun; ...

2017-04-05

Here, we report structural transformation of sixfold vortex domains into two-, four-, and eightfold vortices via a different type of topological defect in hexagonal manganites. Combining high-resolution electron microscopy and Landau-theory-based numerical simulations, we also investigate the remarkable atomic arrangement and the intertwined relationship between the vortex structures and the topological defects. The roles of their displacement field, formation temperature, and nucleation sites are revealed. All conceivable vortices in the system are topologically classified using homotopy group theory, and their origins are identified.

Modification of the crystal structure of gadolinium gallium garnet by helium ion irradiation

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

Ostafiychuk, B. K.; Yaremiy, I. P., E-mail: yaremiy@rambler.ru; Yaremiy, S. I.

2013-12-15

The structure of gadolinium gallium garnet (GGG) single crystals before and after implantation by He{sup +} ions has been investigated using high-resolution X-ray diffraction methods and the generalized dynamic theory of X-ray scattering. The main types of growth defects in GGG single crystals and radiation-induced defects in the ion-implanted layer have been determined. It is established that the concentration of dislocation loops in the GGG surface layer modified by ion implantation increases and their radius decreases with an increase in the implantation dose.

Image-based overlay and alignment metrology through optically opaque media with sub-surface probe microscopy

NASA Astrophysics Data System (ADS)

van Es, Maarten H.; Mohtashami, Abbas; Piras, Daniele; Sadeghian, Hamed

2018-03-01

Nondestructive subsurface nanoimaging through optically opaque media is considered to be extremely challenging and is essential for several semiconductor metrology applications including overlay and alignment and buried void and defect characterization. The current key challenge in overlay and alignment is the measurement of targets that are covered by optically opaque layers. Moreover, with the device dimensions moving to the smaller nodes and the issue of the so-called loading effect causing offsets between between targets and product features, it is increasingly desirable to perform alignment and overlay on product features or so-called on-cell overlay, which requires higher lateral resolution than optical methods can provide. Our recently developed technique known as SubSurface Ultrasonic Resonance Force Microscopy (SSURFM) has shown the capability for high-resolution imaging of structures below a surface based on (visco-)elasticity of the constituent materials and as such is a promising technique to perform overlay and alignment with high resolution in upcoming production nodes. In this paper, we describe the developed SSURFM technique and the experimental results on imaging buried features through various layers and the ability to detect objects with resolution below 10 nm. In summary, the experimental results show that the SSURFM is a potential solution for on-cell overlay and alignment as well as detecting buried defects or voids and generally metrology through optically opaque layers.

Ultrahigh-Resolution Optical Coherence Tomography of Surgically Closed Macular Holes

PubMed Central

Ko, Tony H.; Witkin, Andre J.; Fujimoto, James G.; Chan, Annie; Rogers, Adam H.; Baumal, Caroline R.; Schuman, Joel S.; Drexler, Wolfgang; Reichel, Elias; Duker, Jay S.

2007-01-01

Objective To evaluate retinal anatomy using ultrahigh-resolution optical coherence tomography (OCT) in eyes after successful surgical repair of full-thickness macular hole. Methods Twenty-two eyes of 22 patients were diagnosed as having macular hole, underwent pars plana vitrectomy, and had flat/closed macular anatomy after surgery, as confirmed with biomicroscopic and OCT examination findings. An ultrahigh-resolution–OCT system developed for retinal imaging, with the capability to achieve approximately 3-μm axial resolution, was used to evaluate retinal anatomy after hole repair. Results Despite successful closure of the macular hole, all 22 eyes had macular abnormalities on ultrahigh-resolution–OCT images after surgery. These abnormalities were separated into the following 5 categories: (1) outer foveal defects in 14 eyes (64%), (2) persistent foveal detachment in 4 (18%), (3) moderately reflective foveal lesions in 12 (55%), (4) epiretinal membranes in 14 (64%), and (5) nerve fiber layer defects in 3 (14%). Conclusions With improved visualization of fine retinal architectural features, ultrahigh-resolution OCT can visualize persistent retinal abnormalities despite anatomically successful macular hole surgery. Outer foveal hyporeflective disruptions of the junction between the inner and outer segments of the photoreceptors likely represent areas of foveal photoreceptor degeneration. Moderately reflective lesions likely represent glial cell proliferation at the site of hole reapproximation. Thin epiretinal membranes do not seem to decrease visual acuity and may play a role in reestablishing foveal anatomy after surgery. PMID:16769836

Spatial Variation in Mobility-Lifetime Product in Bulk TlBr and CZT

NASA Astrophysics Data System (ADS)

Phillips, David; Haegel, Nancy; Blaine, Kevin; Kim, Hadong; Ciampi, Guido; Cirignano, Len

2012-02-01

The energy resolution of a semiconductor radiation detector depends on the charge transport properties of the semiconductor, and the mobility-lifetime (μτ) product is a key figure of merit for charge transport. In this work, we investigate the effects of two impurities, Na and Cu, on the μτ product in bulk thallium bromide (TlBr) using cathodoluminescence (CL) and transport imaging. Transport imaging uses a scanning electron microscope to generate a line of charge carriers on the surface of a bulk sample, and the intensity and spatial distribution of the recombination luminescence are recorded. A Green's function approach is used to model the generation, diffusion, and recombination of charge carriers under steady-state conditions. The luminescence distribution is fit to the model to extract the ambipolar diffusion length and the μτ product, providing a high-resolution correlation between the luminescence variations due to dopants/defects and the quantitative transport behavior. The μτ product has been mapped across a 40 μm segment of TlBr at a resolution of 2 μm. Additionally, this approach has been used to locally map variations in ambipolar diffusion length and μτ product due to extended defects in cadmium zinc telluride (CZT).

Design, Construction, and Initial Test of High Spatial Resolution Thermometry Arrays for Detection of Surface Temperature Profiles on SRF Cavities in Super Fluid Helium

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

Ari Palczewski, Rongli Geng, Grigory Eremeev

2011-07-01

We designed and built two high resolution (0.6-0.55mm special resolution [1.1-1.2mm separation]) thermometry arrays prototypes out of the Allen Bradley 90-120 ohm 1/8 watt resistor to measure surface temperature profiles on SRF cavities. One array was designed to be physically flexible and conform to any location on a SRF cavity; the other was modeled after the common G-10/stycast 2850 thermometer and designed to fit on the equator of an ILC (Tesla 1.3GHz) SRF cavity. We will discuss the advantages and disadvantages of each array and their construction. In addition we will present a case study of the arrays performance onmore » a real SRF cavity TB9NR001. TB9NR001 presented a unique opportunity to test the performance of each array as it contained a dual (4mm separation) cat eye defect which conventional methods such as OST (Oscillating Superleak second-sound Transducers) and full coverage thermometry mapping were unable to distinguish between. We will discuss the new arrays ability to distinguish between the two defects and their preheating performance.« less

Immersion lithography defectivity analysis at DUV inspection wavelength

NASA Astrophysics Data System (ADS)

Golan, E.; Meshulach, D.; Raccah, N.; Yeo, J. Ho.; Dassa, O.; Brandl, S.; Schwarz, C.; Pierson, B.; Montgomery, W.

2007-03-01

Significant effort has been directed in recent years towards the realization of immersion lithography at 193nm wavelength. Immersion lithography is likely a key enabling technology for the production of critical layers for 45nm and 32nm design rule (DR) devices. In spite of the significant progress in immersion lithography technology, there remain several key technology issues, with a critical issue of immersion lithography process induced defects. The benefits of the optical resolution and depth of focus, made possible by immersion lithography, are well understood. Yet, these benefits cannot come at the expense of increased defect counts and decreased production yield. Understanding the impact of the immersion lithography process parameters on wafer defects formation and defect counts, together with the ability to monitor, control and minimize the defect counts down to acceptable levels is imperative for successful introduction of immersion lithography for production of advanced DR's. In this report, we present experimental results of immersion lithography defectivity analysis focused on topcoat layer thickness parameters and resist bake temperatures. Wafers were exposed on the 1150i-α-immersion scanner and 1200B Scanner (ASML), defect inspection was performed using a DUV inspection tool (UVision TM, Applied Materials). Higher sensitivity was demonstrated at DUV through detection of small defects not detected at the visible wavelength, indicating on the potential high sensitivity benefits of DUV inspection for this layer. The analysis indicates that certain types of defects are associated with different immersion process parameters. This type of analysis at DUV wavelengths would enable the optimization of immersion lithography processes, thus enabling the qualification of immersion processes for volume production.

The three-dimensional structure of the cellobiohydrolase Cel7A from Aspergillus fumigatus at 1.5 Å resolution

PubMed Central

Moroz, Olga V.; Maranta, Michelle; Shaghasi, Tarana; Harris, Paul V.; Wilson, Keith S.; Davies, Gideon J.

2015-01-01

The enzymatic degradation of plant cell-wall cellulose is central to many industrial processes, including second-generation biofuel production. Key players in this deconstruction are the fungal cellobiohydrolases (CBHs), notably those from family GH7 of the carbohydrate-active enzymes (CAZY) database, which are generally known as CBHI enzymes. Here, three-dimensional structures are reported of the Aspergillus fumigatus CBHI Cel7A solved in uncomplexed and disaccharide-bound forms at resolutions of 1.8 and 1.5 Å, respectively. The product complex with a disaccharide in the +1 and +2 subsites adds to the growing three-dimensional insight into this family of industrially relevant biocatalysts. PMID:25615982

Three-dimensional structure of phosphoribosyl pyrophosphate synthetase from E. coli at 2.71 Å resolution

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

Timofeev, V. I., E-mail: inna@ns.crys.ras.ru, E-mail: tostars@mail.ru, E-mail: ugama@yandex.ru; Abramchik, Yu. A.; Zhukhlistova, N. E.

2016-01-15

Phosphoribosyl pyrophosphate synthetase from Escherichia coli was cloned, purified, and crystallized. Single crystals of the enzyme were grown under microgravity. The X-ray diffraction data set was collected at the Spring-8 synchrotron facility and used to determine the three-dimensional structure of the enzyme by the molecular-replacement method at 2.71 Å resolution. The active and regulatory sites in the molecule of E. coli phosphoribosyl pyrophosphate synthetase were revealed by comparison with the homologous protein from Bacillus subtilis, the structure of which was determined in a complex with functional ligands. The conformations of polypeptide-chain fragments surrounding and composing the active and regulatory sitesmore » were shown to be identical in both proteins.« less

Converting NADH to NAD+ by nicotinamide nucleotide transhydrogenase as a novel strategy against mitochondrial pathologies during aging.

PubMed

Olgun, Abdullah

2009-08-01

Mitochondrial DNA defects are involved supposedly via free radicals in many pathologies including aging and cancer. But, interestingly, free radical production was not found increased in prematurely aging mice having higher mutation rate in mtDNA. Therefore, some other mechanisms like the increase of mitochondrial NADH/NAD(+) and ubiquinol/ubiquinone ratios, can be in action in respiratory chain defects. NADH/NAD(+) ratio can be normalized by the activation or overexpression of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme catalyzing the following very important reaction: NADH + NADP(+ )<--> NADPH + NAD(+). The products NAD(+) and NADPH are required in many critical biological processes, e.g., NAD(+) is used by histone deacetylase Sir2 which regulates longevity in different species. NADPH is used in a number of biosynthesis reactions (e.g., reduced glutathione synthesis), and processes like apoptosis. Increased ubiquinol/ubiquinone ratio interferes the function of dihydroorotate dehydrogenase, the only mitochondrial enzyme involved in ubiquinone mediated de novo pyrimidine synthesis. Uridine and its prodrug triacetyluridine are used to compensate pyrimidine deficiency but their bioavailability is limited. Therefore, the normalization of the ubiquinol/ubiquinone ratio can be accomplished by allotopic expression of alternative oxidase, a mitochondrial ubiquinol oxidase which converts ubiquinol to ubiquinone.

S-Nitrosation destabilizes glutathione transferase P1-1.

PubMed

Balchin, David; Stoychev, Stoyan H; Dirr, Heini W

2013-12-23

Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

Defect Clustering and Nano-phase Structure Characterization of Multicomponent Rare Earth-Oxide-Doped Zirconia-Yttria Thermal Barrier Coatings

NASA Technical Reports Server (NTRS)

Zhu, Dongming; Chen, Yuan L.; Miller, Robert A.

2004-01-01

Advanced thermal barrier coatings (TBCs) have been developed by incorporating multicomponent rare earth oxide dopants into zirconia-based thermal barrier coatings to promote the creation of the thermodynamically stable, immobile oxide defect clusters and/or nanophases within the coating systems. In this paper, the defect clusters, induced by Nd, Gd, and Yb rare earth dopants in the zirconia-yttria thermal barrier coatings, were characterized by high-resolution transmission electron microscopy (TEM). The TEM lattice imaging, selected area diffraction (SAD), and electron energy-loss spectroscopy (EELS) analyses demonstrated that the extensive nanoscale rare earth dopant segregation exists in the plasma-sprayed and electron-physical-vapor-deposited (EB PVD) thermal barrier coatings. The nanoscale concentration heterogeneity and the resulting large lattice distortion promoted the formation of parallel and rotational defective lattice clusters in the coating systems. The presence of the 5-to 100-nm-sized defect clusters and nanophases is believed to be responsible for the significant reduction of thermal conductivity, improved sintering resistance, and long-term high temperature stability of the advanced thermal barrier coating systems.

Photomask quality evaluation using lithography simulation and multi-detector MVM-SEM

NASA Astrophysics Data System (ADS)

Ito, Keisuke; Murakawa, Tsutomu; Fukuda, Naoki; Shida, Soichi; Iwai, Toshimichi; Matsumoto, Jun; Nakamura, Takayuki; Matsushita, Shohei; Hagiwara, Kazuyuki; Hara, Daisuke

2013-06-01

The detection and management of mask defects which are transferred onto wafer becomes more important day by day. As the photomask patterns becomes smaller and more complicated, using Inverse Lithography Technology (ILT) and Source Mask Optimization (SMO) with Optical Proximity Correction (OPC). To evaluate photomask quality, the current method uses aerial imaging by optical inspection tools. This technique at 1Xnm node has a resolution limit because small defects will be difficult to detect. We already reported the MEEF influence of high-end photomask using wide FOV SEM contour data of "E3630 MVM-SEM®" and lithography simulator "TrueMask® DS" of D2S Inc. in the prior paper [1]. In this paper we evaluate the correlation between our evaluation method and optical inspection tools as ongoing assessment. Also in order to reduce the defect classification work, we can compose the 3 Dimensional (3D) information of defects and can judge whether repairs of defects would be required. Moreover, we confirm the possibility of wafer plane CD measurement based on the combination between E3630 MVM-SEM® and 3D lithography simulation.

Investigation of the reconstruction accuracy of guided wave tomography using full waveform inversion

NASA Astrophysics Data System (ADS)

Rao, Jing; Ratassepp, Madis; Fan, Zheng

2017-07-01

Guided wave tomography is a promising tool to accurately determine the remaining wall thicknesses of corrosion damages, which are among the major concerns for many industries. Full Waveform Inversion (FWI) algorithm is an attractive guided wave tomography method, which uses a numerical forward model to predict the waveform of guided waves when propagating through corrosion defects, and an inverse model to reconstruct the thickness map from the ultrasonic signals captured by transducers around the defect. This paper discusses the reconstruction accuracy of the FWI algorithm on plate-like structures by using simulations as well as experiments. It was shown that this algorithm can obtain a resolution of around 0.7 wavelengths for defects with smooth depth variations from the acoustic modeling data, and about 1.5-2 wavelengths from the elastic modeling data. Further analysis showed that the reconstruction accuracy is also dependent on the shape of the defect. It was demonstrated that the algorithm maintains the accuracy in the case of multiple defects compared to conventional algorithms based on Born approximation.

Nucleation and atomic layer reaction in nickel silicide for defect-engineered Si nanochannels.

PubMed

Tang, Wei; Picraux, S Tom; Huang, Jian Yu; Gusak, Andriy M; Tu, King-Ning; Dayeh, Shadi A

2013-06-12

At the nanoscale, defects can significantly impact phase transformation processes and change materials properties. The material nickel silicide has been the industry standard electrical contact of silicon microelectronics for decades and is a rich platform for scientific innovation at the conjunction of materials and electronics. Its formation in nanoscale silicon devices that employ high levels of strain, intentional, and unintentional twins or grain boundaries can be dramatically different from the commonly conceived bulk processes. Here, using in situ high-resolution transmission electron microscopy (HRTEM), we capture single events during heterogeneous nucleation and atomic layer reaction of nickel silicide at various crystalline boundaries in Si nanochannels for the first time. We show through systematic experiments and analytical modeling that unlike other typical face-centered cubic materials such as copper or silicon the twin defects in NiSi2 have high interfacial energies. We observe that these twin defects dramatically change the behavior of new phase nucleation and can have direct implications for ultrascaled devices that are prone to defects or may utilize them to improve device performance.

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

PubMed Central

Oh, Doo-Byoung

2015-01-01

Lysosomal storage diseases (LSDs) are a group of inherent diseases characterized by massive accumulation of undigested compounds in lysosomes, which is caused by genetic defects resulting in the deficiency of a lysosomal hydrolase. Currently, enzyme replacement therapy has been successfully used for treatment of 7 LSDs with 10 approved therapeutic enzymes whereas new approaches such as pharmacological chaperones and gene therapy still await evaluation in clinical trials. While therapeutic enzymes for Gaucher disease have N-glycans with terminal mannose residues for targeting to macrophages, the others require N-glycans containing mannose-6-phosphates that are recognized by mannose-6-phosphate receptors on the plasma membrane for cellular uptake and targeting to lysosomes. Due to the fact that efficient lysosomal delivery of therapeutic enzymes is essential for the clearance of accumulated compounds, the suitable glycan structure and its high content are key factors for efficient therapeutic efficacy. Therefore, glycan remodeling strategies to improve lysosomal targeting and tissue distribution have been highlighted. This review describes the glycan structures that are important for lysosomal targeting and provides information on recent glyco-engineering technologies for the development of therapeutic enzymes with improved efficacy. [BMB Reports 2015; 48(8): 438-444] PMID:25999178

Silica-Immobilized Enzyme Reactors (Postprint)

DTIC Science & Technology

2007-09-01

mode of action of drugs such as aspirin and ibuprofen .[61] Serotonin reuptake inhibitors and monoamine oxidase inhibitors can function as...immobilizing PGA onto chromatography supports and using the enantiomeric selectivity of the enzyme to resolve racemic mixtures.[100] Immobilization onto...column. J. Chroma- togr. B. Biomed. Sci. Appl. 2001, 753, 375–383. 37. Jadaud, P.; Wainer, I.W. The stereochemical resolution of the enantiomers of

Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

PubMed Central

Newby, Andrew C.; Chrambach, Andreas

1979-01-01

1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in specific ratios may be useful in the purification of adenylate cyclase and other intrinsic membrane proteins. ImagesFig. 3. PMID:435255

Searching phase II enzymes inducers, from Michael acceptor-[1,2]dithiolethione hybrids, as cancer chemopreventive agents.

PubMed

Couto, Marcos; de Ovalle, Stefani; Cabrera, Mauricio; Cerecetto, Hugo; González, Mercedes

2015-01-01

Cancer chemoprevention involves the carcinogenic process prevention, delay or reverse by the administration of chemopreventive agents, which are able to suppress or block the carcinogen metabolic activation/formation. The increased activity of phase II detoxification enzymes such as quinone-reductase (QR) and glutation-S-transferase (GST) correlates with the protection against chemically-induced carcinogenesis. It has been shown that synthetic chalcones and 3H-[1,2]-dithiole-3-thiones promote expression of genes involved in chemoprevention. Herein, the induction of phase II enzymes by designed Michael acceptor-dithiolethione hybrids was studied. Hybrids 5 and 7 displayed the induction of quinone-reductase and glutation-S-transferase in vitro in the same order on the wild-type mouse-hepatoma Hepa 1c1c7 and on the aryl-hydrocarbon-nuclear-translocator (Arnt)-defective mutant BPrc1 cells indicating that 7 displays the best chemopreventive potential.

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures.

PubMed

Horrell, Sam; Kekilli, Demet; Sen, Kakali; Owen, Robin L; Dworkowski, Florian S N; Antonyuk, Svetlana V; Keal, Thomas W; Yong, Chin W; Eady, Robert R; Hasnain, S Samar; Strange, Richard W; Hough, Michael A

2018-05-01

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate 'structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a 'top-hat' geometry, which was rapidly transformed to a 'side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Genetic variation in the urea cycle: a model resource for investigating key candidate genes for common diseases.

PubMed

Mitchell, Sabrina; Ellingson, Clint; Coyne, Thomas; Hall, Lynn; Neill, Meaghan; Christian, Natalie; Higham, Catherine; Dobrowolski, Steven F; Tuchman, Mendel; Summar, Marshall

2009-01-01

The urea cycle is the primary means of nitrogen metabolism in humans and other ureotelic organisms. There are five key enzymes in the urea cycle: carbamoyl-phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1). Additionally, a sixth enzyme, N-acetylglutamate synthase (NAGS), is critical for urea cycle function, providing CPS1 with its necessary cofactor. Deficiencies in any of these enzymes result in elevated blood ammonia concentrations, which can have detrimental effects, including central nervous system dysfunction, brain damage, coma, and death. Functional variants, which confer susceptibility for disease or dysfunction, have been described for enzymes within the cycle; however, a comprehensive screen of all the urea cycle enzymes has not been performed. We examined the exons and intron/exon boundaries of the five key urea cycle enzymes, NAGS, and two solute carrier transporter genes (SLC25A13 and SLC25A15) for sequence alterations using single-stranded conformational polymorphism (SSCP) analysis and high-resolution melt profiling. SSCP was performed on a set of DNA from 47 unrelated North American individuals with a mixture of ethnic backgrounds. High-resolution melt profiling was performed on a nonoverlapping DNA set of either 47 or 100 unrelated individuals with a mixture of backgrounds. We identified 33 unarchived polymorphisms in this screen that potentially play a role in the variation observed in urea cycle function. Screening all the genes in the pathway provides a catalog of variants that can be used in investigating candidate diseases. Copyright 2008 Wiley-Liss, Inc.

High resolution crystal structure of rat long chain hydroxy acid oxidase in complex with the inhibitor 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1, 2, 3-thiadiazole. Implications for inhibitor specificity and drug design.

PubMed

Chen, Zhi-wei; Vignaud, Caroline; Jaafar, Adil; Lévy, Bernard; Guéritte, Françoise; Guénard, Daniel; Lederer, Florence; Mathews, F Scott

2012-05-01

Long chain hydroxy acid oxidase (LCHAO) is responsible for the formation of methylguanidine, a toxic compound with elevated serum levels in patients with chronic renal failure. Its isozyme glycolate oxidase (GOX), has a role in the formation of oxalate, which can lead to pathological deposits of calcium oxalate, in particular in the disease primary hyperoxaluria. Inhibitors of these two enzymes may have therapeutic value. These enzymes are the only human members of the family of FMN-dependent l-2-hydroxy acid-oxidizing enzymes, with yeast flavocytochrome b(2) (Fcb2) among its well studied members. We screened a chemical library for inhibitors, using in parallel rat LCHAO, human GOX and the Fcb2 flavodehydrogenase domain (FDH). Among the hits was an inhibitor, CCPST, with an IC(50) in the micromolar range for all three enzymes. We report here the crystal structure of a complex between this compound and LCHAO at 1.3 Å resolution. In comparison with a lower resolution structure of this enzyme, binding of the inhibitor induces a conformational change in part of the TIM barrel loop 4, as well as protonation of the active site histidine. The CCPST interactions are compared with those it forms with human GOX and those formed by two other inhibitors with human GOX and spinach GOX. These compounds differ from CCPST in having the sulfur replaced with a nitrogen in the five-membered ring as well as different hydrophobic substituents. The possible reason for the ∼100-fold difference in affinity between these two series of inhibitors is discussed. The present results indicate that specificity is an issue in the quest for therapeutic inhibitors of either LCHAO or GOX, but they may give leads for this quest. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Improved performance of Yarrowia lipolytica lipase-catalyzed kinetic resolution of (R,S)-2-octanol by an integrated strategy of interfacial activation, bioimprinting and immobilization.

PubMed

Liu, Ying; Guo, Chen; Sun, Xi-Tong; Liu, Chun-Zhao

2013-08-01

Yarrowia lipolytica lipase (YLL) demonstrated an (R)-enantiopreference for efficient resolution of (R,S)-2-octanol. The activity, enantioselectivity, the ratio of substrate to enzyme, acetaldehyde tolerance, and operational stability of YLL were improved by an integrated strategy of interfacial activation, bioimprinting, and immobilization. In comparison with the control, both the enzymatic activity and enantioselectivity increased by a factor of 8.85 and 2.75 by the integrated strategy, respectively. Fifty-one percentage of conversion with 220 of enantioselectivity was obtained using the immobilized YLL prepared by the integrated strategy at a ratio of 104 of substrate to enzyme loaded. The immobilized YLL retained 97% of its initial activity without a decrease in enantioselectivity after 10 successive reuse cycles. Together these results will result in a promising strategy with the YYL for efficient resolution of (R,S)-2-octanol in practice. Copyright © 2013 Elsevier Ltd. All rights reserved.

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis

PubMed Central

Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M.; Bartlam, Mark

2016-01-01

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate. PMID:26919520

Purification, crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis.

PubMed

Zhang, Aili; Guo, Erhong; Qian, Lanfang; Tang, Nga-Yeung; Watt, Rory M; Bartlam, Mark

2016-03-01

Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, β = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.

Investigation of low leakage current radiation detectors on n-type 4H-SiC epitaxial layers

NASA Astrophysics Data System (ADS)

Nguyen, Khai V.; Chaudhuri, Sandeep K.; Mandal, Krishna C.

2014-09-01

The surface leakage current of high-resolution 4H-SiC epitaxial layer Schottky barrier detectors has been improved significantly after surface passivations of 4H-SiC epitaxial layers. Thin (nanometer range) layers of silicon dioxide (SiO2) and silicon nitride (Si3N4) were deposited on 4H-SiC epitaxial layers using plasma enhanced chemical vapor deposition (PECVD) on 20 μm thick n-type 4H-SiC epitaxial layers followed by the fabrication of large area (~12 mm2) Schottky barrier radiation detectors. The fabricated detectors have been characterized through current-voltage (I-V), capacitance-voltage (C-V), and alpha pulse height spectroscopy measurements; the results were compared with that of detectors fabricated without surface passivations. Improved energy resolution of ~ 0.4% for 5486 keV alpha particles was observed after passivation, and it was found that the performance of these detectors were limited by the presence of macroscopic and microscopic crystal defects affecting the charge transport properties adversely. Capacitance mode deep level transient studies (DLTS) revealed the presence of a titanium impurity related shallow level defects (Ec-0.19 eV), and two deep level defects identified as Z1/2 and Ci1 located at Ec-0.62 and ~ Ec-1.40 eV respectively.

[Detection and evaluation of cartilage defects in the canine stifle joint - an ex vivo study using high-field magnetic resonance imaging].

PubMed

Flatz, K M; Glaser, C; Flatz, W H; Reiser, M F; Matis, U

2014-01-01

The aim of our study was to implement and test an imaging protocol for the detection and evaluation of standardised cartilage defects using high-field magnetic resonance imaging (MRI) and to determine its limitations. A total of 84 cartilage defects were created in the femoral condyles of euthanized dogs with a minimum body mass of 25 kg. The cartilage defects had a depth of 0.3 to 1.0 mm and a diameter of 1 to 5 mm. T1-FLASH-3D-WE-sequences with an isotropic voxel size of 0.5 x 0.5 x 0.5 mm and an anisotropic voxel size of 0.3 x 0.3 x 0.8 mm were used. In addition to quantitative evaluation of the cartilage defects, the sig- nal intensities, signal-to-noise ratios and contrast-to-noise ratios of the cartilage were determined. Of special interest were the limita- tions in identifying and delineating the standardised cartilage defects. With the anisotropic voxel size, more cartilage defects were detectable. Our results demonstrated that cartilage defects as small as 3.0 mm in diameter and 0.4 mm in depth were reliably detected using anisotropic settings. Cartilage defects below this size were not reliably detected. We found that for optimal delineation of the joint cartilage and associated defects, a higher in-plane resolution with a larger slice thickness should be used, corresponding to the anisotropic settings employed in this study. For the delineation of larger cartilage defects, both the anisotropic and isotropic imaging methods can be used.

Microbial Gutta-Percha Degradation Shares Common Steps with Rubber Degradation by Nocardia nova SH22a

PubMed Central

Luo, Quan; Hiessl, Sebastian; Poehlein, Anja

2013-01-01

Nocardia nova SH22a, a bacterium capable of degrading gutta-percha (GP) and natural rubber (NR), was used to investigate the GP degradation mechanism and the relations between the GP and NR degradation pathways. For this strain, a protocol of electroporation was systematically optimized, and an efficiency of up to 4.3 × 107 CFU per μg of plasmid DNA was achieved. By applying this optimized protocol to N. nova SH22a, a Tn5096-based transposon mutagenesis library of this bacterium was constructed. Among about 12,000 apramycin-resistant transformants, we identified 76 stable mutants defective in GP or NR utilization. Whereas 10 mutants were specifically defective in GP utilization, the growth of the other 66 mutants was affected on both GP and NR. This indicated that the two degradation pathways are quite similar and share many common steps. The larger number of GP-degrading defective mutants could be explained in one of two ways: either (i) the GP pathway is more complex and harbors more specific steps or (ii) the steps for both pathways are almost identical, but in the case of GP degradation there are fewer enzymes involved in each step. The analysis of transposition loci and genetic studies on interesting genes confirmed the crucial role of an α-methylacyl-coenzyme A racemase in the degradation of both GP and NR. We also demonstrated the probable involvement of enzymes participating in oxidoreduction reactions, β-oxidation, and the synthesis of complex cell envelope lipids in the degradation of GP. PMID:23220954

Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.

PubMed

Prabhu, Yogikala; Blanco, Elias H; Liu, Ming; Peinado, Juan R; Wheeler, Matthew C; Gekakis, Nicholas; Arvan, Peter; Lindberg, Iris

2014-07-01

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3(N222D) mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3(N222D) mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity.

Defective Transport of the Obesity Mutant PC1/3 N222D Contributes to Loss of Function

PubMed Central

Prabhu, Yogikala; Blanco, Elias H.; Liu, Ming; Peinado, Juan R.; Wheeler, Matthew C.; Gekakis, Nicholas; Arvan, Peter

2014-01-01

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3N222D mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3N222D mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity. PMID:24828610

A-to-I RNA editing promotes developmental stage–specific gene and lncRNA expression

PubMed Central

Goldstein, Boaz; Agranat-Tamir, Lily; Light, Dean; Ben-Naim Zgayer, Orna; Fishman, Alla; Lamm, Ayelet T.

2017-01-01

A-to-I RNA editing is a conserved widespread phenomenon in which adenosine (A) is converted to inosine (I) by adenosine deaminases (ADARs) in double-stranded RNA regions, mainly noncoding. Mutations in ADAR enzymes in Caenorhabditis elegans cause defects in normal development but are not lethal as in human and mouse. Previous studies in C. elegans indicated competition between RNA interference (RNAi) and RNA editing mechanisms, based on the observation that worms that lack both mechanisms do not exhibit defects, in contrast to the developmental defects observed when only RNA editing is absent. To study the effects of RNA editing on gene expression and function, we established a novel screen that enabled us to identify thousands of RNA editing sites in nonrepetitive regions in the genome. These include dozens of genes that are edited at their 3′ UTR region. We found that these genes are mainly germline and neuronal genes, and that they are down-regulated in the absence of ADAR enzymes. Moreover, we discovered that almost half of these genes are edited in a developmental-specific manner, indicating that RNA editing is a highly regulated process. We found that many pseudogenes and other lncRNAs are also extensively down-regulated in the absence of ADARs in the embryo but not in the fourth larval (L4) stage. This down-regulation is not observed upon additional knockout of RNAi. Furthermore, levels of siRNAs aligned to pseudogenes in ADAR mutants are enhanced. Taken together, our results suggest a role for RNA editing in normal growth and development by regulating silencing via RNAi. PMID:28031250

A rare case of sterol-C4-methyl oxidase deficiency in a young Italian male: Biochemical and molecular characterization.

PubMed

Frisso, Giulia; Gelzo, Monica; Procopio, Elena; Sica, Concetta; Lenza, Maria Pia; Dello Russo, Antonio; Donati, Maria Alice; Salvatore, Francesco; Corso, Gaetano

2017-08-01

Inborn defects of cholesterol biosynthesis are metabolic disorders presenting with multi-organ and tissue anomalies. An autosomal recessive defect involving the demethylating enzyme C4-methyl sterol (SC4MOL) has been reported in only 4 patients so far. In infancy, all patients were affected by microcephaly, bilateral congenital cataracts, growth delay, psoriasiform dermatitis, immune dysfunction, and intellectual disability. Herein, we describe a new case of SC4MOL deficiency in which a 19-year-old Italian male was affected by bilateral congenital cataracts, growth delay and learning disabilities, behavioral disorders and small stature, but not microcephaly. Our patient had abundant scalp dandruff, without other skin manifestations. Analysis of the blood sterol profile showed accumulation of C4-monomethyl and C4-dimethyl sterols suggesting a deficiency of the SC4MOL enzyme. Sequencing of the MSMO1 gene (also known as the "SC4MOL" gene) confirmed mutations in each allele (c.731A>G, p.Y244C, which is already known, and c.605G>A, p.G202E, which is a novel variant). His father carried c.731A>G mutation, whereas his mother carried c.605G>A. Thus, the combination of multiple skills and methodologies, in particular, blood sterol profiling and genetic analysis, led to the diagnosis of a new case of a very rare defect of cholesterol biosynthesis. Consequently, we suggest that these two analyses should be performed as soon as possible in all undiagnosed patients affected by bilateral cataracts and developmental delay. Copyright © 2017 Elsevier Inc. All rights reserved.

A Simple Method for Rapid Depletion of Rubisco from Soybean (Glycine max) Leaf for Proteomic Analysis of Lower Abundance Proteins

USDA-ARS?s Scientific Manuscript database

2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30 – 50% of the CO2 fixation enzyme Rubisco. Resolution can be improved t...

Metabolite identification of the antimalarial naphthoquine using liquid chromatography-tandem high-resolution mass spectrometry in combination with multiple data-mining tools.

PubMed

Sun, Yanhong; Wang, Shuqi; Ji, Jianbo; Zhai, Guangxi; Xing, Jie

2018-06-01

Naphthoquine (NQ) is one of important partner drugs of artemisinin-based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2-4 h) and has a long elimination half-life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high-resolution LTQ-Orbitrap mass spectrometry (HRMS n ) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid-phase extraction. For data processing, multiple data-mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1-M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N-oxidation. This study demonstrates that LC-HRMS n in combination with multiple data-mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. Copyright © 2018 John Wiley & Sons, Ltd.

High-resolution nondestructive testing of multilayer dielectric materials using wideband microwave synthetic aperture radar imaging

NASA Astrophysics Data System (ADS)

Kim, Tae Hee; James, Robin; Narayanan, Ram M.

2017-04-01

Fiber Reinforced Polymer or Plastic (FRP) composites have been rapidly increasing in the aerospace, automotive and marine industry, and civil engineering, because these composites show superior characteristics such as outstanding strength and stiffness, low weight, as well as anti-corrosion and easy production. Generally, the advancement of materials calls for correspondingly advanced methods and technologies for inspection and failure detection during production or maintenance, especially in the area of nondestructive testing (NDT). Among numerous inspection techniques, microwave sensing methods can be effectively used for NDT of FRP composites. FRP composite materials can be produced using various structures and materials, and various defects or flaws occur due to environmental conditions encountered during operation. However, reliable, low-cost, and easy-to-operate NDT methods have not been developed and tested. FRP composites are usually produced as multilayered structures consisting of fiber plate, matrix and core. Therefore, typical defects appearing in FRP composites are disbondings, delaminations, object inclusions, and certain kinds of barely visible impact damages. In this paper, we propose a microwave NDT method, based on synthetic aperture radar (SAR) imaging algorithms, for stand-off imaging of internal delaminations. When a microwave signal is incident on a multilayer dielectric material, the reflected signal provides a good response to interfaces and transverse cracks. An electromagnetic wave model is introduced to delineate interface widths or defect depths from the reflected waves. For the purpose of numerical analysis and simulation, multilayered composite samples with various artificial defects are assumed, and their SAR images are obtained and analyzed using a variety of high-resolution wideband waveforms.

Evaluation of Chloropentafluorobenzene in a Battery of in vitro Short Term Assays

DTIC Science & Technology

1986-01-01

metabolic activation. The CHO/HGPRT assay measures the ability of a test agent to induce forward mutations at the enzyme hypoxanthine-guanine...phosphoribosyl transferase (HGPRT) locus of Chinese hamster ovary cells on the basis- that presumptive mutants defective in the enzyme HGPRT are unable to...10 0 4.) $4 coc H4 0 o uE p- ’-’ coQ i u ,q10 t GD 4- r ) c41 uH 0 0 41GD c t Ia) U, 40 r. 4 coV P-1- 41 E-4 0 .Z ~-H 1 0) N Cu I0 m : L0 0 z4v- +10

[Hyperammonemia due to ornithine transcarbamylase deficiency--a cause of lethal metabolic crisis during the newborn period and infancy (author's transl)].

PubMed

Schuchmann, L; Colombo, J P; Fischer, H

1980-05-01

A severe hyperammonemia is the characteristic finding in patients with enzyme defects in urea cycle and one of the main causes of the acute metabolic crisis dsuring the newborn period and infancy. A case report is given about two male infants, who died in the age of one and of seven months respectively. In the second child the blood ammonia concentration raised up to 833 micrograms/100 ml, and, OTC deficiency was diagnosed due to enzyme determination in liver biopsie. Probably, the first child, that also died as newborn, suffered from the same disease. In this case, only post mortem findings are available.

The in silico screening and X-ray structure analysis of the inhibitor complex of Plasmodium falciparum orotidine 5'-monophosphate decarboxylase.

PubMed

Takashima, Yasuhide; Mizohata, Eiichi; Krungkrai, Sudaratana R; Fukunishi, Yoshifumi; Kinoshita, Takayoshi; Sakata, Tsuneaki; Matsumura, Hiroyoshi; Krungkrai, Jerapan; Horii, Toshihiro; Inoue, Tsuyoshi

2012-08-01

Orotidine 5'-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) catalyses the final step in the de novo synthesis of uridine 5'-monophosphate (UMP) from orotidine 5'-monophosphate (OMP). A defective PfOMPDC enzyme is lethal to the parasite. Novel in silico screening methods were performed to select 14 inhibitors against PfOMPDC, with a high hit rate of 9%. X-ray structure analysis of PfOMPDC in complex with one of the inhibitors, 4-(2-hydroxy-4-methoxyphenyl)-4-oxobutanoic acid, was carried out to at 2.1 Å resolution. The crystal structure revealed that the inhibitor molecule occupied a part of the active site that overlaps with the phosphate-binding region in the OMP- or UMP-bound complexes. Space occupied by the pyrimidine and ribose rings of OMP or UMP was not occupied by this inhibitor. The carboxyl group of the inhibitor caused a dramatic movement of the L1 and L2 loops that play a role in the recognition of the substrate and product molecules. Combining part of the inhibitor molecule with moieties of the pyrimidine and ribose rings of OMP and UMP represents a suitable avenue for further development of anti-malarial drugs.

Defective B cell tolerance in adenosine deaminase deficiency is corrected by gene therapy

PubMed Central

Sauer, Aisha V.; Morbach, Henner; Brigida, Immacolata; Ng, Yen-Shing; Aiuti, Alessandro; Meffre, Eric

2012-01-01

Adenosine deaminase (ADA) gene defects are among the most common causes of SCID. Restoration of purine metabolism and immune functions can be achieved by enzyme replacement therapy, or more effectively by bone marrow transplant or HSC gene therapy (HSC-GT). However, autoimmune complications and autoantibody production, including anti-nuclear antibodies (ANAs), frequently occur in ADA-SCID patients after treatment. To assess whether ADA deficiency affects the establishment of B cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells of ADA-SCID patients before and after HSC-GT. We found that before HSC-GT, new emigrant/transitional and mature naive B cells from ADA-SCID patients contained more autoreactive and ANA-expressing clones, indicative of defective central and peripheral B cell tolerance checkpoints. We further observed impaired B cell receptor (BCR) and TLR functions in B cells after ADA inhibition, which may underlie the defects in B cell tolerance. Strikingly, after HSC-GT, ADA-SCID patients displayed quasi-normal early B cell tolerance checkpoints, as evidenced by restored removal of developing autoreactive and ANA-expressing B cells. Hence, ADA plays an essential role in controlling autoreactive B cell counterselection by regulating BCR and TLR functions. PMID:22622038

Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31.

PubMed

Lahoda, Maryna; Mesters, Jeroen R; Stsiapanava, Alena; Chaloupkova, Radka; Kuty, Michal; Damborsky, Jiri; Kuta Smatanova, Ivana

2014-02-01

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure

PubMed Central

Nakabayashi, Makoto; Kataoka, Misumi; Mishima, Yumiko; Maeno, Yuka; Ishikawa, Kazuhiko

2014-01-01

β-Glucosidase from Pyrococcus furiosus (BGLPf) is a hyperthermophilic tetrameric enzyme which can degrade cellooligosaccharides to glucose under hyperthermophilic conditions and thus holds promise for the saccharification of lignocellulosic biomass at high temperature. Prior to the production of large amounts of this enzyme, detailed information regarding the oligomeric structure of the enzyme is required. Several crystals of BGLPf have been prepared over the past ten years, but its crystal structure had not been solved until recently. In 2011, the first crystal structure of BGLPf was solved and a model was constructed at somewhat low resolution (2.35 Å). In order to obtain more detailed structural data on BGLPf, the relationship between its tetrameric structure and the quality of the crystal was re-examined. A dimeric form of BGLPf was constructed and its crystal structure was solved at a resolution of 1.70 Å using protein-engineering methods. Furthermore, using the high-resolution crystal structural data for the dimeric form, a monomeric form of BGLPf was constructed which retained the intrinsic activity of the tetrameric form. The thermostability of BGLPf is affected by its oligomeric structure. Here, the biophysical and biochemical properties of engineered dimeric and monomeric BGLPfs are reported, which are promising prototype models to apply to the saccharification reaction. Furthermore, details regarding the oligomeric structures of BGLPf and the reasons why the mutations yielded improved crystal structures are discussed. PMID:24598756

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase.

PubMed

Bueno, Amy N; Shrestha, Rashmi K; Ronau, Judith A; Babar, Aditya; Sheedlo, Michael J; Fuchs, Julian E; Paul, Lake N; Das, Chittaranjan

2015-10-06

The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologues, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggests that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, X-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The X-ray structures of both Phe403Ala and the Phe403Trp, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the hyrdolysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic properties of the flap in the Trp mutant, compared to the wild-type enzyme, manifest as a defect in interactions that facilitate the rate-limiting step. Consistent with this notion, the Trp mutant was able to cleave Lys48-linked and Lys11-linked diubiquitin better than the wild-type enzyme, indicating altered mobility and hence reduced selectivity.

Crystal Structure of the Pseudomonas aeruginosa BEL-1 Extended-Spectrum β-Lactamase and Its Complexes with Moxalactam and Imipenem

PubMed Central

Pozzi, Cecilia; De Luca, Filomena; Benvenuti, Manuela; Poirel, Laurent; Nordmann, Patrice; Rossolini, Gian Maria

2016-01-01

BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower Km values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties. PMID:27671060

Gauging a Hydrocarbon Ruler by an Intrinsic Exciton Probe†

PubMed Central

Khan, M. Adil; Neale, Chris; Michaux, Catherine; Pomés, Régis; Privé, Gilbert G.; Woody, Robert W.; Bishop, Russell E.

2016-01-01

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of Gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid: lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane β-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26–Trp66 phenol–indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton’s expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler–exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment. PMID:17375935

Using FLIM in the study of permeability barrier function of aged and young skin

NASA Astrophysics Data System (ADS)

Xu, P.; Choi, E. H.; Man, M. Q.; Crumrine, D.; Mauro, T.; Elias, P.

2006-02-01

Aged skin commonly is afflicted by inflammatory skin diseases or xerosis/eczema that can be triggered or exacerbated by impaired epidermal permeability barrier homeostasis. It has been previously described a permeability barrier defect in humans of advanced age (> 75 years), which in a murine analog >18 mos, could be attributed to reduced lipid synthesis synthesis. However, the functional abnormality in moderately aged mice is due not to decreased lipid synthesis, but rather to a specific defect in stratum corneum (SC) acidification causing impaired lipid processing processing. Endogenous Na +/H + antiporter (NHE1) level was found declined in moderately aged mouse epidermis. This acidification defect leads to perturbed permeability barrier homeostasis through more than one pathways, we addressed suboptimal activation of the essential, lipid-processing enzyme, β-glucocerebrosidase (BGC) is linked to elevated SC pH. Finally, the importance of the epidermis acidity is shown by the normalization of barrier function after exogenous acidification of moderately aged skin.

SIRT6 deacetylates H3K18Ac at pericentric chromatin to prevent mitotic errors and cell senescence

PubMed Central

Tasselli, Luisa; Xi, Yuanxin; Zheng, Wei; Tennen, Ruth I.; Odrowaz, Zaneta; Simeoni, Federica; Li, Wei; Chua, Katrin F.

2018-01-01

Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing is observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover a pivotal role of the human SIRT6 enzyme in pericentric transcriptional silencing, and show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, histone H3 lysine K18 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, RNAi-depletion of these transcripts rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and H3K18Ac regulation at heterochromatin, and demonstrate the pathogenic role of de-regulated pericentric transcription in aging- and cancer- related cellular dysfunction. PMID:27043296

Crystallization and diffraction analysis of [beta]-N-acetylhexosaminidase from Aspergillus oryzae

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

Vanek, Ondrej; Brynd, Jirí; Hofbauerová, Katerina

2012-05-08

Fungal {beta}-N-acetylhexosaminidases are enzymes that are used in the chemoenzymatic synthesis of biologically interesting oligosaccharides. The enzyme from Aspergillus oryzae was produced and purified from its natural source and crystallized using the hanging-drop vapor-diffusion method. Diffraction data from two crystal forms (primitive monoclinic and primitive tetragonal) were collected to resolutions of 3.2 and 2.4 {angstrom}, respectively. Electrophoretic and quantitative N-terminal protein-sequencing analyses confirmed that the crystals are formed by a complete biologically active enzyme consisting of a glycosylated catalytic unit and a noncovalently attached propeptide.

Techniques for the Cellular and Subcellular Localization of Endocannabinoid Receptors and Enzymes in the Mammalian Brain.

PubMed

Cristino, Luigia; Imperatore, Roberta; Di Marzo, Vincenzo

2017-01-01

This chapter attempts to piece together knowledge about new advanced microscopy techniques to study the neuroanatomical distribution of endocannabinoid receptors and enzymes at the level of cellular and subcellular structures and organelles in the brain. Techniques ranging from light to electron microscopy up to the new advanced LBM, PALM, and STORM super-resolution microscopy will be discussed in the context of their contribution to define the spatial distribution and organization of receptors and enzymes of the endocannabinoid system (ECS), and to better understand ECS brain functions. © 2017 Elsevier Inc. All rights reserved.

Silica-Immobilized Enzyme Reactors

DTIC Science & Technology

2007-08-01

relief from the symptoms of inflammation and pain Silica-IMERs 10 and is the mode of action of drugs such as aspirin and ibuprofen .[61] Serotonin...supports and using the enantiomeric selectivity of the enzyme to resolve racemic mixtures.[100] Immobilization onto supports with various pore sizes and...activity (~37%) and used as a packed- bed IMER to catalyze the racemic resolution of (S)-ketoprofen from its constituent enantiomers . The optically pure (S

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II*♦

PubMed Central

Domsic, John F.; Avvaru, Balendu Sankara; Kim, Chae Un; Gruner, Sol M.; Agbandje-McKenna, Mavis; Silverman, David N.; McKenna, Robert

2008-01-01

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins. PMID:18768466

Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

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

Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie

2005-01-01

The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance tomore » chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.« less

Locating active-site hydrogen atoms in d-xylose isomerase: Time-of-flight neutron diffraction

PubMed Central

Katz, Amy K.; Li, Xinmin; Carrell, H. L.; Hanson, B. Leif; Langan, Paul; Coates, Leighton; Schoenborn, Benno P.; Glusker, Jenny P.; Bunick, Gerard J.

2006-01-01

Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of d-xylose isomerase. This enzyme, from Streptomyces rubiginosus, is one of the largest enzymes studied to date at high resolution (1.8 Å) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which d-xylose is converted to d-xylulose or d-glucose to d-fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an −NH2-terminal group (rather than NH3+). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 Å) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms. PMID:16707576

Characterization of V-shaped defects in 4H-SiC homoepitaxial layers

DOE PAGES

Zhang, Lihua; Su, Dong; Kisslinger, Kim; ...

2014-12-04

Synchrotron white beam x-ray topography images show that faint needle-like surface morphological features observed on the Si-face of 4H-SiC homoepitaxial layers using Nomarski optical microscopy are associated with V shaped stacking faults in the epilayer. KOH etching of the V shaped defect reveals small oval pits connected by a shallow line which corresponding to the surface intersections of two partial dislocations and the stacking fault connecting them. Transmission electron microscopy (TEM) specimens from regions containing the V shaped defects were prepared using focused ion beam milling, and stacking sequences of (85), (50) and (63) are observed at the faulted regionmore » with high resolution TEM. In order to study the formation mechanism of V shaped defect, low dislocation density 4H-SiC substrates were chosen for epitaxial growth, and the corresponding regions before and after epitaxy growth are compared in SWBXT images. It is found that no defects in the substrate are directly associated with the formation of the V shaped defect. Simulation results of the contrast from the two partial dislocations associated with V shaped defect in synchrotron monochromatic beam x-ray topography reveals the opposite sign nature of their Burgers vectors. Therefore, a mechanism of 2D nucleation during epitaxy growth is postulated for the formation of the V shaped defect, which requires elimination of non-sequential 1/4[0001] bilayers from the original structure to create the observed faulted stacking sequence.« less

Characterization of V-shaped defects in 4H-SiC homoepitaxial layers

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

Zhang, Lihua; Su, Dong; Kisslinger, Kim

Synchrotron white beam x-ray topography images show that faint needle-like surface morphological features observed on the Si-face of 4H-SiC homoepitaxial layers using Nomarski optical microscopy are associated with V shaped stacking faults in the epilayer. KOH etching of the V shaped defect reveals small oval pits connected by a shallow line which corresponding to the surface intersections of two partial dislocations and the stacking fault connecting them. Transmission electron microscopy (TEM) specimens from regions containing the V shaped defects were prepared using focused ion beam milling, and stacking sequences of (85), (50) and (63) are observed at the faulted regionmore » with high resolution TEM. In order to study the formation mechanism of V shaped defect, low dislocation density 4H-SiC substrates were chosen for epitaxial growth, and the corresponding regions before and after epitaxy growth are compared in SWBXT images. It is found that no defects in the substrate are directly associated with the formation of the V shaped defect. Simulation results of the contrast from the two partial dislocations associated with V shaped defect in synchrotron monochromatic beam x-ray topography reveals the opposite sign nature of their Burgers vectors. Therefore, a mechanism of 2D nucleation during epitaxy growth is postulated for the formation of the V shaped defect, which requires elimination of non-sequential 1/4[0001] bilayers from the original structure to create the observed faulted stacking sequence.« less

Selection of Streptococcus lactis Mutants Defective in Malolactic Fermentation

PubMed Central

Renault, Pierre P.; Heslot, Henri

1987-01-01

An enrichment medium and a new sensitive medium were developed to detect malolactic variants in different strains of lactic bacteria. Factors such as the concentration of glucose and l-malate, pH level, and the type of indicator dye used are discussed with regard to the kinetics of malic acid conversion to lactic acid. Use of these media allowed a rapid and easier screening of mutagenized streptococcal cells unable to ferment l-malate. A collection of malolactic-negative mutants of Streptococcus lactis induced by UV, nitrosoguanidine, or transposonal mutagenesis were characterized. The results showed that several mutants were apparently defective in the structural gene of malolactic enzyme, whereas others contained mutations which may either inactivate a putative permease or affect a regulatory sequence. PMID:16347282

Investigation of point and extended defects in electron irradiated silicon—Dependence on the particle energy

NASA Astrophysics Data System (ADS)

Radu, R.; Pintilie, I.; Nistor, L. C.; Fretwurst, E.; Lindstroem, G.; Makarenko, L. F.

2015-04-01

This work is focusing on generation, time evolution, and impact on the electrical performance of silicon diodes impaired by radiation induced active defects. n-type silicon diodes had been irradiated with electrons ranging from 1.5 MeV to 27 MeV. It is shown that the formation of small clusters starts already after irradiation with high fluence of 1.5 MeV electrons. An increase of the introduction rates of both point defects and small clusters with increasing energy is seen, showing saturation for electron energies above ˜15 MeV. The changes in the leakage current at low irradiation fluence-values proved to be determined by the change in the configuration of the tri-vacancy (V3). Similar to V3, other cluster related defects are showing bistability indicating that they might be associated with larger vacancy clusters. The change of the space charge density with irradiation and with annealing time after irradiation is fully described by accounting for the radiation induced trapping centers. High resolution electron microscopy investigations correlated with the annealing experiments revealed changes in the spatial structure of the defects. Furthermore, it is shown that while the generation of point defects is well described by the classical Non Ionizing Energy Loss (NIEL), the formation of small defect clusters is better described by the "effective NIEL" using results from molecular dynamics simulations.

Lithography-based automation in the design of program defect masks

NASA Astrophysics Data System (ADS)

Vakanas, George P.; Munir, Saghir; Tejnil, Edita; Bald, Daniel J.; Nagpal, Rajesh

2004-05-01

In this work, we are reporting on a lithography-based methodology and automation in the design of Program Defect masks (PDM"s). Leading edge technology masks have ever-shrinking primary features and more pronounced model-based secondary features such as optical proximity corrections (OPC), sub-resolution assist features (SRAF"s) and phase-shifted mask (PSM) structures. In order to define defect disposition specifications for critical layers of a technology node, experience alone in deciding worst-case scenarios for the placement of program defects is necessary but may not be sufficient. MEEF calculations initiated from layout pattern data and their integration in a PDM layout flow provide a natural approach for improvements, relevance and accuracy in the placement of programmed defects. This methodology provides closed-loop feedback between layout and hard defect disposition specifications, thereby minimizing engineering test restarts, improving quality and reducing cost of high-end masks. Apart from SEMI and industry standards, best-known methods (BKM"s) in integrated lithographically-based layout methodologies and automation specific to PDM"s are scarce. The contribution of this paper lies in the implementation of Design-For-Test (DFT) principles to a synergistic interaction of CAD Layout and Aerial Image Simulator to drive layout improvements, highlight layout-to-fracture interactions and output accurate program defect placement coordinates to be used by tools in the mask shop.

New hits as phase II enzymes inducers from a focused library with heteroatom-heteroatom and Michael-acceptor motives.

PubMed

Cabrera, Mauricio; de Ovalle, Stefani; Bollati-Fogolín, Mariela; Nascimento, Fabiana; Corbelini, Patrícia; Janarelli, Fernanda; Kawano, Daniel; Eifler-Lima, Vera Lucia; González, Mercedes; Cerecetto, Hugo

2015-11-01

The increased activity of phase-II-detoxification enzymes, such as quinone reductase (QR) and glutation S -transferase (GST), correlates with protection against chemically induced carcinogenesis. Herein we studied 11 different chemotypes, pyrazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiazole, 1,3,4-oxathiazole, thienyl hydrazone, α,β-unsaturated-oxime, α,β-unsaturated- N -oxide, coumarin and α,β-unsaturated-carbonyl, as phase-II enzymes inducers in order to identify new pharmacophores with chemopreventive activity. Fifty-four compounds were analyzed on wild-type mouse-hepatoma Hepa-1c1c7 and on the aryl-hydrocarbon-nuclear-translocator (Arnt)-defective mutant BpRc1 cells. New monofunctional inducers of QR and GST were identified, the 1,2,5-oxadiazol-2-oxide (3) , the 1,2,4-triazine-4-oxides (23) and (32) and the tetrahydropyrimidinones (28) and (49) . It was confirmed that Nrf2 nuclear translocation is the operative molecular mechanism that allows compound (3) to exert protective effects via expression of downstream phase-II enzymes.

Lipid-induced NOX2 activation inhibits autophagic flux by impairing lysosomal enzyme activity[S

PubMed Central

Jaishy, Bharat; Zhang, Quanjiang; Chung, Heaseung S.; Riehle, Christian; Soto, Jamie; Jenkins, Stephen; Abel, Patrick; Cowart, L. Ashley; Van Eyk, Jennifer E.; Abel, E. Dale

2015-01-01

Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCβII. These findings reveal a novel mechanism linking lipotoxicity with a PKCβ-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs. PMID:25529920

New hits as phase II enzymes inducers from a focused library with heteroatom–heteroatom and Michael-acceptor motives

PubMed Central

Cabrera, Mauricio; de Ovalle, Stefani; Bollati-Fogolín, Mariela; Nascimento, Fabiana; Corbelini, Patrícia; Janarelli, Fernanda; Kawano, Daniel; Eifler-Lima, Vera Lucia; González, Mercedes; Cerecetto, Hugo

2015-01-01

The increased activity of phase-II-detoxification enzymes, such as quinone reductase (QR) and glutation S-transferase (GST), correlates with protection against chemically induced carcinogenesis. Herein we studied 11 different chemotypes, pyrazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiazole, 1,3,4-oxathiazole, thienyl hydrazone, α,β-unsaturated-oxime, α,β-unsaturated-N-oxide, coumarin and α,β-unsaturated-carbonyl, as phase-II enzymes inducers in order to identify new pharmacophores with chemopreventive activity. Fifty-four compounds were analyzed on wild-type mouse-hepatoma Hepa-1c1c7 and on the aryl-hydrocarbon-nuclear-translocator (Arnt)-defective mutant BpRc1 cells. New monofunctional inducers of QR and GST were identified, the 1,2,5-oxadiazol-2-oxide (3), the 1,2,4-triazine-4-oxides (23) and (32) and the tetrahydropyrimidinones (28) and (49). It was confirmed that Nrf2 nuclear translocation is the operative molecular mechanism that allows compound (3) to exert protective effects via expression of downstream phase-II enzymes. PMID:28031894

Efficient creation of dipolar coupled nitrogen-vacancy spin qubits in diamond

NASA Astrophysics Data System (ADS)

Jakobi, I.; Momenzadeh, S. A.; Fávaro de Oliveira, F.; Michl, J.; Ziem, F.; Schreck, M.; Neumann, P.; Denisenko, A.; Wrachtrup, J.

2016-09-01

Coherently coupled pairs or multimers of nitrogen-vacancy defect electron spins in diamond have many promising applications especially in quantum information processing (QIP) but also in nanoscale sensing applications. Scalable registers of spin qubits are essential to the progress of QIP. Ion implantation is the only known technique able to produce defect pairs close enough to allow spin coupling via dipolar interaction. Although several competing methods have been proposed to increase the resulting resolution of ion implantation, the reliable creation of working registers is still to be demonstrated. The current limitation are residual radiation-induced defects, resulting in degraded qubit performance as trade-off for positioning accuracy. Here we present an optimized estimation of nanomask implantation parameters that are most likely to produce interacting qubits under standard conditions. We apply our findings to a well-established technique, namely masks written in electron-beam lithography, to create coupled defect pairs with a reasonable probability. Furthermore, we investigate the scaling behavior and necessary improvements to efficiently engineer interacting spin architectures.

Atomically resolved structural determination of graphene and its point defects via extrapolation assisted phase retrieval

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

Latychevskaia, Tatiana; Fink, Hans-Werner

Previously reported crystalline structures obtained by an iterative phase retrieval reconstruction of their diffraction patterns seem to be free from displaying any irregularities or defects in the lattice, which appears to be unrealistic. We demonstrate here that the structure of a nanocrystal including its atomic defects can unambiguously be recovered from its diffraction pattern alone by applying a direct phase retrieval procedure not relying on prior information of the object shape. Individual point defects in the atomic lattice are clearly apparent. Conventional phase retrieval routines assume isotropic scattering. We show that when dealing with electrons, the quantitatively correct transmission functionmore » of the sample cannot be retrieved due to anisotropic, strong forward scattering specific to electrons. We summarize the conditions for this phase retrieval method and show that the diffraction pattern can be extrapolated beyond the original record to even reveal formerly not visible Bragg peaks. Such extrapolated wave field pattern leads to enhanced spatial resolution in the reconstruction.« less

Quasiparticle Scattering in the Rashba Semiconductor BiTeBr: The Roles of Spin and Defect Lattice Site.

PubMed

Butler, Christopher John; Yang, Po-Ya; Sankar, Raman; Lien, Yen-Neng; Lu, Chun-I; Chang, Luo-Yueh; Chen, Chia-Hao; Wei, Ching-Ming; Chou, Fang-Cheng; Lin, Minn-Tsong

2016-09-28

Observations of quasiparticle interference have been used in recent years to examine exotic carrier behavior at the surfaces of emergent materials, connecting carrier dispersion and scattering dynamics to real-space features with atomic resolution. We observe quasiparticle interference in the strongly Rashba split 2DEG-like surface band found at the tellurium termination of BiTeBr and examine two mechanisms governing quasiparticle scattering: We confirm the suppression of spin-flip scattering by comparing measured quasiparticle interference with a spin-dependent elastic scattering model applied to the calculated spectral function. We also use atomically resolved STM maps to identify point defect lattice sites and spectro-microscopy imaging to discern their varying scattering strengths, which we understand in terms of the calculated orbital characteristics of the surface band. Defects on the Bi sublattice cause the strongest scattering of the predominantly Bi 6p derived surface band, with other defects causing nearly no scattering near the conduction band minimum.

Automated spot defect characterization in a field portable night vision goggle test set

NASA Astrophysics Data System (ADS)

Scopatz, Stephen; Ozten, Metehan; Aubry, Gilles; Arquetoux, Guillaume

2018-05-01

This paper discusses a new capability developed for and results from a field portable test set for Gen 2 and Gen 3 Image Intensifier (I2) tube-based Night Vision Goggles (NVG). A previous paper described the test set and the automated and semi-automated tests supported for NVGs including a Knife Edge MTF test to replace the operator's interpretation of the USAF 1951 resolution chart. The major improvement and innovation detailed in this paper is the use of image analysis algorithms to automate the characterization of spot defects of I² tubes with the same test set hardware previously presented. The original and still common Spot Defect Test requires the operator to look through the NVGs at target of concentric rings; compare the size of the defects to a chart and manually enter the results into a table based on the size and location of each defect; this is tedious and subjective. The prior semi-automated improvement captures and displays an image of the defects and the rings; allowing the operator determine the defects with less eyestrain; while electronically storing the image and the resulting table. The advanced Automated Spot Defect Test utilizes machine vision algorithms to determine the size and location of the defects, generates the result table automatically and then records the image and the results in a computer-generated report easily usable for verification. This is inherently a more repeatable process that ensures consistent spot detection independent of the operator. Results of across several NVGs will be presented.

Quantitative Proteome Analysis of Mouse Liver Lysosomes Provides Evidence for Mannose 6-phosphate-independent Targeting Mechanisms of Acid Hydrolases in Mucolipidosis II.

PubMed

Markmann, Sandra; Krambeck, Svenja; Hughes, Christopher J; Mirzaian, Mina; Aerts, Johannes M F G; Saftig, Paul; Schweizer, Michaela; Vissers, Johannes P C; Braulke, Thomas; Damme, Markus

2017-03-01

The efficient receptor-mediated targeting of soluble lysosomal proteins to lysosomes requires the modification with mannose 6-phosphate (M6P) residues. Although the absence of M6P results in misrouting and hypersecretion of lysosomal enzymes in many cells, normal levels of lysosomal enzymes have been reported in liver of patients lacking the M6P-generating phosphotransferase (PT). The identity of lysosomal proteins depending on M6P has not yet been comprehensively analyzed. In this study we purified lysosomes from liver of PT-defective mice and 67 known soluble lysosomal proteins were identified that illustrated quantitative changes using an ion mobility-assisted data-independent label-free LC-MS approach. After validation of various differentially expressed lysosomal components by Western blotting and enzyme activity assays, the data revealed a small number of lysosomal proteins depending on M6P, including neuraminidase 1, cathepsin F, Npc2, and cathepsin L, whereas the majority reach lysosomes by alternative pathways. These data were compared with findings on cultured hepatocytes and liver sinusoid endothelial cells isolated from the liver of wild-type and PT-defective mice. Our findings show that the relative expression, targeting efficiency and lysosomal localization of lysosomal proteins tested in cultured hepatic cells resemble their proportion in isolated liver lysosomes. Hypersecretion of newly synthesized nonphosphorylated lysosomal proteins suggest that secretion-recapture mechanisms contribute to maintain major lysosomal functions in liver. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

X-ray-induced mutation of Bacillus sp. MR10 for manno-oligosaccharides production from copra meal.

PubMed

Chaikaew, Siriporn; Kanpiengjai, Apinun; Intatep, Jenjira; Unban, Kridsada; Wongputtisin, Pairote; Takata, Goro; Khanongnuch, Chartchai

2017-04-21

The present study demonstrates the effectiveness of X-ray radiation in strain improvement for defective lipase production by Bacillus sp. MR10 for further application in the fermentative production of manno-oligosaccharides (MOS) from agricultural by-product, defatted copra meal (DCM). The mutants obtained were screened based on their defective lipase activity together with their β-mannanase production performance. Among 10 selected mutants, the strain M7 was the highest promising mutant regarding the smallest lipase activity (0.05 U/ml) and the retained β-mannanase activity similar to the parental strain (22 U/ml) were detected. The mutant M7 effectively hydrolyzed DCM to MOS with low-degree of polymerization (DP) oligomers including mannotriose (M3), mannotetraose (M4), and mannopentose (M5) as the main products. Although the pattern of DCM hydrolysis products of mutant M7 was distinctly different from wild type, the biochemical and catalytic properties of purified β-mannanase of mutant were similar to those of wild type. Both purified β-mannanases with apparent molecular mass of 38 kDa displayed optimal activity at pH 5-7 and 45-55°C. Co 2+ and Hg 2+ nearly completely inhibited activities of both enzymes, whereas Ba 2+ , Fe 3+ , and 2-mercaptoethanol obviously activated enzyme activities. Both enzymes showed high specificity for locust bean gum, konjac mannan, DCM, and guar gum. Thus, the mutant M7 has a potential for commercial production of high-quality MOS from low-cost DCM for further application in the feed industry.

Mutations in mitochondrial enzyme GPT2 cause metabolic dysfunction and neurological disease with developmental and progressive features

PubMed Central

Ouyang, Qing; Nakayama, Tojo; Baytas, Ozan; Davidson, Shawn M.; Yang, Chendong; Schmidt, Michael; Lizarraga, Sofia B.; Mishra, Sasmita; EI-Quessny, Malak; Niaz, Saima; Gul Butt, Mirrat; Imran Murtaza, Syed; Javed, Afzal; Chaudhry, Haroon Rashid; Vaughan, Dylan J.; Hill, R. Sean; Partlow, Jennifer N.; Yoo, Seung-Yun; Lam, Anh-Thu N.; Nasir, Ramzi; Al-Saffar, Muna; Barkovich, A. James; Schwede, Matthew; Nagpal, Shailender; Rajab, Anna; DeBerardinis, Ralph J.; Housman, David E.; Mochida, Ganeshwaran H.; Morrow, Eric M.

2016-01-01

Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and ∼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms. PMID:27601654

Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase.

PubMed

Beassoni, Paola R; Berti, Federico Pérez de; Otero, Lisandro H; Risso, Valeria A; Ferreyra, Raul G; Lisa, Angela T; Domenech, Carlos E; Ermácora, Mario R

2010-06-01

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.

Roles of Type 1A Topoisomerases in Genome Maintenance in Escherichia coli

PubMed Central

Usongo, Valentine; Drolet, Marc

2014-01-01

In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the E. coli topo I (topA) and III (topB) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from oriC (topo I alone) and R-loops and (ii) by acting with RecQ. PMID:25102178

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals.

PubMed

Sarker, Suprovath Kumar; Islam, Md Tarikul; Eckhoff, Grace; Hossain, Mohammad Amir; Qadri, Syeda Kashfi; Muraduzzaman, A K M; Bhuyan, Golam Sarower; Shahidullah, Mohammod; Mannan, Mohammad Abdul; Tahura, Sarabon; Hussain, Manzoor; Akhter, Shahida; Nahar, Nazmun; Shirin, Tahmina; Qadri, Firdausi; Mannoor, Kaiissar

2016-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh.

Molecular Analysis of Glucose-6-Phosphate Dehydrogenase Gene Mutations in Bangladeshi Individuals

PubMed Central

Sarker, Suprovath Kumar; Hossain, Mohammad Amir; Qadri, Syeda Kashfi; Muraduzzaman, A. K. M.; Bhuyan, Golam Sarower; Shahidullah, Mohammod; Mannan, Mohammad Abdul; Tahura, Sarabon; Hussain, Manzoor; Akhter, Shahida; Nahar, Nazmun; Shirin, Tahmina; Qadri, Firdausi; Mannoor, Kaiissar

2016-01-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X-linked human enzyme defect of red blood cells (RBCs). Individuals with this gene defect appear normal until exposed to oxidative stress which induces hemolysis. Consumption of certain foods such as fava beans, legumes; infection with bacteria or virus; and use of certain drugs such as primaquine, sulfa drugs etc. may result in lysis of RBCs in G6PD deficient individuals. The genetic defect that causes G6PD deficiency has been identified mostly as single base missense mutations. One hundred and sixty G6PD gene mutations, which lead to amino acid substitutions, have been described worldwide. The purpose of this study was to detect G6PD gene mutations in hospital-based settings in the local population of Dhaka city, Bangladesh. Qualitative fluorescent spot test and quantitative enzyme activity measurement using RANDOX G6PDH kit were performed for analysis of blood specimens and detection of G6PD-deficient participants. For G6PD-deficient samples, PCR was done with six sets of primers specific for G6PD gene. Automated Sanger sequencing of the PCR products was performed to identify the mutations in the gene. Based on fluorescence spot test and quantitative enzyme assay followed by G6PD gene sequencing, 12 specimens (11 males and one female) among 121 clinically suspected patient-specimens were found to be deficient, suggesting a frequency of 9.9% G6PD deficiency. Sequencing of the G6PD-deficient samples revealed c.C131G substitution (exon-3: Ala44Gly) in six samples, c.G487A substitution (exon-6:Gly163Ser) in five samples and c.G949A substitution (exon-9: Glu317Lys) of coding sequence in one sample. These mutations either affect NADP binding or disrupt protein structure. From the study it appears that Ala44Gly and Gly163Ser are the most common G6PD mutations in Dhaka, Bangladesh. This is the first study of G6PD mutations in Bangladesh. PMID:27880809

Observation of ‘hidden’ planar defects in boron carbide nanowires and identification of their orientations

PubMed Central

2014-01-01

The physical properties of nanostructures strongly depend on their structures, and planar defects in particular could significantly affect the behavior of the nanowires. In this work, planar defects (twins or stacking faults) in boron carbide nanowires are extensively studied by transmission electron microscopy (TEM). Results show that these defects can easily be invisible, i.e., no presence of characteristic defect features like modulated contrast in high-resolution TEM images and streaks in diffraction patterns. The simplified reason of this invisibility is that the viewing direction during TEM examination is not parallel to the (001)-type planar defects. Due to the unique rhombohedral structure of boron carbide, planar defects are only distinctive when the viewing direction is along the axial or short diagonal directions ([100], [010], or 1¯10) within the (001) plane (in-zone condition). However, in most cases, these three characteristic directions are not parallel to the viewing direction when boron carbide nanowires are randomly dispersed on TEM grids. To identify fault orientations (transverse faults or axial faults) of those nanowires whose planar defects are not revealed by TEM, a new approach is developed based on the geometrical analysis between the projected preferred growth direction of a nanowire and specific diffraction spots from diffraction patterns recorded along the axial or short diagonal directions out of the (001) plane (off-zone condition). The approach greatly alleviates tedious TEM examination of the nanowire and helps to establish the reliable structure–property relations. Our study calls attention to researchers to be extremely careful when studying nanowires with potential planar defects by TEM. Understanding the true nature of planar defects is essential in tuning the properties of these nanostructures through manipulating their structures. PMID:24423258

Observation of 'hidden' planar defects in boron carbide nanowires and identification of their orientations.

PubMed

Guan, Zhe; Cao, Baobao; Yang, Yang; Jiang, Youfei; Li, Deyu; Xu, Terry T

2014-01-15

The physical properties of nanostructures strongly depend on their structures, and planar defects in particular could significantly affect the behavior of the nanowires. In this work, planar defects (twins or stacking faults) in boron carbide nanowires are extensively studied by transmission electron microscopy (TEM). Results show that these defects can easily be invisible, i.e., no presence of characteristic defect features like modulated contrast in high-resolution TEM images and streaks in diffraction patterns. The simplified reason of this invisibility is that the viewing direction during TEM examination is not parallel to the (001)-type planar defects. Due to the unique rhombohedral structure of boron carbide, planar defects are only distinctive when the viewing direction is along the axial or short diagonal directions ([100], [010], or 1¯10) within the (001) plane (in-zone condition). However, in most cases, these three characteristic directions are not parallel to the viewing direction when boron carbide nanowires are randomly dispersed on TEM grids. To identify fault orientations (transverse faults or axial faults) of those nanowires whose planar defects are not revealed by TEM, a new approach is developed based on the geometrical analysis between the projected preferred growth direction of a nanowire and specific diffraction spots from diffraction patterns recorded along the axial or short diagonal directions out of the (001) plane (off-zone condition). The approach greatly alleviates tedious TEM examination of the nanowire and helps to establish the reliable structure-property relations. Our study calls attention to researchers to be extremely careful when studying nanowires with potential planar defects by TEM. Understanding the true nature of planar defects is essential in tuning the properties of these nanostructures through manipulating their structures.

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis*

PubMed Central

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M.

2010-01-01

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed. PMID:20382731

Single-molecule force spectroscopy approach to enzyme catalysis.

PubMed

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M

2010-06-18

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.

Monomer structure of a hyperthermophilic β-glucosidase mutant forming a dodecameric structure in the crystal form

PubMed Central

Nakabayashi, Makoto; Kataoka, Misumi; Watanabe, Masahiro; Ishikawa, Kazuhiko

2014-01-01

One of the β-glucosidases from Pyrococcus furiosus (BGLPf) is found to be a hyperthermophilic tetrameric enzyme that can degrade cellooligosaccharides. Recently, the crystal structures of the tetrameric and dimeric forms were solved. Here, a new monomeric form of BGLPf was constructed by removing the C-terminal region of the enzyme and its crystal structure was solved at a resolution of 2.8 Å in space group P1. It was discovered that the mutant enzyme forms a unique dodecameric structure consisting of two hexameric rings in the asymmetric unit of the crystal. Under biological conditions, the mutant enzyme forms a monomer. This result helps explain how BGLPf has attained its oligomeric structure and thermostability. PMID:25005077

Rational protein engineering in action: The first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

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

Evdokimov, Artem G.; Mekel, Marlene; Hutchings, Kim

2008-07-08

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality -- they only diffracted X-rays to 3--5 {angstrom} resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 {angstrom} or better. This methodology is discussed andmore » contrasted with the more traditional domain truncation approach.« less

Characterization technique for detection of atom-size crystalline defects and strains using two-dimensional fast-Fourier-transform sampling Moiré method

NASA Astrophysics Data System (ADS)

Kodera, Masako; Wang, Qinghua; Ri, Shien; Tsuda, Hiroshi; Yoshioka, Akira; Sugiyama, Toru; Hamamoto, Takeshi; Miyashita, Naoto

2018-04-01

Recently, we have developed a two-dimensional (2D) fast-Fourier-transform (FFT) sampling Moiré technique to visually and quantitatively determine the locations of minute defects in a transmission electron microscopy (TEM) image. We applied this technique for defect detection with GaN high electron mobility transistor (HEMT) devices, and successfully and clearly visualized atom-size defects in AlGaN/GaN crystalline structures. The defect density obtained in the AlGaN/GaN structures is ∼1013 counts/cm2. In addition, we have successfully confirmed that the distribution and number of defects closely depend on the process conditions. Thus, this technique is quite useful for a device development. Moreover, the strain fields in an AlGaN/GaN crystal were effectively calculated with nm-scale resolution using this method. We also demonstrated that this sampling Moiré technique is applicable to silicon devices, which have principal directions different from those of AlGaN/GaN crystals. As a result, we believe that the 2D FFT sampling Moiré method has great potential applications to the discovery of new as yet unknown phenomena occurring between the characteristics of a crystalline material and device performance.

Growth of defect-free GaAsSbN axial nanowires via self-catalyzed molecular beam epitaxy

NASA Astrophysics Data System (ADS)

Sharma, Manish; Deshmukh, Prithviraj; Kasanaboina, Pavan; Reynolds, C. Lewis, Jr.; Liu, Yang; Iyer, Shanthi

2017-12-01

Bandgap reduction of 10% by incorporation of a dilute amount of N is reported for the first time, in axial GaAsSb nanowires (NWs) grown on Si (111) via Ga-assisted molecular beam epitaxy. Impact of N incorporation on the surface morphology, NW growth kinetics, and their structural and optical properties were examined. Dilute nitride NWs with Sb composition of 7 at% did not exhibit any noticeable planar defects, as revealed by the absence of satellite twin peaks in the selected-area diffraction pattern and high-resolution transmission electron microscopy imaging. Point defects were also minimal in as-grown dilute nitride NWs, as ascertained from the comparison of low-temperature photoluminescence spectra as well as the shape and shift of Raman modes, with in situ annealed NWs in different ambients. Evidence of enhanced incorporation of N was found in the NWs in situ annealed in N ambient, but with deteriorated optical quality due to simultaneous creation of N-induced defects. The lack of any noticeable defects in the as-grown GaAsSbN NWs demonstrates the advantage of the vapor-liquid-solid mechanism responsible for growth of axial configuration over the vapor-solid growth mechanism for core-shell NWs as well as their thin film counterpart, which commonly exhibit N-induced point defects.

Topological defects in electric double layers of ionic liquids at carbon interfaces

DOE PAGES

Black, Jennifer M.; Okatan, Mahmut Baris; Feng, Guang; ...

2015-06-07

The structure and properties of the electrical double layer in ionic liquids is of interest in a wide range of areas including energy storage, catalysis, lubrication, and many more. Theories describing the electrical double layer for ionic liquids have been proposed, however a full molecular level description of the double layer is lacking. To date, studies have been predominantly focused on ion distributions normal to the surface, however the 3D nature of the electrical double layer in ionic liquids requires a full picture of the double layer structure not only normal to the surface, but also in plane. Here wemore » utilize 3D force mapping to probe the in plane structure of an ionic liquid at a graphite interface and report the direct observation of the structure and properties of topological defects. The observation of ion layering at structural defects such as step-edges, reinforced by molecular dynamics simulations, defines the spatial resolution of the method. Observation of defects allows for the establishment of the universality of ionic liquid behavior vs. separation from the carbon surface and to map internal defect structure. In conclusion, these studies offer a universal pathway for probing the internal structure of topological defects in soft condensed matter on the nanometer level in three dimensions.« less

Lattice disorder produced in GaN by He-ion implantation

NASA Astrophysics Data System (ADS)

Han, Yi; Peng, Jinxin; Li, Bingsheng; Wang, Zhiguang; Wei, Kongfang; Shen, Tielong; Sun, Jianrong; Zhang, Limin; Yao, Cunfeng; Gao, Ning; Gao, Xing; Pang, Lilong; Zhu, Yabin; Chang, Hailong; Cui, Minghuan; Luo, Peng; Sheng, Yanbin; Zhang, Hongpeng; Zhang, Li; Fang, Xuesong; Zhao, Sixiang; Jin, Jin; Huang, Yuxuan; Liu, Chao; Tai, Pengfei; Wang, Dong; He, Wenhao

2017-09-01

The lattice disorders induced by He-ion implantation in GaN epitaxial films to fluences of 2 × 1016, 5 × 1016 and 1 × 1017 cm-2 at room temperature (RT) have been investigated by a combination of Raman spectroscopy, high-resolution X-ray diffraction (HRXRD), nano-indentation, and transmission electron microscopy (TEM). The experimental results present that Raman intensity decreases with increasing fluence. Raman frequency "red shift" occurs after He-ion implantation. Strain increases with increasing fluence. The hardness of the highly damaged layer increases monotonically with increasing fluence. Microstructural results demonstrate that the width of the damage band and the number density of observed dislocation loops increases with increasing fluence. High-resolution TEM images exhibit that He-ion implantation lead to the formation of planar defects and most of the lattice defects are of interstitial-type basal loops. The relationships of Raman intensity, lattice strain, swelling and hardness with He-implantation-induced lattice disorders are discussed.

In situ mitigation of subsurface and peripheral focused ion beam damage via simultaneous pulsed laser heating

DOE PAGES

Stanford, Michael G.; Lewis, Brett B.; Iberi, Vighter O.; ...

2016-02-16

Focused helium and neon ion (He(+)/Ne(+) ) beam processing has recently been used to push resolution limits of direct-write nanoscale synthesis. The ubiquitous insertion of focused He(+) /Ne(+) beams as the next-generation nanofabrication tool-of-choice is currently limited by deleterious subsurface and peripheral damage induced by the energetic ions in the underlying substrate. The in situ mitigation of subsurface damage induced by He(+)/Ne(+) ion exposures in silicon via a synchronized infrared pulsed laser-assisted process is demonstrated. The pulsed laser assist provides highly localized in situ photothermal energy which reduces the implantation and defect concentration by greater than 90%. The laser-assisted exposuremore » process is also shown to reduce peripheral defects in He(+) patterned graphene, which makes this process an attractive candidate for direct-write patterning of 2D materials. In conclusion, these results offer a necessary solution for the applicability of high-resolution direct-write nanoscale material processing via focused ion beams.« less

An Improved Unsupervised Image Segmentation Evaluation Approach Based on - and Over-Segmentation Aware

NASA Astrophysics Data System (ADS)

Su, Tengfei

2018-04-01

In this paper, an unsupervised evaluation scheme for remote sensing image segmentation is developed. Based on a method called under- and over-segmentation aware (UOA), the new approach is improved by overcoming the defect in the part of estimating over-segmentation error. Two cases of such error-prone defect are listed, and edge strength is employed to devise a solution to this issue. Two subsets of high resolution remote sensing images were used to test the proposed algorithm, and the experimental results indicate its superior performance, which is attributed to its improved OSE detection model.

Uranium Hydride Nucleation and Growth Model FY'16 ESC Annual Report

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

Hill, Mary Ann; Richards, Andrew Walter; Holby, Edward F.

2016-12-20

Uranium hydride corrosion is of great interest to the nuclear industry. Uranium reacts with water and/or hydrogen to form uranium hydride which adversely affects material performance. Hydride nucleation is influenced by thermal history, mechanical defects, oxide thickness, and chemical defects. Information has been gathered from past hydride experiments to formulate a uranium hydride model to be used in a Canned Subassembly (CSA) lifetime prediction model. This multi-scale computer modeling effort started in FY’13, and the fourth generation model is now complete. Additional high-resolution experiments will be run to further test the model.

Detection of 6-demethoxyubiquinone in CoQ10 deficiency disorders: Insights into enzyme interactions and identification of potential therapeutics.

PubMed

Herebian, Diran; Seibt, Annette; Smits, Sander H J; Bünning, Gisela; Freyer, Christoph; Prokisch, Holger; Karall, Daniela; Wredenberg, Anna; Wedell, Anna; López, Luis C; Mayatepek, Ertan; Distelmaier, Felix

2017-07-01

Coenzyme Q 10 (CoQ 10 ) is an essential cofactor of the mitochondrial oxidative phosphorylation (OXPHOS) system and its deficiency has important implications for several inherited metabolic disorders of childhood. The biosynthesis of CoQ 10 is a complicated process, which involves at least 12 different enzymes. One of the metabolic intermediates that are formed during CoQ 10 biosynthesis is the molecule 6-demethoxyubiquinone (6-DMQ). This CoQ precursor is processed at the level of COQ7 and COQ9. We selected this metabolite as a marker substance for metabolic analysis of cell lines with inherited genetic defects (COQ2, COQ4, COQ7 and COQ9) or siRNA knockdown in CoQ biosynthesis enzymes using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). In COQ4, COQ7 and COQ9 deficient cell lines, we detected significantly elevated levels of 6-DMQ. This suggests a functional interplay of these proteins. However, additional siRNA studies demonstrated that elevated 6-DMQ levels are not an exclusive marker of the COQ7/COQ9 enzymatic step of CoQ 10 biosynthesis but constitute a more general phenomenon that occurs in disorders impairing the function or stability of the CoQ-synthome. To further investigate the interdependence of CoQ 10 biosynthesis enzyme expression, we performed immunoblotting in various cell lines with CoQ 10 deficiency, indicating that COQ4, COQ7 and COQ9 protein expression levels are highly regulated depending on the underlying defect. Supplementation of cell lines with synthetic CoQ precursor compounds demonstrated beneficial effects of 2,4-dihydroxybenzoic acid in COQ7 and COQ9 deficiency. Moreover, vanillic acid selectively stimulated CoQ 10 biosynthesis and improved cell viability in COQ9 deficiency. However, compounds tested in this study failed to rescue COQ4 deficiency. Copyright © 2017 Elsevier Inc. All rights reserved.

Fabrication Quality Analysis of a Fiber Optic Refractive Index Sensor Created by CO2 Laser Machining

PubMed Central

Chen, Chien-Hsing; Yeh, Bo-Kuan; Tang, Jaw-Luen; Wu, Wei-Te

2013-01-01

This study investigates the CO2 laser-stripped partial cladding of silica-based optic fibers with a core diameter of 400 μm, which enables them to sense the refractive index of the surrounding environment. However, inappropriate treatments during the machining process can generate a number of defects in the optic fiber sensors. Therefore, the quality of optic fiber sensors fabricated using CO2 laser machining must be analyzed. The results show that analysis of the fiber core size after machining can provide preliminary defect detection, and qualitative analysis of the optical transmission defects can be used to identify imperfections that are difficult to observe through size analysis. To more precisely and quantitatively detect fabrication defects, we included a tensile test and numerical aperture measurements in this study. After a series of quality inspections, we proposed improvements to the existing CO2 laser machining parameters, namely, a vertical scanning pathway, 4 W of power, and a feed rate of 9.45 cm/s. Using these improved parameters, we created optical fiber sensors with a core diameter of approximately 400 μm, no obvious optical transmission defects, a numerical aperture of 0.52 ± 0.019, a 0.886 Weibull modulus, and a 1.186 Weibull-shaped parameter. Finally, we used the optical fiber sensor fabricated using the improved parameters to measure the refractive indices of various solutions. The results show that a refractive-index resolution of 1.8 × 10−4 RIU (linear fitting R2 = 0.954) was achieved for sucrose solutions with refractive indices ranging between 1.333 and 1.383. We also adopted the particle plasmon resonance sensing scheme using the fabricated optical fibers. The results provided additional information, specifically, a superior sensor resolution of 5.73 × 10−5 RIU, and greater linearity at R2 = 0.999. PMID:23535636

Enzymatic Resolution of 1-Phenylethanol and Formation of a Diastereomer: An Undergraduate [superscript 1]H NMR Experiment to Introduce Chiral Chemistry

ERIC Educational Resources Information Center

Faraldos, Juan A.; Giner, Jos-Luis; Smith, David H.; Wilson, Mark; Ronhovde, Kyla; Wilson, Erin; Clevette, David; Holmes, Andrea E.; Rouhier, Kerry

2011-01-01

This organic laboratory experiment introduces students to stereoselective enzyme reactions, resolution of enantiomers, and NMR analysis of diastereomers. The reaction between racemic 1-phenylethanol and vinyl acetate in hexane to form an ester is catalyzed by acylase I. The unreacted alcohol is then treated with a chiral acid and the resulting…

An Evanescent Microwave Probe for Super-Resolution Nondestructive Imaging of Metals, Semiconductors, Dielectrics, Composites and Biological Specimens

NASA Technical Reports Server (NTRS)

Pathak, P. S.; Tabib-Azar, M.; Ponchak, G.

1998-01-01

Using evanescent microwaves with decay lengths determined by a combination of microwave wavelength (lambda) and waveguide termination geometry, we have imaged and mapped material non-uniformities and defects with a resolving capability of lambda/3800=79 microns at 1 GHz. In our method a microstrip quarter wavelength resonator was used to generate evanescent microwaves. We imaged materials with a wide range of conductivities. Carbon composites, dielectrics (Duroid, polymers), semiconductors (3C-SiC, polysilicon, natural diamond), metals (tungsten alloys, copper, zinc, steel), high-temperature superconductors, and botanical samples were scanned for defects, residual stresses, integrity of brazed junctions, subsurface features, areas of different film thickness and moisture content. The evanescent microwave probe is a versatile tool and it can be used to perform very fast, large scale mapping of a wide range of materials. This method of characterization compares favorably with ultrasound testing, which has a resolution of about 0.1 mm and suffers from high absorption in composite materials and poor transmission across boundaries. Eddy current methods which can have a resolution on the order of 50 microns are restricted to evaluating conducting materials. Evanescent microwave imaging, with careful choice of operating frequency and probe geometry, can have a resolution of up to 1 micron. In this method we can scan hot and moving objects, sample preparation is not required, testing is non-destructive, non-invasive and non-contact, and can be done in air, in liquid or in vacuum.

Electrical characterisation of defects in wide bandgap semiconductors

NASA Astrophysics Data System (ADS)

Elsherif, Osama S.

Defects usually have a very large influence on the semiconductor material properties and hence on fabricated electronic devices. The nature and properties of defects in semiconducting materials can be investigated by applying electrical characterization techniques such as thermal admittance spectroscopy (TAS), deep level transient spectroscopy (DLTS) and high resolution Laplace-DLTS measurements. This dissertation presents the electrical characterisation of two different wide bandgap semiconducting materials (polycrystalline diamond and GaN) which have both recently attracted a great deal of attention because of their potential applications in the fields of power electronics and optoelectronics. Raman spectroscopy, I-V and C-V measurements were carried out as supporting experiments for the above investigations. The first part of this work focuses on studying the effect of B concentration on the electronic states in polycrystalline diamond thin films grown on silicon by the hot filament chemical vapour deposition method. A combination of high-resolution LDLTS and direct-capture cross-section measurements was used to investigate whether the deep electronic states present in the layers originated from point or extended defects. There was good agreement between data on deep electronic levels obtained from DLTS and TAS experiments. A number of hole traps have been detected; the majority of these levels show an unusual dependence of the DLTS signal on the fill pulse duration which is interpreted as possibly the levels are part of extended defects within the grain boundaries. In contrast, a defect level found in a more highly doped film, with an activation energy of -0.37 eV, exhibited behaviour characteristic of an isolated point defect, which we attribute to B-related centres in the bulk diamond, away from the dislocations. The second part of this thesis presents electrical measurements carried out at temperatures up to 450 K in order to study the electronic states associated with Mg in Mg-doped GaN films grown on sapphire by metalorganic vapour phase epitaxy, and to determine how these are affected by the threading dislocation density (TDD). Two different buffer layer schemes between the film and the sapphire substrate were used, giving rise to different TDDs in the GaN. Admittance spectroscopy of the films finds a single impurity-related acceptor level. It is observed in theses experiments that admittance spectroscopy detects no traps that can be attributed to extended defects, despite the fact that the dislocations are well-known to be active recombination centres. This unexpected finding is discussed in detail.

Electrical characterisation of defects in wide bandgap semiconductors

NASA Astrophysics Data System (ADS)

Elsherif, Osama S.

Defects usually have a very large influence on the semiconductor material properties and hence on fabricated electronic devices. The nature and properties of defects in semiconducting materials can be investigated by applying electrical characterization techniques such as thermal admittance spectroscopy (TAS), deep level transient spectroscopy (DLTS) and high resolution Laplace-DLTS measurements. This dissertation presents the electrical characterisation of two different wide bandgap semiconducting materials (polycrystalline diamond and GaN) which have both recently attracted a great deal of attention because of their potential applications in the fields of power electronics and optoelectronics. Raman spectroscopy, I-V and C-V measurements were carried out as supporting experiments for the above investigations.The first part of this work focuses on studying the effect of B concentration on the electronic states in polycrystalline diamond thin films grown on silicon by the hot filament chemical vapour deposition method. A combination of high-resolution LDLTS and direct-capture cross-section measurements was used to investigate whether the deep electronic states present in the layers originated from point or extended defects. There was good agreement between data on deep electronic levels obtained from DLTS and TAS experiments. A number of hole traps have been detected; the majority of these levels show an unusual dependence of the DLTS signal on the fill pulse duration which is interpreted as possibly the levels are part of extended defects within the grain boundaries. In contrast, a defect level found in a more highly doped film, with an activation energy of -0.37 eV, exhibited behaviour characteristic of an isolated point defect, which we attribute to B-related centres in the bulk diamond, away from the dislocations.The second part of this thesis presents electrical measurements carried out at temperatures up to 450 K in order to study the electronic states associated with Mg in Mg-doped GaN films grown on sapphire by metalorganic vapour phase epitaxy, and to determine how these are affected by the threading dislocation density (TDD). Two different buffer layer schemes between the film and the sapphire substrate were used, giving rise to different TDDs in the GaN. Admittance spectroscopy of the films finds a single impurity-related acceptor level. It is observed in theses experiments that admittance spectroscopy detects no traps that can be attributed to extended defects, despite the fact that the dislocations are well-known to be active recombination centres. This unexpected finding is discussed in detail.

High-Resolution X-Ray Structures of Two Functionally Distinct Members of the Cyclic Amide Hydrolase Family of Toblerone Fold Enzymes

PubMed Central

Peat, Thomas S.; Balotra, Sahil; Wilding, Matthew; Hartley, Carol J.; Newman, Janet

2017-01-01

ABSTRACT The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+ or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity. IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences. PMID:28235873

Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal.

PubMed

Horrell, Sam; Antonyuk, Svetlana V; Eady, Robert R; Hasnain, S Samar; Hough, Michael A; Strange, Richard W

2016-07-01

Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07-1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a 'catalytic reaction movie' highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.

High-Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into the Catalytic Mechanism and Inhibition by Aldehydes

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

French, Jarrod B.; Cen, Yana; Sauve, Anthony A.

2010-11-11

Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD{sup +} in most prokaryotes and most single-cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD{sup +} homeostasis has stimulated interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD{sup +}-consuming enzymes, such as the NAD{sup +}-dependent deacetylases (sirtuins). Here, we report several high-resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. Themore » structure of the C136S mutant in complex with nicotinamide provides details about substrate binding, while a trapped nicotinoyl thioester in a complex with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features, including a metal ion that coordinates the substrate and the catalytically relevant water molecule and an oxyanion hole that both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence of several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.« less

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner*

PubMed Central

Gagnon, Susannah M. L.; Meloncelli, Peter J.; Zheng, Ruixiang B.; Haji-Ghassemi, Omid; Johal, Asha R.; Borisova, Svetlana N.; Lowary, Todd L.; Evans, Stephen V.

2015-01-01

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the “tucked under” conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB. PMID:26374898

Longitudinal in vivo evaluation of bone regeneration by combined measurement of multi-pinhole SPECT and micro-CT for tissue engineering

NASA Astrophysics Data System (ADS)

Lienemann, Philipp S.; Metzger, Stéphanie; Kiveliö, Anna-Sofia; Blanc, Alain; Papageorgiou, Panagiota; Astolfo, Alberto; Pinzer, Bernd R.; Cinelli, Paolo; Weber, Franz E.; Schibli, Roger; Béhé, Martin; Ehrbar, Martin

2015-05-01

Over the last decades, great strides were made in the development of novel implants for the treatment of bone defects. The increasing versatility and complexity of these implant designs request for concurrent advances in means to assess in vivo the course of induced bone formation in preclinical models. Since its discovery, micro-computed tomography (micro-CT) has excelled as powerful high-resolution technique for non-invasive assessment of newly formed bone tissue. However, micro-CT fails to provide spatiotemporal information on biological processes ongoing during bone regeneration. Conversely, due to the versatile applicability and cost-effectiveness, single photon emission computed tomography (SPECT) would be an ideal technique for assessing such biological processes with high sensitivity and for nuclear imaging comparably high resolution (<1 mm). Herein, we employ modular designed poly(ethylene glycol)-based hydrogels that release bone morphogenetic protein to guide the healing of critical sized calvarial bone defects. By combined in vivo longitudinal multi-pinhole SPECT and micro-CT evaluations we determine the spatiotemporal course of bone formation and remodeling within this synthetic hydrogel implant. End point evaluations by high resolution micro-CT and histological evaluation confirm the value of this approach to follow and optimize bone-inducing biomaterials.

Spatial Resolution Versus Data Acquisition Efficiency in Mapping an Inhomogeneous System with Species Diffusion

DOE PAGES

Chen, Fengxiang; Zhang, Yong; Gfroerer, T. H.; ...

2015-06-02

Traditionally, spatially-resolved photoluminescence (PL) has been performed using a point-by-point scan mode with both excitation and detection occurring at the same spatial location. But with the availability of high quality detector arrays like CCDs, an imaging mode has become popular for performing spatially-resolved PL. By illuminating the entire area of interest and collecting the data simultaneously from all spatial locations, the measurement efficiency can be greatly improved. However, this new approach has proceeded under the implicit assumption of comparable spatial resolution. We show here that when carrier diffusion is present, the spatial resolution can actually differ substantially between the twomore » modes, with the less efficient scan mode being far superior. We apply both techniques in investigation of defects in a GaAs epilayer – where isolated singlet and doublet dislocations can be identified. A superposition principle is developed for solving the diffusion equation to extract the intrinsic carrier diffusion length, which can be applied to a system with arbitrarily distributed defects. The understanding derived from this work is significant for a broad range of problems in physics and beyond (for instance biology) – whenever the dynamics of generation, diffusion, and annihilation of species can be probed with either measurement mode.« less

Bragg coherent diffractive imaging of single-grain defect dynamics in polycrystalline films

NASA Astrophysics Data System (ADS)

Yau, Allison; Cha, Wonsuk; Kanan, Matthew W.; Stephenson, G. Brian; Ulvestad, Andrew

2017-05-01

Polycrystalline material properties depend on the distribution and interactions of their crystalline grains. In particular, grain boundaries and defects are crucial in determining their response to external stimuli. A long-standing challenge is thus to observe individual grains, defects, and strain dynamics inside functional materials. Here we report a technique capable of revealing grain heterogeneity, including strain fields and individual dislocations, that can be used under operando conditions in reactive environments: grain Bragg coherent diffractive imaging (gBCDI). Using a polycrystalline gold thin film subjected to heating, we show how gBCDI resolves grain boundary and dislocation dynamics in individual grains in three-dimensional detail with 10-nanometer spatial and subangstrom displacement field resolution. These results pave the way for understanding polycrystalline material response under external stimuli and, ideally, engineering particular functions.

Determination of atomic vacancies in InAs/GaSb strained-layer superlattices by atomic strain

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

Kim, Honggyu; Meng, Yifei; Kwon, Ji-Hwan

Determining vacancy in complex crystals or nanostructures represents an outstanding crystallographic problem that has a large impact on technology, especially for semiconductors, where vacancies introduce defect levels and modify the electronic structure. However, vacancy is hard to locate and its structure is difficult to probe experimentally. Reported here are atomic vacancies in the InAs/GaSb strained-layer superlattice (SLS) determined by atomic-resolution strain mapping at picometre precision. It is shown that cation and anion vacancies in the InAs/GaSb SLS give rise to local lattice relaxations, especially the nearest atoms, which can be detected using a statistical method and confirmed by simulation. Themore » ability to map vacancy defect-induced strain and identify its location represents significant progress in the study of vacancy defects in compound semiconductors.« less

Diffraction and Imaging Study of Imperfections of Protein Crystals with Coherent X-rays

NASA Technical Reports Server (NTRS)

Hu, Z. W.; Thomas, B. R.; Chernov, A. A.; Chu, Y. S.; Lai, B.

2004-01-01

High angular-resolution x-ray diffraction and phase contrast x-ray imaging were combined to study defects and perfection of protein crystals. Imperfections including line defects, inclusions and other microdefects were observed in the diffraction images of a uniformly grown lysozyme crystal. The observed line defects carry distinct dislocation features running approximately along the <110> growth front and have been found to originate mostly in a central growth area and occasionally in outer growth regions. Slow dehydration led to the broadening of a fairly symmetric 4 4 0 rocking curve by a factor of approximately 2.6, which was primarily attributed to the dehydration-induced microscopic effects that are clearly shown in diffraction images. X-ray imaging and diffraction characterization of the quality of apoferritin crystals will also be discussed in the presentation.

Determination of atomic vacancies in InAs/GaSb strained-layer superlattices by atomic strain

DOE PAGES

Kim, Honggyu; Meng, Yifei; Kwon, Ji-Hwan; ...

2018-01-01

Determining vacancy in complex crystals or nanostructures represents an outstanding crystallographic problem that has a large impact on technology, especially for semiconductors, where vacancies introduce defect levels and modify the electronic structure. However, vacancy is hard to locate and its structure is difficult to probe experimentally. Reported here are atomic vacancies in the InAs/GaSb strained-layer superlattice (SLS) determined by atomic-resolution strain mapping at picometre precision. It is shown that cation and anion vacancies in the InAs/GaSb SLS give rise to local lattice relaxations, especially the nearest atoms, which can be detected using a statistical method and confirmed by simulation. Themore » ability to map vacancy defect-induced strain and identify its location represents significant progress in the study of vacancy defects in compound semiconductors.« less

Bragg coherent diffractive imaging of single-grain defect dynamics in polycrystalline films

DOE PAGES

Yau, Allison; Cha, Wonsuk; Kanan, Matthew W.; ...

2017-05-19

Polycrystalline material properties depend on the distribution and interactions of their crystalline grains. In particular, grain boundaries and defects are crucial in determining their response to external stimuli. A long-standing challenge is thus to observe individual grains, defects, and strain dynamics inside functional materials. Here we report a technique capable of revealing grain heterogeneity, including strain fields and individual dislocations, that can be used under operando conditions in reactive environments: grain Bragg coherent diffractive imaging (gBCDI). Using a polycrystalline gold thin film subjected to heating, we show how gBCDI resolves grain boundary and dislocation dynamics in individual grains in three-dimensionalmore » detail with 10-nanometer spatial and subangstrom displacement field resolution. Finally, these results pave the way for understanding polycrystalline material response under external stimuli and, ideally, engineering particular functions.« less

Imaging charge carriers in potential-induced degradation defects of c-Si solar cells by scanning capacitance microscopy

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

Jiang, C. -S.; Xiao, C.; Moutinho, H. R.

We report on nm-resolution imaging of charge-carrier distribution around local potential-induced degradation (PID) shunting defects using scanning capacitance microscopy. We imaged on cross sections of heavily field-degraded module areas, cored out and selected by mm-scale photoluminescence imaging. We found localized areas with abnormal carrier behavior induced by the PID defects: the apparent n-type carrier extends vertically into the absorber to ~1-2 um from the cell surface, and laterally in similar lengths; in defect-free areas, the n-type carrier extends ~0.5 um, which is consistent with the junction depth. For comparison, we also investigated areas of the same module exhibiting the leastmore » PID stress, and we found no such heavily damaged junction area. Instead, we found slightly abnormal carrier behavior, where the carrier-type inversion in the absorber did not occur, but the p-type carrier concentration changed slightly in a much smaller lateral length of ~300 nm. These nano-electrical findings may indicate a possible mechanism that the existing extended defects, which may not be significantly harmful to cell performance, can be changed by PID to heavily damaged junction areas.« less

Imaging charge carriers in potential-induced degradation defects of c-Si solar cells by scanning capacitance microscopy

DOE PAGES

Jiang, C. -S.; Xiao, C.; Moutinho, H. R.; ...

2018-02-13

We report on nm-resolution imaging of charge-carrier distribution around local potential-induced degradation (PID) shunting defects using scanning capacitance microscopy. We imaged on cross sections of heavily field-degraded module areas, cored out and selected by mm-scale photoluminescence imaging. We found localized areas with abnormal carrier behavior induced by the PID defects: the apparent n-type carrier extends vertically into the absorber to ~1-2 um from the cell surface, and laterally in similar lengths; in defect-free areas, the n-type carrier extends ~0.5 um, which is consistent with the junction depth. For comparison, we also investigated areas of the same module exhibiting the leastmore » PID stress, and we found no such heavily damaged junction area. Instead, we found slightly abnormal carrier behavior, where the carrier-type inversion in the absorber did not occur, but the p-type carrier concentration changed slightly in a much smaller lateral length of ~300 nm. These nano-electrical findings may indicate a possible mechanism that the existing extended defects, which may not be significantly harmful to cell performance, can be changed by PID to heavily damaged junction areas.« less

Pseudotumor of the distal common bile duct at endoscopic retrograde cholangiopancreatography

PubMed Central

Tan, Justin H.; Coakley, Fergus V; Wang, Zhen J.; Poder, Liina; Webb, Emily; Yeh, Benjamin M.

2010-01-01

Background Prior studies have described a pseudocalculus appearance in the distal common bile duct as a normal variant at cholangiography. The objective of this study is to describe the occurrence of pseudotumor in the distal common bile duct at endoscopic retrograde cholangiopancreatography (ERCP). Methods Nine patients who underwent ERCP between May 2004 and July 2008 were identified as having a transient eccentric mural-based filling defect in the distal common bile duct. A single reader systematically reviewed all studies and recorded the imaging findings. Results The mean diameter of the filling defect was 9 mm (range, 5 to 11). Eight patients had resolution of the filling defect during the same ERCP or on a subsequent ERCP, and in 2 of these patients the inferior border of the filling defect was not well visualized. The other patient underwent surgical resection of a presumed tumor with no evidence of malignancy on surgical pathology. Conclusion An eccentric mural-based filling defect in the distal common bile duct can be artifactual in nature and may reflect transient contraction of the sphincter of Oddi. Recognition of this pseudotumor may help avoid unnecessary surgery. PMID:21724120

Racemic resolution of some DL-amino acids using Aspergillus fumigatus L-amino acid oxidase.

PubMed

Singh, Susmita; Gogoi, Binod K; Bezbaruah, Rajib L

2011-07-01

The ability of Aspergillus fumigatus L-amino acid oxidase (L-aao) to cause the resolution of racemic mixtures of DL-amino acids was investigated with DL-alanine, DL-phenylalanine, DL-tyrosine, and DL-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three DL-amino acids resulting in the production of optically pure D-alanine (100% resolution), D-phenylalanine (80.2%), and D-tyrosine (84.1%), respectively. The optically pure D-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus L: -amino acid oxidase for racemic resolution of DL-amino acids.

Concept for room temperature single-spin tunneling force microscopy with atomic spatial resolution

NASA Astrophysics Data System (ADS)

Payne, Adam

A study of a force detected single-spin magnetic resonance measurement concept with atomic spatial resolution is presented. The method is based upon electrostatic force detection of spin-selection rule controlled single electron tunneling between two electrically isolated paramagnetic states. Single-spin magnetic resonance detection is possible by measuring the force detected tunneling charge noise on and off spin resonance. Simulation results of this charge noise, based upon physical models of the tunneling and spin physics, are directly compared to measured atomic force microscopy (AFM) system noise. The results show that the approach could provide single-spin measurement of electrically isolated defect states with atomic spatial resolution at room temperature.

Krebs cycle metabolon: structural evidence of substrate channeling revealed by cross-linking and mass spectrometry.

PubMed

Wu, Fei; Minteer, Shelley

2015-02-02

It has been hypothesized that the high metabolic flux in the mitochondria is due to the self-assembly of enzyme supercomplexes (called metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle metabolon was accomplished by in vivo cross-linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low-resolution structure for the three-enzyme complex was achieved, as well as the two-fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein-protein association, which is the first structural evidence of substrate channeling in the Krebs cycle metabolon. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa M.; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.

2003-01-01

The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha (sub 2) beta (sub 2)-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the C(sup alpha) -C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-Angstrom resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-Angstrom shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.

Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.

PubMed

Gao, Hua-De; Thanasekaran, Pounraj; Chiang, Chao-Wei; Hong, Jia-Lin; Liu, Yen-Chun; Chang, Yu-Hsu; Lee, Hsien-Ming

2015-07-28

Photoactivatable (caged) bioeffectors provide a way to remotely trigger or disable biochemical pathways in living organisms at a desired time and location with a pulse of light (uncaging), but the phototoxicity of ultraviolet (UV) often limits its application. In this study, we have demonstrated the near-infrared (NIR) photoactivatable enzyme platform using protein kinase A (PKA), an important enzyme in cell biology. We successfully photoactivated PKA using NIR to phosphorylate its substrate, and this induced a downstream cellular response in living cells with high spatiotemporal resolution. In addition, this system allows NIR to selectively activate the caged enzyme immobilized on the nanoparticle surface without activating other caged proteins in the cytosol. This NIR-responsive enzyme-nanoparticle system provides an innovative approach to remote-control proteins and enzymes, which can be used by researchers who need to avoid direct UV irradiation or use UV as a secondary channel to turn on a bioeffector.

Microanalysis of tool steel and glass with laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Loebe, Klaus; Uhl, Arnold; Lucht, Hartmut

2003-10-01

A laser microscope system for the microanalytical characterization of complex materials is described. The universal measuring principle of laser-induced breakdown spectroscopy (LIBS) in combination with echelle optics permits a fast simultaneous multielement analysis with a possible spatial resolution below 10 pm. The developed system features completely UV-transparent optics for the laser-microscope coupling and the emission beam path and enables parallel signal detection within the wavelength range of 200-800 nm with a spectral resolution of a few picometers. Investigations of glass defects and tool steels were performed. The characterization of a glass defect in a tumbler by a micro-LIBS line scan, with use of a 266-nm diode-pumped Nd:YAG laser for excitation, is possible by simple comparison of plasma spectra of the defect and the surrounding area. Variations in the main elemental composition as well as impurities by trace elements are detected at the same time. Through measurement of the calibration samples with the known concentration of the corresponding element, a correlation between the intensity of spectral lines and the element concentration was also achieved. The change of elemental composition at the transient stellite solder of tool steels has been determined by an area scan. The two-dimensional pictures show abrupt changes of the element distribution along the solder edge and allow fundamental researches of dynamic modifications (e.g., diffusion) in steel.

Enhanced photoluminescence from single nitrogen-vacancy defects in nanodiamonds coated with phenol-ionic complexes

NASA Astrophysics Data System (ADS)

Bray, Kerem; Previdi, Rodolfo; Gibson, Brant C.; Shimoni, Olga; Aharonovich, Igor

2015-03-01

Fluorescent nanodiamonds are attracting major attention in the field of bio-sensing and bio-labeling. In this work we demonstrate a robust approach to achieve an encapsulation of individual nanodiamonds with phenol-ionic complexes that enhance the photoluminescence from single nitrogen vacancy (NV) centers. We show that single NV centres in the coated nanodiamonds also exhibit shorter lifetimes, opening another channel for high resolution sensing. We propose that the nanodiamond encapsulation reduces the non-radiative decay pathways of the NV color centers. Our results provide a versatile and assessable way to enhance photoluminescence from nanodiamond defects that can be used in a variety of sensing and imaging applications.Fluorescent nanodiamonds are attracting major attention in the field of bio-sensing and bio-labeling. In this work we demonstrate a robust approach to achieve an encapsulation of individual nanodiamonds with phenol-ionic complexes that enhance the photoluminescence from single nitrogen vacancy (NV) centers. We show that single NV centres in the coated nanodiamonds also exhibit shorter lifetimes, opening another channel for high resolution sensing. We propose that the nanodiamond encapsulation reduces the non-radiative decay pathways of the NV color centers. Our results provide a versatile and assessable way to enhance photoluminescence from nanodiamond defects that can be used in a variety of sensing and imaging applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07510b

Determination of Phenylalanine and Tyrosine by High Performance Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Peat, Judy; Garg, Uttam

2016-01-01

Hyperphenylalaninemia/phenylketonuria (PKU) is one of the most common inborn errors of amino acid metabolism affecting about 1:15,000 infants in the United States. PKU is an autosomal recessive disorder that if untreated results in mental retardation. The most common cause of PKU is deficiency of the enzyme phenylalanine hydroxylase that converts phenylalanine to tyrosine. Tyrosine deficiency results in impaired synthesis of catecholamines and thyroxine. Less commonly, it can result from defects in the synthesis or regeneration of tetrahydrobiopterin (BH4), an essential cofactor for the enzyme phenylalanine hydroxylase. Increased phenylalanine and decreased tyrosine in blood are used in the diagnosis and follow-up of patients with PKU. LC/MS/MS method is described for the quantification of phenylalanine and tyrosine.

Clinical and Biochemical Pitfalls in the Diagnosis of Peroxisomal Disorders.

PubMed

Klouwer, Femke C C; Huffnagel, Irene C; Ferdinandusse, Sacha; Waterham, Hans R; Wanders, Ronald J A; Engelen, Marc; Poll-The, Bwee Tien

2016-08-01

Peroxisomal disorders are a heterogeneous group of genetic metabolic disorders, caused by a defect in peroxisome biogenesis or a deficiency of a single peroxisomal enzyme. The peroxisomal disorders include the Zellweger spectrum disorders, the rhizomelic chondrodysplasia punctata spectrum disorders, X-linked adrenoleukodystrophy, and multiple single enzyme deficiencies. There are several core phenotypes caused by peroxisomal dysfunction that clinicians can recognize. The diagnosis is suggested by biochemical testing in blood and urine and confirmed by functional assays in cultured skin fibroblasts, followed by mutation analysis. This review describes the phenotype of the main peroxisomal disorders and possible pitfalls in (laboratory) diagnosis to aid clinicians in the recognition of this group of diseases. Georg Thieme Verlag KG Stuttgart · New York.

Unplugging the callose plug from sieve pores.

PubMed

Xie, Bo; Hong, Zonglie

2011-04-01

The presence of callose in sieve plates has been known for a long time, but how this polysaccharide plug is synthesized has remained unsolved. Two independent laboratories have recently reported the identification of callose synthase 7 (CalS7), also known as glucan synthase-like 7 (GSL7), as the enzyme responsible for callose deposition in sieve plates. Mutant plants defective in this enzyme failed to synthesize callose in developing sieve plates during phloem formation and were unable to accumulate callose in sieve pores in response to stress treatments. The mutant plants developed less open pores per sieve plate and the pores were smaller in diameter. As a result, phloem conductivity was reduced significantly and the mutant plants were shorter and set fewer seeds.

Unplugging the callose plug from sieve pores

PubMed Central

Xie, Bo

2011-01-01

The presence of callose in sieve plates has been known for a long time, but how this polysaccharide plug is synthesized has remained unsolved. Two independent laboratories have recently reported the identification of callose synthase 7 (CalS7), also known as glucan synthase-like 7 (GSL7), as the enzyme responsible for callose deposition in sieve plates. Mutant plants defective in this enzyme failed to synthesize callose in developing sieve plates during phloem formation and were unable to accumulate callose in sieve pores in response to stress treatments. The mutant plants developed less open pores per sieve plate and the pores were smaller in diameter. As a result, phloem conductivity was reduced significantly and the mutant plants were shorter and set fewer seeds. PMID:21386663

Phosphoserine-modified calcium phosphate cements: bioresorption and substitution.

PubMed

Offer, Liliana; Veigel, Bastian; Pavlidis, Theodoros; Heiss, Christian; Gelinsky, Michael; Reinstorf, Antje; Wenisch, Sabine; Lips, Katrin Susanne; Schnettler, Reinhard

2011-01-01

This work reports the effects of phosphoserine addition on the biodegradability of calcium phosphate cements. The characteristics of a phosphoserine-modified calcium phosphate cement without collagen in a large animal model are presented here for the first time. Critical size bone defects in the proximal tibia of 10 sheep were filled with the bone cement, and five sheep with empty defects were included as controls. The sheep were sacrificed after either 10 days or 12 weeks, and bones were processed for histological, histomorphometric and enzyme histochemical analyses as well as transmission electron microscopic examination. After 12 weeks, there was no significant reduction in either the implant or the bone defect cross-sectional area. Different amounts of fibrous tissue were observed around the implant and in the bone defect after 12 weeks. The direct bone-implant contact decreased after 12 weeks (p = 0.034). Although the implanted material properly filled the defect and promoted an initial activation of macrophages and osteoblasts, the resorption and simultaneous substitution did not reach expected levels during the experimental time course. Although other studies have shown that the addition of phosphoserine to calcium phosphate cements that have already been modified with collagen I resulted in an acceleration of cement resorption and bone regeneration, this study demonstrates that phosphoserine-modified calcium phosphate cements without collagen perform poorly in the treatment of bone defects. Efforts to use phosphoserine in the development of new composites should take into consideration the need to improve osteoconduction simultaneously via other means. Copyright © 2010 John Wiley & Sons, Ltd.

Exclusion of aldose reductase as a mediator of ERG deficits in a mouse model of diabetic eye disease.

PubMed

Samuels, Ivy S; Lee, Chieh-Allen; Petrash, J Mark; Peachey, Neal S; Kern, Timothy S

2012-11-01

Streptozotocin (STZ)-induced diabetes is associated with reductions in the electrical response of the outer retina and retinal pigment epithelium (RPE) to light. Aldose reductase (AR) is the first enzyme required in the polyol-mediated metabolism of glucose, and AR inhibitors have been shown to improve diabetes-induced electroretinogram (ERG) defects. Here, we used control and AR -/- mice to determine if genetic inactivation of this enzyme likewise inhibits retinal electrophysiological defects observed in a mouse model of type 1 diabetes. STZ was used to induce hyperglycemia and type 1 diabetes. Diabetic and age-matched nondiabetic controls of each genotype were maintained for 22 weeks, after which ERGs were used to measure the light-evoked components of the RPE (dc-ERG) and the neural retina (a-wave, b-wave). In comparison to their nondiabetic controls, wildtype (WT) and AR -/- diabetic mice displayed significant decreases in the c-wave, fast oscillation, and off response components of the dc-ERG but not in the light peak response. Nondiabetic AR -/- mice displayed larger ERG component amplitudes than did nondiabetic WT mice; however, the amplitude of dc-ERG components in diabetic AR -/- animals were similar to WT diabetics. ERG a-wave amplitudes were not reduced in either diabetic group, but b-wave amplitudes were lower in WT and AR -/-diabetic mice. These findings demonstrate that the light-induced responses of the RPE and outer retina are disrupted in diabetic mice, but these defects are not due to photoreceptor dysfunction, nor are they ameliorated by deletion of AR. This latter finding suggests that benefits observed in other studies utilizing pharmacological inhibitors of AR might have been secondary to off-target effects of the drugs.

Deficiency of Sbds in the mouse pancreas leads to features of Shwachman-Diamond syndrome, with loss of zymogen granules.

PubMed

Tourlakis, Marina E; Zhong, Jian; Gandhi, Rikesh; Zhang, Siyi; Chen, Lingling; Durie, Peter R; Rommens, Johanna M

2012-08-01

Shwachman-Diamond syndrome (SDS) is the second leading cause of hereditary exocrine pancreatic dysfunction. More than 90% of patients with SDS have biallelic loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene, which encodes a factor involved in ribosome function. We investigated whether mutations in Sbds lead to similar pancreatic defects in mice. Pancreas-specific knock-out mice were generated using a floxed Sbds allele and bred with mice carrying a null or disease-associated missense Sbds allele. Cre recombinase, regulated by the pancreatic transcription factor 1a promoter, was used to disrupt Sbds specifically in the pancreas. Models were assessed for pancreatic dysfunction and growth impairment. Disruption of Sbds in the mouse pancreas was sufficient to recapitulate SDS phenotypes. Pancreata of mice with Sbds mutations had decreased mass, fat infiltration, but general preservation of ductal and endocrine compartments. Pancreatic extracts from mutant mice had defects in formation of the 80S ribosomal complex. The exocrine compartment of mutant mice was hypoplastic and individual acini produced few zymogen granules. The null Sbds allele resulted in an earlier onset of phenotypes as well as endocrine impairment. Mutant mice had reduced serum levels of digestive enzymes and overall growth impairment. We developed a mouse model of SDS with pancreatic phenotypes similar to those of the human disease. This model could be used to investigate organ-specific consequences of Sbds-associated ribosomopathy. Sbds genotypes correlated with phenotypes. Defects developed specifically in the pancreata of mice, reducing growth of mice and production of digestive enzymes. SBDS therefore appears to be required for normal pancreatic development and function. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

A-to-I RNA editing promotes developmental stage-specific gene and lncRNA expression.

PubMed

Goldstein, Boaz; Agranat-Tamir, Lily; Light, Dean; Ben-Naim Zgayer, Orna; Fishman, Alla; Lamm, Ayelet T

2017-03-01

A-to-I RNA editing is a conserved widespread phenomenon in which adenosine (A) is converted to inosine (I) by adenosine deaminases (ADARs) in double-stranded RNA regions, mainly noncoding. Mutations in ADAR enzymes in Caenorhabditis elegans cause defects in normal development but are not lethal as in human and mouse. Previous studies in C. elegans indicated competition between RNA interference (RNAi) and RNA editing mechanisms, based on the observation that worms that lack both mechanisms do not exhibit defects, in contrast to the developmental defects observed when only RNA editing is absent. To study the effects of RNA editing on gene expression and function, we established a novel screen that enabled us to identify thousands of RNA editing sites in nonrepetitive regions in the genome. These include dozens of genes that are edited at their 3' UTR region. We found that these genes are mainly germline and neuronal genes, and that they are down-regulated in the absence of ADAR enzymes. Moreover, we discovered that almost half of these genes are edited in a developmental-specific manner, indicating that RNA editing is a highly regulated process. We found that many pseudogenes and other lncRNAs are also extensively down-regulated in the absence of ADARs in the embryo but not in the fourth larval (L4) stage. This down-regulation is not observed upon additional knockout of RNAi. Furthermore, levels of siRNAs aligned to pseudogenes in ADAR mutants are enhanced. Taken together, our results suggest a role for RNA editing in normal growth and development by regulating silencing via RNAi. © 2017 Goldstein et al.; Published by Cold Spring Harbor Laboratory Press.

Pathophysiology of hypophosphatasia and the potential role of asfotase alfa.

PubMed

Orimo, Hideo

2016-01-01

Hypophosphatasia (HPP) is an inherited systemic bone disease that is characterized by bone hypomineralization. HPP is classified into six forms according to the age of onset and severity as perinatal (lethal), perinatal benign, infantile, childhood, adult, and odontohypophosphatasia. The causative gene of the disease is the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP is expressed ubiquitously, and its physiological role is apparent in bone mineralization. A defect in bone mineralization can manifest in several ways, including rickets or osteomalacia in HPP patients. Patients with severe forms suffer from respiratory failure because of hypoplastic chest, which is the main cause of death. They sometimes present with seizures due to a defect in vitamin B6 metabolism resulting from the lack of alkaline phosphatase activity in neuronal cells, which is also lethal. Patients with a mild form of the disease exhibit rickets or osteomalacia and a functional defect of exercise. Odontohypophosphatasia shows only dental manifestations. To date, 302 mutations in the ALPL gene have been reported, mainly single-nucleotide substitutions, and the relationships between phenotype and genotype have been partially elucidated. An established treatment for HPP was not available until the recent development of enzyme replacement therapy. The first successful enzyme replacement therapy in model mice using a modified human TNAP protein (asfotase alfa) was reported in 2008, and subsequently success in patients with severe form of the disease was reported in 2012. In 2015, asfotase alfa was approved in Japan in July, followed by in the EU and Canada in August, and then by the US Food and Drug Administration in the USA in October. It is expected that therapy with asfotase alfa will drastically change treatments and prognosis of HPP.

Sinorhizobium meliloti requires a cobalamin-dependent ribonucleotide reductase for symbiosis with its plant host

PubMed Central

Taga, Michiko E.; Walker, Graham C.

2010-01-01

Vitamin B12 (cobalamin) is a critical cofactor for animals and protists, yet its biosynthesis is limited to prokaryotes. We previously showed that the symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti requires cobalamin to establish a symbiotic relationship with its plant host, Medicago sativa (alfalfa). Here, the specific requirement for cobalamin in the S. meliloti-alfalfa symbiosis was investigated. Of the three known cobalamin-dependent enzymes in S. meliloti, the methylmalonyl CoA mutase (BhbA) does not affect symbiosis whereas disruption of the metH gene encoding the cobalamin-dependent methionine synthase causes a significant defect in symbiosis. Expression of the cobalamin-independent methionine synthase MetE alleviates this symbiotic defect, indicating that the requirement for methionine synthesis does not reflect a need for the cobalamin-dependent enzyme. To investigate the function of the cobalamin-dependent ribonucleotide reductase (RNR) encoded by nrdJ, S. meliloti was engineered to express an Escherichia coli cobalamin-independent (Class Ia) RNR instead of nrdJ. This strain is severely defective in symbiosis. Electron micrographs show that these cells can penetrate alfalfa nodules but are unable to differentiate into nitrogen-fixing bacteroids and instead are lysed in the plant cytoplasm. Flow cytometry analysis indicates that these bacteria are largely unable to undergo endoreduplication. These phenotypes may be due to the inactivation of the Class Ia RNR by reactive oxygen species and/or inadequate oxygen availability in the nodule. These results show that the critical role of the cobalamin-dependent RNR for survival of S. meliloti in its plant host can account for the considerable resources that S. meliloti dedicates to cobalamin biosynthesis. PMID:20698752

In vivo monitoring of urea cycle activity with (13)C-acetate as a tracer of ureagenesis.

PubMed

Opladen, Thomas; Lindner, Martin; Das, Anibh M; Marquardt, Thorsten; Khan, Aneal; Emre, Sukru H; Burton, Barbara K; Barshop, Bruce A; Böhm, Thea; Meyburg, Jochen; Zangerl, Kathrin; Mayorandan, Sebene; Burgard, Peter; Dürr, Ulrich H N; Rosenkranz, Bernd; Rennecke, Jörg; Derbinski, Jens; Yudkoff, Marc; Hoffmann, Georg F

2016-01-01

The hepatic urea cycle is the main metabolic pathway for detoxification of ammonia. Inborn errors of urea cycle function present with severe hyperammonemia and a high case fatality rate. Long-term prognosis depends on the residual activity of the defective enzyme. A reliable method to estimate urea cycle activity in-vivo does not exist yet. The aim of this study was to evaluate a practical method to quantify (13)C-urea production as a marker for urea cycle function in healthy subjects, patients with confirmed urea cycle defect (UCD) and asymptomatic carriers of UCD mutations. (13)C-labeled sodium acetate was applied orally in a single dose to 47 subjects (10 healthy subjects, 28 symptomatic patients, 9 asymptomatic carriers). The oral (13)C-ureagenesis assay is a safe method. While healthy subjects and asymptomatic carriers did not differ with regards to kinetic variables for urea cycle flux, symptomatic patients had lower (13)C-plasma urea levels. Although the (13)C-ureagenesis assay revealed no significant differences between individual urea cycle enzyme defects, it reflected the heterogeneity between different clinical subgroups, including male neonatal onset ornithine carbamoyltransferase deficiency. Applying the (13)C-urea area under the curve can differentiate between severe from more mildly affected neonates. Late onset patients differ significantly from neonates, carriers and healthy subjects. This study evaluated the oral (13)C-ureagenesis assay as a sensitive in-vivo measure for ureagenesis capacity. The assay has the potential to become a reliable tool to differentiate UCD patient subgroups, follow changes in ureagenesis capacity and could be helpful in monitoring novel therapies of UCD. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional roles of three cutin biosynthetic acyltransferases in cytokinin responses and skotomorphogenesis.

PubMed

Wu, Lei; Zhou, Zhao-Yang; Zhang, Chun-Guang; Chai, Juan; Zhou, Qin; Wang, Li; Hirnerová, Eva; Mrvková, Michaela; Novák, Ondřej; Guo, Guang-Qin

2015-01-01

Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR) genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis.

Functional Roles of Three Cutin Biosynthetic Acyltransferases in Cytokinin Responses and Skotomorphogenesis

PubMed Central

Chai, Juan; Zhou, Qin; Wang, Li; Hirnerová, Eva; Mrvková, Michaela; Novák, Ondřej; Guo, Guang-Qin

2015-01-01

Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR) genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis. PMID:25803274

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies.

PubMed

Feichtinger, René G; Oláhová, Monika; Kishita, Yoshihito; Garone, Caterina; Kremer, Laura S; Yagi, Mikako; Uchiumi, Takeshi; Jourdain, Alexis A; Thompson, Kyle; D'Souza, Aaron R; Kopajtich, Robert; Alston, Charlotte L; Koch, Johannes; Sperl, Wolfgang; Mastantuono, Elisa; Strom, Tim M; Wortmann, Saskia B; Meitinger, Thomas; Pierre, Germaine; Chinnery, Patrick F; Chrzanowska-Lightowlers, Zofia M; Lightowlers, Robert N; DiMauro, Salvatore; Calvo, Sarah E; Mootha, Vamsi K; Moggio, Maurizio; Sciacco, Monica; Comi, Giacomo P; Ronchi, Dario; Murayama, Kei; Ohtake, Akira; Rebelo-Guiomar, Pedro; Kohda, Masakazu; Kang, Dongchon; Mayr, Johannes A; Taylor, Robert W; Okazaki, Yasushi; Minczuk, Michal; Prokisch, Holger

2017-10-05

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp -/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp -/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fatty Acid Synthase Activity as a Target for c-Met Driven Prostate Cancer

DTIC Science & Technology

2013-07-01

to aid future studies. Identification is a highly significant finding with regard to the potential for future therapeutic development targeted at...Met trafficking, stability, and ultimately oncogenic potential . Palmitoylation defective mutants will be used in animal models of c-Met driven tumor...growth (Aim 2). In addition, future work toward identifying the enzyme responsible for palmitoylation of c- Met will provide a new specific target

Defective Priming of CD4+ T Cell Responses During Pre-patent Schistosome Infection

DTIC Science & Technology

2011-11-26

maculopapular rash [11]. Symptoms of acute infection are usually not seen in residents of endemic areas, likely because of desensitization developed in...secreting proteolytic enzymes and enter the circulation after a few days [11]. They migrate through the capillaries of the lung and after approximately...highlighting the potential for resistance [34]. However, there are limitations to chemotherapeutic control, including the expense of the drug itself

A molecular chaperone for mitochondrial complex I assembly is mutated in a progressive encephalopathy

PubMed Central

Ogilvie, Isla; Kennaway, Nancy G.; Shoubridge, Eric A.

2005-01-01

NADH:ubiquinone oxidoreductase (complex I) deficiency is a common cause of mitochondrial oxidative phosphorylation disease. It is associated with a wide range of clinical phenotypes in infants, including Leigh syndrome, cardiomyopathy, and encephalomyopathy. In at least half of patients, enzyme deficiency results from a failure to assemble the holoenzyme complex; however, the molecular chaperones required for assembly of the mammalian enzyme remain unknown. Using whole genome subtraction of yeasts with and without a complex I to generate candidate assembly factors, we identified a paralogue (B17.2L) of the B17.2 structural subunit. We found a null mutation in B17.2L in a patient with a progressive encephalopathy and showed that the associated complex I assembly defect could be completely rescued by retroviral expression of B17.2L in patient fibroblasts. An anti-B17.2L antibody did not associate with the holoenzyme complex but specifically recognized an 830-kDa subassembly in several patients with complex I assembly defects and coimmunoprecipitated a subset of complex I structural subunits from normal human heart mitochondria. These results demonstrate that B17.2L is a bona fide molecular chaperone that is essential for the assembly of complex I and for the normal function of the nervous system. PMID:16200211

Plastidial Folate Prevents Starch Biosynthesis Triggered by Sugar Influx into Non-Photosynthetic Plastids of Arabidopsis

PubMed Central

Hayashi, Makoto; Tanaka, Mina; Yamamoto, Saki; Nakagawa, Taro; Kanai, Masatake; Anegawa, Aya; Ohnishi, Miwa; Mimura, Tetsuro; Nishimura, Mikio

2017-01-01

Abstract Regulation of sucrose–starch interconversion in plants is important to maintain energy supplies necessary for viability and growth. Arabidopsis mutants were screened for aberrant responses to sucrose to identify candidates with a defect in the regulation of starch biosynthesis. One such mutant, fpgs1-4, accumulated substantial amounts of starch in non-photosynthetic cells. Dark-grown mutant seedlings exhibited shortened hypocotyls and accumulated starch in etioplasts when supplied with exogenous sucrose/glucose. Similar starch accumulation from exogenous sucrose was observed in mutant chloroplasts, when photosynthesis was prevented by organ culture in darkness. Molecular genetic analyses revealed that the mutant was defective in plastidial folylpolyglutamate synthetase, one of the enzymes engaged in folate biosynthesis. Active folate derivatives are important biomolecules that function as cofactors for a variety of enzymes. Exogenously supplied 5-formyl-tetrahydrofolate abrogated the mutant phenotypes, indicating that the fpgs1-4 mutant produced insufficient folate derivative levels. In addition, the antifolate agents methotrexate and 5-fluorouracil induced starch accumulation from exogenously supplied sucrose in dark-grown seedlings of wild-type Arabidopsis. These results indicate that plastidial folate suppresses starch biosynthesis triggered by sugar influx into non-photosynthetic cells, demonstrating a hitherto unsuspected link between plastidial folate and starch metabolism. PMID:28586467

A glycogene mutation map for discovery of diseases of glycosylation

PubMed Central

Hansen, Lars; Lind-Thomsen, Allan; Joshi, Hiren J; Pedersen, Nis Borbye; Have, Christian Theil; Kong, Yun; Wang, Shengjun; Sparso, Thomas; Grarup, Niels; Vester-Christensen, Malene Bech; Schjoldager, Katrine; Freeze, Hudson H; Hansen, Torben; Pedersen, Oluf; Henrissat, Bernard; Mandel, Ulla; Clausen, Henrik; Wandall, Hans H; Bennett, Eric P

2015-01-01

Glycosylation of proteins and lipids involves over 200 known glycosyltransferases (GTs), and deleterious defects in many of the genes encoding these enzymes cause disorders collectively classified as congenital disorders of glycosylation (CDGs). Most known CDGs are caused by defects in glycogenes that affect glycosylation globally. Many GTs are members of homologous isoenzyme families and deficiencies in individual isoenzymes may not affect glycosylation globally. In line with this, there appears to be an underrepresentation of disease-causing glycogenes among these larger isoenzyme homologous families. However, genome-wide association studies have identified such isoenzyme genes as candidates for different diseases, but validation is not straightforward without biomarkers. Large-scale whole-exome sequencing (WES) provides access to mutations in, for example, GT genes in populations, which can be used to predict and/or analyze functional deleterious mutations. Here, we constructed a draft of a functional mutational map of glycogenes, GlyMAP, from WES of a rather homogenous population of 2000 Danes. We cataloged all missense mutations and used prediction algorithms, manual inspection and in case of carbohydrate-active enzymes family GT27 experimental analysis of mutations to map deleterious mutations. GlyMAP (http://glymap.glycomics.ku.dk) provides a first global view of the genetic stability of the glycogenome and should serve as a tool for discovery of novel CDGs. PMID:25267602

An efficient screen for peroxisome-deficient mutants of Pichia pastoris.

PubMed Central

Liu, H; Tan, X; Veenhuis, M; McCollum, D; Cregg, J M

1992-01-01

We describe a rapid and efficient screen for peroxisome-deficient (per) mutants in the yeast Pichia pastoris. The screen relies on the unusual ability of P. pastoris to grow on two carbon sources, methanol and oleic acid, both of which absolutely require peroxisomes to be metabolized. A collection of 280 methanol utilization-defective (Mut-) P. pastoris mutants was isolated, organized into 46 complementation groups, and tested for those that were also oleate-utilization defective (Out-) but still capable of growth on ethanol and glucose. Mutants in 10 groups met this phenotypic description, and 8 of these were observed by electron microscopy to be peroxisome deficient (Per-). In each per mutant, Mut-, Out-, and Per- phenotypes were tightly linked and therefore were most likely due to a mutation at a single locus. Subcellular fractionation experiments indicated that the peroxisomal marker enzyme catalase was mislocalized to the cytosol in both methanol- and oleate-induced cultures of the mutants. In contrast, alcohol oxidase, a peroxisomal methanol utilization pathway enzyme, was virtually absent from per mutant cells. The relative ease of per mutant isolation in P. pastoris, in conjunction with well-developed procedures for its molecular and genetic manipulation, makes this organism an attractive system for studies on peroxisome biogenesis. Images PMID:1629154

Nkx2.5 regulates Endothelin Converting Enzyme-1 during pharyngeal arch patterning

PubMed Central

Iklé, Jennifer M.; Tavares, Andre L. P.; King, Marisol; Ding, Hailei; Colombo, Sophie; Firulli, Beth A.; Firulli, Anthony B.; Targoff, Kimara L.; Yelon, Deborah; Clouthier, David E.

2017-01-01

In gnathostomes, dorsoventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches is crucial for the development of hinged jaws. One of the key signals that mediates this process is Endothelin-1 (EDN1). Loss of EDN1 binding to the Endothelin-A receptor (EDNRA) results in loss of EDNRA signaling and subsequent facial birth defects in humans, mice and zebrafish. A rate-limiting step in this crucial signaling pathway is the conversion of immature EDN1 into a mature active form by Endothelin converting enzyme-1 (ECE1). However, surprisingly little is known about how Ece1 transcription is induced or regulated. We show here that Nkx2.5 is required for proper craniofacial development in zebrafish and acts in part by upregulating ece1 expression. Disruption of nkx2.5 in zebrafish embryos results in defects in both ventral and dorsal pharyngeal arch-derived elements, with changes in ventral arch gene expression consistent with a disruption in Ednra signaling. ece1 mRNA rescues the nkx2.5 morphant phenotype, indicating that Nkx2.5 functions through modulating Ece1 expression or function. These studies illustrate a new function for Nkx2.5 in embryonic development and provide new avenues with which to pursue potential mechanisms underlying human facial disorders. PMID:28109039

Cerebellar Alterations and Gait Defects as Therapeutic Outcome Measures for Enzyme Replacement Therapy in α-Mannosidosis

PubMed Central

Damme, Markus; Stroobants, Stijn; Walkley, Steven U.; Lüllmann-Rauch, Renate; D`Hooge, Rudi; Fogh, Jens; Saftig, Paul; Lübke, Torben; Blanz, Judith

2011-01-01

α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyl-linked oligosaccharides in cells throughout the body, most notably in the CNS. This leads to a broad spectrum of neurological manifestations, including progressive intellectual impairment, disturbed motor functions and cerebellar atrophy. To develop therapeutic outcome measures for enzyme replacement therapy (ERT) that could be used for human patients, a gene knockout model of α-mannosidosis in mice was analyzed for CNS pathology and motor deficits. In the cerebellar molecular layer, α-mannosidosis mice display clusters of activated Bergman glia, infiltration of phagocytic macrophages and accumulation of free cholesterol and gangliosides (GM1), notably in regions lacking Purkinje cells. α-mannosidosis brain lysates also displayed increased expression of Lamp1 and hyperglycosylation of the cholesterol binding protein NPC2. Detailed assessment of motor function revealed age-dependent gait defects in the mice that resemble the disturbed motor function in human patients. Short-term ERT partially reversed the observed cerebellar pathology with fewer activated macrophages and astrocytes but unchanged levels of hyperglycosylated NPC2, gangliosides and cholesterol. The present study demonstrates cerebellar alterations in α-mannosidosis mice that relate to the motor deficits and pathological changes seen in human patients and can be used as therapeutic outcome measures. PMID:21157375

Low dose trichloroethylene alters cytochrome P450 - 2C subfamily expression in the developing chick heart

PubMed Central

Makwana, Om; Ahles, Lauren; Lencinas, Alejandro; Selmin, Ornella I.; Runyan, Raymond B.

2013-01-01

Trichloroethylene (TCE) is an organic solvent and common environmental contaminant. TCE exposure is associated with heart defects in humans and animal models. Primary metabolism of TCE in adult rodent models is by specific hepatic cytochrome P450 enzymes (Lash et al., 2000). As association of TCE exposure with cardiac defects is in exposed embryos prior to normal liver development, we investigated metabolism of TCE in the early embryo. Developing chick embryos were dosed in ovo with environmentally relevant doses of TCE (8 ppb and 800 ppb) and RNA was extracted from cardiac and extra-cardiac tissue (whole embryo without heart). Real time PCR showed upregulation of CYP2H1 transcripts in response to TCE exposure in the heart. No detectable cytochrome expression was found in extra-cardiac tissue. As seen previously, the dose response was non-monotonic and 8ppb elicited stronger upregulation than 800 ppb. Immunostaining for CYP2C subfamily expression confirmed protein expression and showed localization in both myocardium and endothelium. TCE exposure increased protein expression in both tissues. These data demonstrate that the earliest embryonic expression of phase I detoxification enzymes is in the developing heart. Expression of these CYPs is likely to be relevant to the susceptibility of the developing heart to environmental teratogens. PMID:22855351

Heparan Sulfate Biosynthesis Enzyme, Ext1, Contributes to Outflow Tract Development of Mouse Heart via Modulation of FGF Signaling.

PubMed

Zhang, Rui; Cao, Peijuan; Yang, Zhongzhou; Wang, Zhenzhen; Wu, Jiu-Lin; Chen, Yan; Pan, Yi

2015-01-01

Glycosaminoglycans are important regulators of multiple signaling pathways. As a major constituent of the heart extracellular matrix, glycosaminoglycans are implicated in cardiac morphogenesis through interactions with different signaling morphogens. Ext1 is a glycosyltransferase responsible for heparan sulfate synthesis. Here, we evaluate the function of Ext1 in heart development by analyzing Ext1 hypomorphic mutant and conditional knockout mice. Outflow tract alignment is sensitive to the dosage of Ext1. Deletion of Ext1 in the mesoderm induces a cardiac phenotype similar to that of a mutant with conditional deletion of UDP-glucose dehydrogenase, a key enzyme responsible for synthesis of all glycosaminoglycans. The outflow tract defect in conditional Ext1 knockout(Ext1f/f:Mesp1Cre) mice is attributable to the reduced contribution of second heart field and neural crest cells. Ext1 deletion leads to downregulation of FGF signaling in the pharyngeal mesoderm. Exogenous FGF8 ameliorates the defects in the outflow tract and pharyngeal explants. In addition, Ext1 expression in second heart field and neural crest cells is required for outflow tract remodeling. Our results collectively indicate that Ext1 is crucial for outflow tract formation in distinct progenitor cells, and heparan sulfate modulates FGF signaling during early heart development.

Regulation of intracellular formaldehyde toxicity during methanol metabolism of the methylotrophic yeast Pichia methanolica.

PubMed

Wakayama, Keishi; Yamaguchi, Sakiko; Takeuchi, Akihito; Mizumura, Tasuku; Ozawa, Shotaro; Tomizuka, Noboru; Hayakawa, Takashi; Nakagawa, Tomoyuki

2016-11-01

In this study we found that the methylotrophic yeast Pichia methanolica showed impaired growth on high methanol medium (>5%, or 1.56 M, methanol). In contrast, P. methanolica grew well on glucose medium containing 5% methanol, but the growth defects reappeared on glucose medium supplemented with 5 mM formaldehyde. During methanol growth of P. methanolica, formaldehyde accumulated in the medium up to 0.3 mM before it was consumed rapidly based on cell growth. These findings indicate that the growth defect of P. methanolica on high methanol media is not caused directly by methanol toxicity, but rather by formaldehyde, which is a key toxic intermediate of methanol metabolism. Moreover, during methanol growth of P. methanolica, expression of enzymes in the methanol-oxidation pathway were induced before the alcohol oxidase isozymes Mod1p and Mod2p, and Mod1p expression was induced before Mod2p. These results suggest that to avoid excess accumulation of formaldehyde-the toxic intermediate of methanol metabolism-P. methanolica grown on methanol strictly regulates the order in which methanol-metabolizing enzymes are expressed. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Changes is genes coding for laccases 1 and 2 may contribute to deformation and reduction of wings in apollo butterfly (Parnassius apollo, Lepidoptera: Papilionidae) from the isolated population in Pieniny National Park (Poland).

PubMed

Łukasiewicz, Kinga; Węgrzyn, Grzegorz

2016-01-01

An isolated population of apollo butterfly (Parnassius apollo, Lepidoptera: Papilionidae) occurs in Pieniny National Park (Poland). Deformations and reductions of wings in a relatively large number of individuals from this population is found, yet the reasons for these defects are unknown. During studies devoted to identify cause(s) of this phenomenon, we found that specific regions of genes coding of enzymes laccases 1 and 2 could not be amplified from DNA samples isolated from large fractions of malformed insects while expected PCR products were detected in almost all (with one exception) normal butterflies. Laccases (p-diphenol:dioxygen oxidoreductases) are oxidases containing several copper atoms. They catalyse single-electron oxidations of phenolic or other compounds with concomitant reduction of oxygen to water. In insects, their enzymatic activities were found previously in epidermis, midgut, Malpighian tubules, salivary glands, and reproductive tissues. Therefore, we suggest that defects in genes coding for laccases might contribute to deformation and reduction of wings in apollo butterflies, though it seems obvious that deficiency in these enzymes could not be the sole cause of these developmental improperties in P. apollo from Pieniny National Park.

Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope.

PubMed

Hieckmann, Ellen; Nacke, Markus; Allardt, Matthias; Bodrov, Yury; Chekhonin, Paul; Skrotzki, Werner; Weber, Jörg

2016-05-28

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications of semiconductor materials. However, it is still under debate how the defect structure determines the band structure, and therefore, the recombination behavior of electron-hole pairs responsible for the optical and electrical properties of the extended defects. The present paper is a survey of procedures for the spatially resolved investigation of structural and of physical properties of extended defects in semiconductor materials with a scanning electron microscope (SEM). Representative examples are given for crystalline silicon. The luminescence behavior of extended defects can be investigated by cathodoluminescence (CL) measurements. They are particularly valuable because spectrally and spatially resolved information can be obtained simultaneously. For silicon, with an indirect electronic band structure, CL measurements should be carried out at low temperatures down to 5 K due to the low fraction of radiative recombination processes in comparison to non-radiative transitions at room temperature. For the study of the electrical properties of extended defects, the electron beam induced current (EBIC) technique can be applied. The EBIC image reflects the local distribution of defects due to the increased charge-carrier recombination in their vicinity. The procedure for EBIC investigations is described for measurements at room temperature and at low temperatures. Internal strain fields arising from extended defects can be determined quantitatively by cross-correlation electron backscatter diffraction (ccEBSD). This method is challenging because of the necessary preparation of the sample surface and because of the quality of the diffraction patterns which are recorded during the mapping of the sample. The spatial resolution of the three experimental techniques is compared.

Comprehensive Characterization of Extended Defects in Semiconductor Materials by a Scanning Electron Microscope

PubMed Central

Hieckmann, Ellen; Nacke, Markus; Allardt, Matthias; Bodrov, Yury; Chekhonin, Paul; Skrotzki, Werner; Weber, Jörg

2016-01-01

Extended defects such as dislocations and grain boundaries have a strong influence on the performance of microelectronic devices and on other applications of semiconductor materials. However, it is still under debate how the defect structure determines the band structure, and therefore, the recombination behavior of electron-hole pairs responsible for the optical and electrical properties of the extended defects. The present paper is a survey of procedures for the spatially resolved investigation of structural and of physical properties of extended defects in semiconductor materials with a scanning electron microscope (SEM). Representative examples are given for crystalline silicon. The luminescence behavior of extended defects can be investigated by cathodoluminescence (CL) measurements. They are particularly valuable because spectrally and spatially resolved information can be obtained simultaneously. For silicon, with an indirect electronic band structure, CL measurements should be carried out at low temperatures down to 5 K due to the low fraction of radiative recombination processes in comparison to non-radiative transitions at room temperature. For the study of the electrical properties of extended defects, the electron beam induced current (EBIC) technique can be applied. The EBIC image reflects the local distribution of defects due to the increased charge-carrier recombination in their vicinity. The procedure for EBIC investigations is described for measurements at room temperature and at low temperatures. Internal strain fields arising from extended defects can be determined quantitatively by cross-correlation electron backscatter diffraction (ccEBSD). This method is challenging because of the necessary preparation of the sample surface and because of the quality of the diffraction patterns which are recorded during the mapping of the sample. The spatial resolution of the three experimental techniques is compared. PMID:27285177

High-speed atomic force microscopy and peak force tapping control

NASA Astrophysics Data System (ADS)

Hu, Shuiqing; Mininni, Lars; Hu, Yan; Erina, Natalia; Kindt, Johannes; Su, Chanmin

2012-03-01

ITRS Roadmap requires defect size measurement below 10 nanometers and challenging classifications for both blank and patterned wafers and masks. Atomic force microscope (AFM) is capable of providing metrology measurement in 3D at sub-nanometer accuracy but has long suffered from drawbacks in throughput and limitation of slow topography imaging without chemical information. This presentation focus on two disruptive technology developments, namely high speed AFM and quantitative nanomechanical mapping, which enables high throughput measurement with capability of identifying components through concurrent physical property imaging. The high speed AFM technology has allowed the imaging speed increase by 10-100 times without loss of the data quality. Such improvement enables the speed of defect review on a wafer to increase from a few defects per hour to nearly 100 defects an hour, approaching the requirements of ITRS Roadmap. Another technology development, Peak Force Tapping, substantially simplified the close loop system response, leading to self-optimization of most challenging samples groups to generate expert quality data. More importantly, AFM also simultaneously provides a series of mechanical property maps with a nanometer spatial resolution during defect review. These nanomechanical maps (including elastic modulus, hardness, and surface adhesion) provide complementary information for elemental analysis, differentiate defect materials by their physical properties, and assist defect classification beyond topographic measurements. This paper will explain the key enabling technologies, namely high speed tip-scanning AFM using innovative flexure design and control algorithm. Another critical element is AFM control using Peak Force Tapping, in which the instantaneous tip-sample interaction force is measured and used to derive a full suite of physical properties at each imaging pixel. We will provide examples of defect review data on different wafers and media disks. The similar AFM-based defect review capacity was also applied to EUV masks.

Microwave Assisted Enzymatic Kinetic Resolution of (±)-1-Phenyl-2-propyn-1-ol in Nonaqueous Media

PubMed Central

Devendran, Saravanan; Yadav, Ganapati D.

2014-01-01

Kinetic resolution of 1-phenyl-2-propyn-1-ol, an important chiral synthon, was studied through trans-esterification with acyl acetate to investigate synergism between microwave irradiation and enzyme catalysis. Lipases from different microbial origins were employed for the kinetic resolution of (R/S)-1-phenyl-2-propyn-1-ol, among which Candida antarctica lipase B, immobilized on acrylic resin (Novozym 435), was found to be the best catalyst in n-hexane as solvent. Vinyl acetate was the most effective among different acyl esters studied. The effect of various parameters was studied in a systematic manner. Definite synergism between microwave and enzyme was observed. The initial rate was improved around 1.28 times under microwave irradiation than conventional heating. Under optimum conditions, maximum conversion (48.78%) and high enantiomeric excess (93.25%) were obtained in 2 h. From modeling studies, it is concluded that the reaction follows the Ping-Pong bi-bi mechanism with dead end alcohol inhibition. Kinetic parameters were obtained by using nonlinear regression. This process is green, clean, and easily scalable as compared to the chemical process. PMID:24707487

Low-Temperature Single Carbon Nanotube Spectroscopy of sp 3 Quantum Defects

DOE PAGES

He, Xiaowei; Gifford, Brendan J.; Hartmann, Nicolai F.; ...

2017-09-28

Aiming to unravel the relationship between chemical configuration and electronic structure of sp3 defects of aryl-functionalized (6,5) single-walled carbon nanotubes (SWCNTs), we perform low-temperature single nanotube photoluminescence (PL) spectroscopy studies and correlate our observations with quantum chemistry simulations. Here, we observe sharp emission peaks from individual defect sites that are spread over an extremely broad, 1000-1350 nm, spectral range. Our simulations allow us to attribute this spectral diversity to the occurrence of six chemically and energetically distinct defect states resulting from topological variation in the chemical binding configuration of the monovalent aryl groups. Both PL emission efficiency and spectral linemore » width of the defect states are strongly influenced by the local dielectric environment. Wrapping the SWCNT with a polyfluorene polymer provides the best isolation from the environment and yields the brightest emission with near-resolution limited spectral line width of 270 ueV, as well as spectrally resolved emission wings associated with localized acoustic phonons. Pump-dependent studies further revealed that the defect states are capable of emitting single, sharp, isolated PL peaks over 3 orders of magnitude increase in pump power, a key characteristic of two-level systems and an important prerequisite for single-photon emission with high purity. Our findings point to the tremendous potential of sp3 defects in development of room temperature quantum light sources capable of operating at telecommunication wavelengths as the emission of the defect states can readily be extended to this range via use of larger diameter SWCNTs.« less

Low-Temperature Single Carbon Nanotube Spectroscopy of sp 3 Quantum Defects

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

He, Xiaowei; Gifford, Brendan J.; Hartmann, Nicolai F.

Aiming to unravel the relationship between chemical configuration and electronic structure of sp3 defects of aryl-functionalized (6,5) single-walled carbon nanotubes (SWCNTs), we perform low-temperature single nanotube photoluminescence (PL) spectroscopy studies and correlate our observations with quantum chemistry simulations. Here, we observe sharp emission peaks from individual defect sites that are spread over an extremely broad, 1000-1350 nm, spectral range. Our simulations allow us to attribute this spectral diversity to the occurrence of six chemically and energetically distinct defect states resulting from topological variation in the chemical binding configuration of the monovalent aryl groups. Both PL emission efficiency and spectral linemore » width of the defect states are strongly influenced by the local dielectric environment. Wrapping the SWCNT with a polyfluorene polymer provides the best isolation from the environment and yields the brightest emission with near-resolution limited spectral line width of 270 ueV, as well as spectrally resolved emission wings associated with localized acoustic phonons. Pump-dependent studies further revealed that the defect states are capable of emitting single, sharp, isolated PL peaks over 3 orders of magnitude increase in pump power, a key characteristic of two-level systems and an important prerequisite for single-photon emission with high purity. Our findings point to the tremendous potential of sp3 defects in development of room temperature quantum light sources capable of operating at telecommunication wavelengths as the emission of the defect states can readily be extended to this range via use of larger diameter SWCNTs.« less

Advances in Stereoconvergent Catalysis from 2005–2015: Transition-Metal-Mediated Stereoablative Reactions, Dynamic Kinetic Resolutions, and Dynamic Kinetic Asymmetric Transformations

PubMed Central

Bhat, Vikram; Welin, Eric R.; Guo, Xuelei; Stoltz, Brian M.

2017-01-01

An important subset of asymmetric synthesis is dynamic kinetic resolution, dynamic kinetic asymmetric processes and stereoablative transformations. Initially, only enzymes were known to catalyze dynamic kinetic processes but recently various synthetic catalysts have been developed. This review summarizes major advances in non-enzymatic, transition metal promoted dynamic asymmetric transformations reported between 2005 and 2015. PMID:28164696

Enantiomeric scaffolding of α-tetralone and related scaffolds by EKR (enzymatic kinetic resolution) and stereoselective ketoreduction with ketoreductases.

PubMed

Bhuniya, Rajib; Nanda, Samik

2012-01-21

Stereochemically pure compounds containing an all carbon quaternary stereocenter based on 1-tetralone, 1-indanone and 4-chromanone scaffolds have been synthesized by employing Lipase PS (Burkholderia cepacia) catalyzed kinetic resolution. These scaffolds are further functionalized by microbial ketoreductase enzymes (Geotrichum candidum, Candida parapsilosis and Aspergillus niger) to access stereochemically pure diols which, on further synthetic manipulation, yield novel cyclic compounds.

NMR Crystallography of Enzyme Active Sites: Probing Chemically-Detailed, Three-Dimensional Structure in Tryptophan Synthase

PubMed Central

Dunn, Michael F.

2013-01-01

Conspectus NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry – offers unprecedented insight into three-dimensional, chemically-detailed structure. From its initial role in refining diffraction data of organic and inorganic solids, NMR crystallography is now being developed for application to active sites in biomolecules, where it reveals chemically-rich detail concerning the interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. For example, typical X-ray crystal structures (1.5 to 2.5 Å resolution) of enzyme-bound intermediates identify possible hydrogen-bonding interactions between site residues and substrate, but do not directly identify the protonation state of either. Solid-state NMR can provide chemical shifts for selected atoms of enzyme-substrate complexes, but without a larger structural framework in which to interpret them, only empirical correlations with local chemical structure are possible. Ab initio calculations and molecular mechanics can build models for enzymatic processes, but rely on chemical details that must be specified. Together, however, X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry can provide consistent and testable models for structure and function of enzyme active sites: X-ray crystallography provides a coarse framework upon which models of the active site can be developed using computational chemistry; these models can be distinguished by comparison of their calculated NMR chemical shifts with the results of solid-state NMR spectroscopy experiments. Conceptually, each technique is a puzzle piece offering a generous view of the big picture. Only when correctly pieced together, however, can they reveal the big picture at highest resolution. In this Account, we detail our first steps in the development of NMR crystallography for application to enzyme catalysis. We begin with a brief introduction to NMR crystallography and then define the process that we have employed to probe the active site in the β-subunit of tryptophan synthase with unprecedented atomic-level resolution. This approach has resulted in a novel structural hypothesis for the protonation state of the quinonoid intermediate in tryptophan synthase and its surprising role in directing the next step in the catalysis of L-Trp formation. PMID:23537227

Femtosecond laser micro-inscription of optical coherence tomography resolution test artifacts.

PubMed

Tomlins, Peter H; Smith, Graham N; Woolliams, Peter D; Rasakanthan, Janarthanan; Sugden, Kate

2011-04-25

Optical coherence tomography (OCT) systems are becoming more commonly used in biomedical imaging and, to enable continued uptake, a reliable method of characterizing their performance and validating their operation is required. This paper outlines the use of femtosecond laser subsurface micro-inscription techniques to fabricate an OCT test artifact for validating the resolution performance of a commercial OCT system. The key advantage of this approach is that by utilizing the nonlinear absorption a three dimensional grid of highly localized point and line defects can be written in clear fused silica substrates.

X-Ray crystal structure of GarR—tartronate semialdehyde reductase from Salmonella typhimurium

PubMed Central

Osipiuk, J.; Zhou, M.; Moy, S.; Collart, F.

2009-01-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related β-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 Å resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme. PMID:19184529

X-ray crystal structure of GarR-tartronate semialdehyde reductase from Salmonella typhimurium.

PubMed

Osipiuk, J; Zhou, M; Moy, S; Collart, F; Joachimiak, A

2009-09-01

Tartronate semialdehyde reductases (TSRs), also known as 2-hydroxy-3-oxopropionate reductases, catalyze the reduction of tartronate semialdehyde using NAD as cofactor in the final stage of D-glycerate biosynthesis. These enzymes belong to family of structurally and mechanically related beta-hydroxyacid dehydrogenases which differ in substrate specificity and catalyze reactions in specific metabolic pathways. Here, we present the crystal structure of GarR a TSR from Salmonella typhimurium determined by the single-wavelength anomalous diffraction method and refined to 1.65 A resolution. The active site of the enzyme contains L-tartrate which most likely mimics a position of a glycerate which is a product of the enzyme reaction. The analysis of the TSR structure shows also a putative NADPH binding site in the enzyme.

A 405-kb cosmid contig and HindIII restriction map of the progressive myoclonus epilepsy type 1 (EPM1) candidate region in 21q22.3

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

Lafreniere, R.G.; Rouleau, G.A.; De Jong, P.J.

1995-09-01

As a step toward identifying the molecular defect in patients afflicted with progressive myoclonus epilepsy type 1 (EPM1), we have assembled a cosmid contig of the candidate EPM1 region in 21q22.3. The contig constitutes a collection of 87 different cosmids spanning 405 kb based on a derived HindIII restriction map. Potential CpG-rich islands have been identified based on the restriction map generated from eight different rare-cutting enzymes. This contig contains the genetic material required for the isolation of expressed sequences and the identification of the gene defective in EPM1 and possibly other disorders mapping to this region. 15 refs., 1more » fig.« less

MMP20 Overexpression Disrupts Molar Ameloblast Polarity and Migration.

PubMed

Shin, M; Chavez, M B; Ikeda, A; Foster, B L; Bartlett, J D

2018-07-01

Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20 +/+ Tg + ) were assessed by multiscale analyses, combining several approaches from high-resolution micro-computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20 +/+ Tg + mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20 +/+ Tg + molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20 +/+ Tg + mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.

X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Å resolution

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

Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.

2010-01-12

Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging tomore » this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.« less

A method of extending the depth of focus of the high-resolution X-ray imaging system employing optical lens and scintillator: a phantom study.

PubMed

Li, Guang; Luo, Shouhua; Yan, Yuling; Gu, Ning

2015-01-01

The high-resolution X-ray imaging system employing synchrotron radiation source, thin scintillator, optical lens and advanced CCD camera can achieve a resolution in the range of tens of nanometers to sub-micrometer. Based on this advantage, it can effectively image tissues, cells and many other small samples, especially the calcification in the vascular or in the glomerulus. In general, the thickness of the scintillator should be several micrometers or even within nanometers because it has a big relationship with the resolution. However, it is difficult to make the scintillator so thin, and additionally thin scintillator may greatly reduce the efficiency of collecting photons. In this paper, we propose an approach to extend the depth of focus (DOF) to solve these problems. We develop equation sets by deducing the relationship between the high-resolution image generated by the scintillator and the degraded blur image due to defect of focus first, and then we adopt projection onto convex sets (POCS) and total variation algorithm to get the solution of the equation sets and to recover the blur image. By using a 20 μm thick unmatching scintillator to replace the 1 μm thick matching one, we simulated a high-resolution X-ray imaging system and got a degraded blur image. Based on the algorithm proposed, we recovered the blur image and the result in the experiment showed that the proposed algorithm has good performance on the recovery of image blur caused by unmatching thickness of scintillator. The method proposed is testified to be able to efficiently recover the degraded image due to defect of focus. But, the quality of the recovery image especially of the low contrast image depends on the noise level of the degraded blur image, so there is room for improving and the corresponding denoising algorithm is worthy for further study and discussion.

A method of extending the depth of focus of the high-resolution X-ray imaging system employing optical lens and scintillator: a phantom study

PubMed Central

2015-01-01

Background The high-resolution X-ray imaging system employing synchrotron radiation source, thin scintillator, optical lens and advanced CCD camera can achieve a resolution in the range of tens of nanometers to sub-micrometer. Based on this advantage, it can effectively image tissues, cells and many other small samples, especially the calcification in the vascular or in the glomerulus. In general, the thickness of the scintillator should be several micrometers or even within nanometers because it has a big relationship with the resolution. However, it is difficult to make the scintillator so thin, and additionally thin scintillator may greatly reduce the efficiency of collecting photons. Methods In this paper, we propose an approach to extend the depth of focus (DOF) to solve these problems. We develop equation sets by deducing the relationship between the high-resolution image generated by the scintillator and the degraded blur image due to defect of focus first, and then we adopt projection onto convex sets (POCS) and total variation algorithm to get the solution of the equation sets and to recover the blur image. Results By using a 20 μm thick unmatching scintillator to replace the 1 μm thick matching one, we simulated a high-resolution X-ray imaging system and got a degraded blur image. Based on the algorithm proposed, we recovered the blur image and the result in the experiment showed that the proposed algorithm has good performance on the recovery of image blur caused by unmatching thickness of scintillator. Conclusions The method proposed is testified to be able to efficiently recover the degraded image due to defect of focus. But, the quality of the recovery image especially of the low contrast image depends on the noise level of the degraded blur image, so there is room for improving and the corresponding denoising algorithm is worthy for further study and discussion. PMID:25602532

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation

PubMed Central

Wu, Qiong; Madany, Pasil; Dobson, Jason R.; Schnabl, Jake M.; Sharma, Soni; Smith, Tara C.; van Wijnen, Andre J.; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.; Muthuswami, Rohini; Imbalzano, Anthony N.; Nickerson, Jeffrey A.

2016-01-01

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer. PMID:27223259

Pharmacogenetics of drug-metabolizing enzymes: implications for a safer and more effective drug therapy

PubMed Central

Ingelman-Sundberg, Magnus; Rodriguez-Antona, Cristina

2005-01-01

The majority of phase I- and phase II-dependent drug metabolism is carried out by polymorphic enzymes which can cause abolished, quantitatively or qualitatively decreased or enhanced drug metabolism. Several examples exist where subjects carrying certain alleles do not benefit from drug therapy due to ultrarapid metabolism caused by multiple genes or by induction of gene expression or, alternatively, suffer from adverse effects of the drug treatment due to the presence of defective alleles. It is likely that future predictive genotyping for such enzymes might benefit 15–25% of drug treatments, and thereby allow prevention of adverse drug reactions and causalities, and thus improve the health of a significant fraction of the patients. However, it will take time before this will be a reality within the clinic. We describe some important aspects in the field with emphasis on cytochrome P450 and discuss also polymorphic aspects of foetal expression of CYP3A5 and CYP3A7. PMID:16096104

5,10 Methylenetetrahydrofolate reductase genetic polymorphism as a risk factor for neural tube defects

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

Ou, C.Y.; Brown, V.K.; Khoury, M.J.

1996-06-28

Persons with a thermolabile form of the enzyme 5,10 methylenetetrahydrofolate reductase (MTHFR) have reduced enzyme activity and increased plasma homocysteine which can be lowered by supplemental folic acid. Thermolability of the enzyme has recently been shown to be caused by a common mutation (677C{sup {r_arrow}}T) in the MTHFR gene. We studied 41 fibroblast cultures from NTD-affected fetuses and compared their genotypes with those of 109 blood specimens from individuals in the general population. 677C{sup {r_arrow}}T homozygosity was associated with a 7.2 fold increased risk for NTDs (95% confidence interval: 1.8-30.3; p value: 0.001). These preliminary data suggest that the 677C{supmore » {r_arrow}}T polymorphism of the MTHFR gene is a risk factor for spina bifida and anencephaly that may provide a partial biologic explanation for why folic acid prevents these types of NTD. 13 refs., 1 fig., 1 tab.« less

Structural Basis for Flip-Flop Action of Thiamin-Dependent Enzymes Revealed by Crystal Structure of Human Pyruvate Dehydrogenase

NASA Technical Reports Server (NTRS)

Ciszak, Ewa; Korotchkina, Lioubov G.; Dominiak, Paulina M.; Sidhu, Sukdeep; Patel, Mulchand S.

2003-01-01

The biologically active derivative of vitamin B1; thiamin pyrophosphate; is used as cofactor by many enzymes that perform a wide range of catalytic functions in the pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, the first catalytic component enzyme of human pyruvate dehydrogenase complex, this cofactor is used to cleave the C(sup alpha)-C(=0) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase, the second catalytic component of the complex. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites have puzzled researchers from earlier functional studies of this enzyme. In order to gain insight into the mechanism of action of this enzyme, we determined the crystal structure of the holoform of human pyruvate dehydrogenase at 1.958, resolution. We propose a kinetic model for the flip-flop action of this enzyme through the concerted approx. 2A, shuttle-like motion of the heterodimers. The similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase and other functionally related enzymes suggests this newly defined mechanism of shuttle-like motion of domains to be common for the family of thiamin pyrophosphate-dependent enzymes.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3-independent mechanism.

PubMed

Gwynn, B; Ciciotte, S L; Hunter, S J; Washburn, L L; Smith, R S; Andersen, S G; Swank, R T; Dell'Angelica, E C; Bonifacino, J S; Eicher, E M; Peters, L L

2000-12-15

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes of cno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3-independent mechanism critical to the biogenesis of lysosome-related organelles. (Blood. 2000;96:4227-4235)

Succinate dehydrogenase (SDHx) mutations in pituitary tumors: could this be a new role for mitochondrial complex II and/or Krebs cycle defects?

PubMed

Xekouki, Paraskevi; Stratakis, Constantine A

2012-12-01

Succinate dehydrogenase (SDH) or mitochondrial complex II is a multimeric enzyme that is bound to the inner membrane of mitochondria and has a dual role as it serves both as a critical step of the tricarboxylic acid or Krebs cycle and as a member of the respiratory chain that transfers electrons directly to the ubiquinone pool. Mutations in SDH subunits have been implicated in the formation of familial paragangliomas (PGLs) and/or pheochromocytomas (PHEOs) and in Carney-Stratakis syndrome. More recently, SDH defects were associated with predisposition to a Cowden disease phenotype, renal, and thyroid cancer. We recently described a kindred with the coexistence of familial PGLs and an aggressive GH-secreting pituitary adenoma, harboring an SDHD mutation. The pituitary tumor showed loss of heterozygosity at the SDHD locus, indicating the possibility that SDHD's loss was causatively linked to the development of the neoplasm. In total, 29 cases of pituitary adenomas presenting in association with PHEOs and/or extra-adrenal PGLs have been reported in the literature since 1952. Although a number of other genetic defects are possible in these cases, we speculate that the association of PHEOs and/or PGLs with pituitary tumors is a new syndromic association and a novel phenotype for SDH defects.

Succinate dehydrogenase (SDHx) mutations in pituitary tumors: could this be a new role for mitochondrial complex II and/or Krebs cycle defects?

PubMed Central

Xekouki, Paraskevi; Stratakis, Constantine A

2013-01-01

Succinate dehydrogenase (SDH) or mitochondrial complex II is a multimeric enzyme that is bound to the inner membrane of mitochondria and has a dual role as it serves both as a critical step of the tricarboxylic acid or Krebs cycle and as a member of the respiratory chain that transfers electrons directly to the ubiquinone pool. Mutations in SDH subunits have been implicated in the formation of familial paragangliomas (PGLs) and/or pheochromocytomas (PHEOs) and in Carney–Stratakis syndrome. More recently, SDH defects were associated with predisposition to a Cowden disease phenotype, renal, and thyroid cancer. We recently described a kindred with the coexistence of familial PGLs and an aggressive GH-secreting pituitary adenoma, harboring an SDHD mutation. The pituitary tumor showed loss of heterozygosity at the SDHD locus, indicating the possibility that SDHD’s loss was causatively linked to the development of the neoplasm. In total, 29 cases of pituitary adenomas presenting in association with PHEOs and/or extra-adrenal PGLs have been reported in the literature since 1952. Although a number of other genetic defects are possible in these cases, we speculate that the association of PHEOs and/or PGLs with pituitary tumors is a new syndromic association and a novel phenotype for SDH defects. PMID:22889736

Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

PubMed

Suzuki, Sho W; Onodera, Jun; Ohsumi, Yoshinori

2011-02-25

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

Evolution of Metastable Defects and Its Effect on the Electronic Properties of MoS2 Films.

PubMed

Precner, M; Polaković, T; Qiao, Qiao; Trainer, D J; Putilov, A V; Di Giorgio, C; Cone, I; Zhu, Y; Xi, X X; Iavarone, M; Karapetrov, G

2018-04-30

We report on structural and electronic properties of defects in chemical vapor-deposited monolayer and few-layer MoS 2 films. Scanning tunneling microscopy, Kelvin probe force microscopy, and transmission electron microscopy were used to obtain high resolution images and quantitative measurements of the local density of states, work function and nature of defects in MoS 2 films. We track the evolution of defects that are formed under heating and electron beam irradiation. We observe formation of metastable domains with different work function values after annealing the material in ultra-high vacuum to moderate temperatures. We attribute these metastable values of the work function to evolution of crystal defects forming during the annealing. The experiments show that sulfur vacancies formed after exposure to elevated temperatures diffuse, coalesce, and migrate bringing the system from a metastable to equilibrium ground state. The process could be thermally or e-beam activated with estimated energy barrier for sulfur vacancy migration of 0.6 eV in single unit cell MoS 2 . Even at equilibrium conditions, the work function and local density of states values are strongly affected near grain boundaries and edges. The results provide initial estimates of the thermal budgets available for reliable fabrication of MoS 2 -based integrated electronics and indicate the importance of defect control and layer passivation.

DGCR6 at the proximal part of the DiGeorge critical region is involved in conotruncal heart defects

PubMed Central

Gao, Wenming; Higaki, Takashi; Eguchi-Ishimae, Minenori; Iwabuki, Hidehiko; Wu, Zhouying; Yamamoto, Eiichi; Takata, Hidemi; Ohta, Masaaki; Imoto, Issei; Ishii, Eiichi; Eguchi, Mariko

2015-01-01

Cardiac anomaly is one of the hallmarks of DiGeorge syndrome (DGS), observed in approximately 80% of patients. It often shows a characteristic morphology, termed as conotruncal heart defects. In many cases showing only the conotruncal heart defect, deletion of 22q11.2 region cannot be detected by fluorescence in situ hybridization (FISH), which is used to detect deletion in DGS. We investigated the presence of genomic aberrations in six patients with congenital conotruncal heart defects, who show no deletion at 22q11.2 in an initial screening by FISH. In these patients, no abnormalities were identified in the coding region of the TBX1 gene, one of the key genes responsible for the phenotype of DGS. However, when copy number alteration was analyzed by high-resolution array analysis, a small deletion or duplication in the proximal end of DiGeorge critical region was detected in two patients. The affected region contains the DGCR6 and PRODH genes. DGCR6 has been reported to affect the expression of the TBX1 gene. Our results suggest that altered dosage of gene(s) other than TBX1, possibly DGCR6, may also be responsible for the development of conotruncal heart defects observed in patients with DGS and, in particular, in those with stand-alone conotruncal heart defects. PMID:27081520

Evolution of Metastable Defects and Its Effect on the Electronic Properties of MoS 2 Films

DOE PAGES

Precner, Marian; Polakovic, T.; Qiao, Qiao; ...

2018-04-30

Here, we report on structural and electronic properties of defects in chemical vapor-deposited monolayer and few-layer MoS 2 films. Scanning tunneling microscopy, Kelvin probe force microscopy, and transmission electron microscopy were used to obtain high resolution images and quantitative measurements of the local density of states, work function and nature of defects in MoS 2 films. We track the evolution of defects that are formed under heating and electron beam irradiation. We observe formation of metastable domains with different work function values after annealing the material in ultra-high vacuum to moderate temperatures. We attribute these metastable values of the workmore » function to evolution of crystal defects forming during the annealing. The experiments show that sulfur vacancies formed after exposure to elevated temperatures diffuse, coalesce, and migrate bringing the system from a metastable to equilibrium ground state. The process could be thermally or e-beam activated with estimated energy barrier for sulfur vacancy migration of 0.6 eV in single unit cell MoS 2. Even at equilibrium conditions, the work function and local density of states values are strongly affected near grain boundaries and edges. The results provide initial estimates of the thermal budgets available for reliable fabrication of MoS 2-based integrated electronics and indicate the importance of defect control and layer passivation.« less

Evolution of Metastable Defects and Its Effect on the Electronic Properties of MoS 2 Films

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

Precner, Marian; Polakovic, T.; Qiao, Qiao

Here, we report on structural and electronic properties of defects in chemical vapor-deposited monolayer and few-layer MoS 2 films. Scanning tunneling microscopy, Kelvin probe force microscopy, and transmission electron microscopy were used to obtain high resolution images and quantitative measurements of the local density of states, work function and nature of defects in MoS 2 films. We track the evolution of defects that are formed under heating and electron beam irradiation. We observe formation of metastable domains with different work function values after annealing the material in ultra-high vacuum to moderate temperatures. We attribute these metastable values of the workmore » function to evolution of crystal defects forming during the annealing. The experiments show that sulfur vacancies formed after exposure to elevated temperatures diffuse, coalesce, and migrate bringing the system from a metastable to equilibrium ground state. The process could be thermally or e-beam activated with estimated energy barrier for sulfur vacancy migration of 0.6 eV in single unit cell MoS 2. Even at equilibrium conditions, the work function and local density of states values are strongly affected near grain boundaries and edges. The results provide initial estimates of the thermal budgets available for reliable fabrication of MoS 2-based integrated electronics and indicate the importance of defect control and layer passivation.« less

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

Sinnecker, G.H.G; Hiort, O.; Kruse, K.

Conversion of testosterone (T) to dihydrotestosterone (DHT) in genital tissue is catalysed by the enzyme 5{alpha}-reductase 2, which is encoded by the SRD5A2 gene. The potent androgen DHT is required for full masculinization of the external genitalia. Mutations of the SRD5A2 gene inhibit enzyme activity, diminish DHT formation, and hence cause masculinization defects of varying degree. The classical syndrome, formerly described as pseudovaginal perineoscrotal hypospadias, is characterized by a predominantly female phenotype at birth and significant virilization without gynecomastia at puberty. We investigated nine patients with steroid 5{alpha}-reductase 2 deficiency (SRD). T/DHT-ratios were highly increased in the classical syndrome, butmore » variable in the less severe affected patients. Mutations in the SRD5A2 gene had been characterized using PCR-SSCP analysis and direct DNA sequencing. A small deletion was encountered in two patients, while all other patients had single base mutations which result in amino acid substitutions. We conclude that phenotypes may vary widely in patients with SRD5A2 gene mutations spanning the whole range from completely female to normal male without distinctive clinical signs of the disease. Hence, steroid 5{alpha}-reductase deficiency should be considered not only in sex reversed patients with female or ambiguous phenotypes, but also in those with mild symptoms of undermasculinization as encountered in patients with hypospadias and/or micropenis. A classification based on the severity of the masculinization defect may be used for correlation of phenotypes with enzyme activities and genotypes, and for comparisons of phenotypes between different patients as the basis for clinical decisions to be made in patients with pseudohermaphroditism due to steroid 5{alpha}-reductase 2 deficiency. 22 refs., 2 figs., 2 tabs.« less

Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

PubMed Central

Allombert, Julie; Lazzaroni, Jean-Claude; Baïlo, Nathalie; Gilbert, Christophe; Charpentier, Xavier; Doublet, Patricia

2014-01-01

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. PMID:24379287

In silico approach to explore the disruption in the molecular mechanism of human hyaluronidase 1 by mutant E268K that directs Natowicz syndrome.

PubMed

Meshach Paul, D; Rajasekaran, R

2017-03-01

Natowicz syndrome (mucopolysaccharidoses type 9) is a lysosomal storage disorder caused by deficient or defective human hyaluronidase 1. The disorder is not well studied at the molecular level. Therefore, a new in silico approach was proposed to study the molecular basis on which one clinically observed mutation, Glu268Lys, results in a defective enzyme. The native and mutant structures were subjected to comparative analyses using a conformational sampling approach for geometrical variables viz, RMSF, RMSD, and Ramachandran plot. In addition, the strength of a Cys207-Cys221 disulfide bond and electrostatic interaction between Arg265 and Asp206 were studied, as they are known to be involved in the catalytic activity of the enzyme. Native and mutant E268K showed statistically significant variations with p < 0.05 in RMSD, Ramachandran plot, strengths of disulfide bond, and electrostatic interactions. Further, single model analysis showed variations between native and mutant structures in terms of intra-protein interactions, hydrogen bond dilution, secondary structure, and dihedral angles. Docking analysis predicted the mutant to have a less favorable substrate binding energy compared to the native protein. Additionally, steered MD analysis indicated that the substrate should have more affinity to the native than mutant enzymes. The observed changes theoretically explain the less favorable binding energy of substrate towards mutant E268K, thereby providing a structural basis for its reduced catalytic activity. Hence, our study provides a basis for understanding the disruption in the molecular mechanism of human hyaluronidase 1 by mutation E268K, which may prove useful for the development of synthetic chaperones as a treatment option for Natowicz syndrome.

Filamentous invasive growth of mutants of the genes encoding ammonia-metabolizing enzymes in the fission yeast Schizosaccharomyces pombe.

PubMed

Sasaki, Yoshie; Kojima, Ayumi; Shibata, Yuriko; Mitsuzawa, Hiroshi

2017-01-01

The fission yeast Schizosaccharomyces pombe undergoes a switch from yeast to filamentous invasive growth in response to certain environmental stimuli. Among them is ammonium limitation. Amt1, one of the three ammonium transporters in this yeast, is required for the ammonium limitation-induced morphological transition; however, the underlying molecular mechanism remains to be understood. Cells lacking Amt1 became capable of invasive growth upon increasing concentrations of ammonium in the medium, suggesting that the ammonium taken up into the cell or a metabolic intermediate in ammonium assimilation might serve as a signal for the ammonium limitation-induced morphological transition. To investigate the possible role of ammonium-metabolizing enzymes in the signaling process, deletion mutants were constructed for the gdh1, gdh2, gln1, and glt1 genes, which were demonstrated by enzyme assays to encode NADP-specific glutamate dehydrogenase, NAD-specific glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, respectively. Growth tests on various nitrogen sources revealed that a gln1Δ mutant was a glutamine auxotroph and that a gdh1Δ mutant had a defect in growth on ammonium, particularly at high concentrations. The latter observation indicates that the NADP-specific glutamate dehydrogenase of S. pombe plays a major role in ammonium assimilation under high ammonium concentrations. Invasive growth assays showed that gdh1Δ and glt1Δ mutants underwent invasive growth to a lesser extent than did wild-type strains. Increasing the ammonium concentration in the medium suppressed the invasive growth defect of the glt1Δ mutant, but not the gdh1Δ mutant. These results suggest that the nitrogen status of the cell is important in the induction of filamentous invasive growth in S. pombe.

Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

PubMed Central

Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

2016-01-01

Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

Defective mitochondrial RNA processing due to PNPT1 variants causes Leigh syndrome.

PubMed

Matilainen, Sanna; Carroll, Christopher J; Richter, Uwe; Euro, Liliya; Pohjanpelto, Max; Paetau, Anders; Isohanni, Pirjo; Suomalainen, Anu

2017-09-01

Leigh syndrome is a severe infantile encephalopathy with an exceptionally variable genetic background. We studied the exome of a child manifesting with Leigh syndrome at one month of age and progressing to death by the age of 2.4 years, and identified novel compound heterozygous variants in PNPT1, encoding the polynucleotide phosphorylase (PNPase). Expression of the wild type PNPT1 in the subject's myoblasts functionally complemented the defects, and the pathogenicity was further supported by structural predictions and protein and RNA analyses. PNPase is a key enzyme in mitochondrial RNA metabolism, with suggested roles in mitochondrial RNA import and degradation. The variants were predicted to locate in the PNPase active site and disturb the RNA processing activity of the enzyme. The PNPase trimer formation was not affected, but specific RNA processing intermediates derived from mitochondrial transcripts of the ND6 subunit of Complex I, as well as small mRNA fragments, accumulated in the subject's myoblasts. Mitochondrial RNA processing mediated by the degradosome consisting of hSUV3 and PNPase is poorly characterized, and controversy on the role and location of PNPase within human mitochondria exists. Our evidence indicates that PNPase activity is essential for the correct maturation of the ND6 transcripts, and likely for the efficient removal of degradation intermediates. Loss of its activity will result in combined respiratory chain deficiency, and a classic respiratory chain-deficiency-associated disease, Leigh syndrome, indicating an essential role for the enzyme for normal function of the mitochondrial respiratory chain. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Infrared absorption study of neutron-transmutation-doped germanium

NASA Technical Reports Server (NTRS)

Park, I. S.; Haller, E. E.

1988-01-01

Using high-resolution far-infrared Fourier transform absorption spectroscopy and Hall effect measurements, the evolution of the shallow acceptor and donor impurity levels in germanium during and after the neutron transmutation doping process was studied. The results show unambiguously that the gallium acceptor level concentration equals the concentration of transmutated Ge-70 atoms during the whole process indicating that neither recoil during transmutation nor gallium-defect complex formation play significant roles. The arsenic donor levels appear at full concentration only after annealing for 1 h at 450 C. It is shown that this is due to donor-radiation-defect complex formation. Again, recoil does not play a significant role.