Construction and enzymatic degradation of multilayered poly-l-lysine/DNA films.

PubMed

Ren, Kefeng; Ji, Jian; Shen, Jiacong

2006-03-01

The layer-by-layer (LbL) self-assembly of poly-l-lysine (PLL) and deoxyribonucleic acid (DNA) was used to construct the enzymatic biodegradable multilayered films. The LbL build up of DNA multilayers was monitored by UV-vis spectrometry, and atomic force microscopy (AFM). AFM, UV-vis spectrometry and fluorescence spectrometry measurements indicated that 90% of DNA within the films was released almost linearly under 5 U mL(-1)alpha-chymotrypsin in PBS at 37 degrees C in 35 h. TEM and zeta potential experiments revealed that the released DNA molecules were condensed into the slight positive complexes with size from 20 to several hundred nanometers. The well-structured, easy processed enzymatic biodegradable multilayered film may have great potential for gene applications in tissue engineering, medical implants, etc.

Key Building Blocks via Enzyme-Mediated Synthesis

NASA Astrophysics Data System (ADS)

Fischer, Thomas; Pietruszka, Jörg

Biocatalytic approaches to valuable building blocks in organic synthesis have emerged as an important tool in the last few years. While first applications were mainly based on hydrolases, other enzyme classes such as oxidoreductases or lyases moved into the focus of research. Nowadays, a vast number of biotransformations can be found in the chemical and pharmaceutical industries delivering fine chemicals or drugs. The mild reaction conditions, high stereo-, regio-, and chemoselectivities, and the often shortened reaction pathways lead to economical and ecological advantages of enzymatic conversions. Due to the enormous number of enzyme-mediated syntheses, the present chapter is not meant to be a complete review, but to deliver comprehensive insights into well established enzymatic systems and recent advances in the application of enzymes in natural product synthesis. Furthermore, it is focused on the most frequently used enzymes or enzyme classes not covered elsewhere in the present volume.

Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

PubMed

Nongonierma, Alice B; FitzGerald, Richard J

2018-06-01

Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

Influence of enzymatic extrusion liquefaction pretreatment for Chinese rice wine on the volatiles generated from extruded rice.

PubMed

Xu, Enbo; Li, Hongyan; Wu, Zhengzong; Wang, Fang; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

2015-01-01

Volatile compounds in enzymatic extruded rice, produced under different conditions of varying barrel temperature (BT), α-amylase concentration (AC) and moisture content (MC), were extracted and identified by headspace solid phase microextraction (HS-SPME) and gas chromatography-linked mass spectrometry (GC-MS). Statistical analyses reflected that the Maillard reaction could be inhibited both by the mild extrusion conditions and the enhanced hydrolysis caused by thermostable α-amylase. Relative amounts of total volatiles in enzymatic extruded rice were far less than those in severe processed extruded rice. Reverse-phase high-performance liquid chromatography (RP-HPLC) showed that the amino acids (AAs) involved in Maillrad reaction were utmostly preserved in extruded rice with highest amylase concentration by comparison of total AA content of different extrudates. These results suggest that enzymatic extrusion liquefaction is an effective way to control the generation of volatiles from extruded rice for Chinese rice wine production. © 2014 Institute of Food Technologists®

Modeling Atmospheric Reactive Nitrogen

EPA Science Inventory

Nitrogen is an essential building block of all proteins and thus an essential nutrient for all life. Reactive nitrogen, which is naturally produced via enzymatic reactions, forest fires and lightning, is continually recycled and cascades through air, water, and soil media. Human ...

Chemical functionalization of surfaces for building three-dimensional engineered biosensors

NASA Astrophysics Data System (ADS)

Marques, Marco E.; Mansur, Alexandra A. P.; Mansur, Herman S.

2013-06-01

This study presents a new approach for developing biosensors based on enzymatic systems with designed three-dimensional structures. Silica glass slides were chemically functionalized at surfaces by reacting with organosilanes, 3-mercaptopropyltriethoxysilane (MPTES), and 3-aminopropyltriethoxysilane (APTES), using sol-gel process at room temperature. The functionalization of the supports was characterized by contact angle measurements and FTIR spectroscopy. The first enzyme layer was covalently immobilized to the support by a bi-functional linker (glutaraldehyde). The second enzyme layer was deposited using the protein conjugation method based on the high affinity "avidin-biotin" interactions. Each enzyme was biotinylated before being added to the nanostructured system and avidin was used as the binder between consecutive enzyme layers. The biochemical response was assayed at all stages to certify that the enzymatic bioactivity was retained throughout the entire layer-by-layer (LBL) process. The model of building 3D-enzymatic systems was evaluated using the enzymatic structure with glucose oxidase (GOx) and horseradish peroxidase (HRP). It was verified that the amino-modified support presented the highest bioactivity response compared to the other chemical functionalities. Moreover, the bienzyme nanostructure demonstrated relevant biochemical activity upon injecting the glucose substrate into the system. Finally, as a proof of concept, the bienzyme systems were assayed using real samples of regular and sugar-free soft drinks where they effectively behaved as structured biosensor for glucose with the built-in 3D hybrid architecture. Based on the results, it can be foreseen the development of promising new nanomaterials for several analytical applications such as monitoring the quality of food and beverages for nutrition purposes.

Fuel ethanol production from corn stover under optimized dilute phosphoric acid pretreatment and enzymatic hydrolysis

USDA-ARS?s Scientific Manuscript database

Ethanol is a renewable oxygenated fuel. Dilute acid pretreatment is a promising pretreatment technology for conversion of lignocellulosic biomass to fuel ethanol. Generation of fermentable sugars from corn stover involves pretreatment and enzymatic saccharification. Pretreatment is crucial as nat...

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors

PubMed Central

Rodriguez, Sofia B.; Leyh, Thomas S.

2014-01-01

The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation. PMID:25153179

Application of a low level, uniform ultrasound field for the acceleration of enzymatic bio-processing of cotton

USDA-ARS?s Scientific Manuscript database

Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. Our research has found that th...

Application of Low Level, Uniform Ultrasound Field for Acceleration of Enzymatic Bio-processing of Cotton

USDA-ARS?s Scientific Manuscript database

Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. Our research has found that th...

The Closure of the Cycle: Enzymatic Synthesis and Functionalization of Bio-Based Polyesters.

PubMed

Pellis, Alessandro; Herrero Acero, Enrique; Ferrario, Valerio; Ribitsch, Doris; Guebitz, Georg M; Gardossi, Lucia

2016-04-01

The polymer industry is under pressure to mitigate the environmental cost of petrol-based plastics. Biotechnologies contribute to the gradual replacement of petrol-based chemistry and the development of new renewable products, leading to the closure of carbon circle. An array of bio-based building blocks is already available on an industrial scale and is boosting the development of new generations of sustainable and functionally competitive polymers, such as polylactic acid (PLA). Biocatalysts add higher value to bio-based polymers by catalyzing not only their selective modification, but also their synthesis under mild and controlled conditions. The ultimate aim is the introduction of chemical functionalities on the surface of the polymer while retaining its bulk properties, thus enlarging the spectrum of advanced applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Self-interrupted synthesis of sterically hindered aliphatic polyamide dendrimers

PubMed Central

Jishkariani, Davit; Timsina, Yam N.; Grama, Silvia; Gillani, Syeda S.; Divar, Masoumeh; Yadavalli, Srujana S.; Moussodia, Ralph-Olivier; Leowanawat, Pawaret; Berrios Camacho, Angely M.; Walter, Ricardo; Goulian, Mark; Klein, Michael L.; Percec, Virgil

2017-01-01

2,2-Bis(azidomethyl)propionic acid was prepared in four steps and 85% yield from the commercially available 2,2-bis(hydroxymethyl)propionic acid and used as the starting building block for the divergent, convergent, and double-stage convergent–divergent iterative methods for the synthesis of dendrimers and dendrons containing ethylenediamine (EDA), piperazine (PPZ), and methyl 2,2-bis(aminomethyl)propionate (COOMe) cores. These cores have the same multiplicity but different conformations. A diversity of synthetic methods were used for the synthesis of dendrimers and dendrons. Regardless of the method used, a self-interruption of the synthesis was observed at generation 4 for the dendrimer with an EDA core and at generation 5 for the one with a PPZ core, whereas for the COOMe core, self-interruption was observed at generation 6 dendron, which is equivalent to generation 5 dendrimer. Molecular modeling and molecular-dynamics simulations demonstrated that the observed self-interruption is determined by the backfolding of the azide groups at the periphery of the dendrimer. The latter conformation inhibits completely the heterogeneous hydrogenation of the azide groups catalyzed by 10% Pd/carbon as well as homogeneous hydrogenation by the Staudinger method. These self-terminated polyamide dendrimers are enzymatically and hydrolytically stable and also exhibit antimicrobial activity. Thus, these nanoscale constructs open avenues for biomedical applications. PMID:28270599

Carpatizine, a novel bridged oxazine derivative generated by non-enzymatic reactions.

PubMed

Fu, Peng; MacMillan, John B

2017-06-27

Carpatizine (1), a new bridged oxazine derivative, was isolated from a marine-derived Streptomyces strain SNE-011. The structure was fully determined by spectroscopic analysis, ECD calculations and chemical methods. A plausible non-enzymatic reaction mechanism from daryamide D leading to carpatizine was presented, which was confirmed by chemical transformation.

The Effects of a Low-Level Uniform Ultrasound Field on Enzymatic Bio-Processing of Cotton: An Investigation of Three Fabric Weights

USDA-ARS?s Scientific Manuscript database

Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. Our research has found that t...

Mechanistic investigation in ultrasound induced enhancement of enzymatic hydrolysis of invasive biomass species.

PubMed

Borah, Arup Jyoti; Agarwal, Mayank; Poudyal, Manisha; Goyal, Arun; Moholkar, Vijayanand S

2016-08-01

This study has assessed four invasive weeds, viz. Saccharum spontaneum (SS), Mikania micrantha (MM), Lantana camara (LC) and Eichhornia crassipes (EC) for enzymatic hydrolysis prior to bioalcohol fermentation. Enzymatic hydrolysis of pretreated biomasses of weeds has been conducted with mechanical agitation and sonication under constant (non-optimum) conditions. Profiles of total reducible sugar release have been fitted to HCH-1 model of enzymatic hydrolysis using Genetic Algorithm. Trends in parameters of this model reveal physical mechanism of ultrasound-induced enhancement of enzymatic hydrolysis. Sonication accelerates hydrolysis kinetics by ∼10-fold. This effect is contributed by several causes, attributed to intense micro-convection generated during sonication: (1) increase in reaction velocity, (2) increase in enzyme-substrate affinity, (3) reduction in product inhibition, and (4) enhancement of enzyme activity due to conformational changes in its secondary structure. Enhancement effect of sonication is revealed to be independent of conditions of enzymatic hydrolysis - whether optimum or non-optimum. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modeling and Recent Shift in the Composition of Atmospheric Reactive Nitrogen

EPA Science Inventory

Nitrogen is an essential building block of all proteins and thus an essential nutrient for all life. Reactive nitrogen, which is naturally produced via enzymatic reactions, forest fires and lightning, is continually recycled and cascades through air, water, and soil media. Human ...

Design, fabrication, and operation of hybrid bionanodevices for biomedical applications

NASA Astrophysics Data System (ADS)

Tucker, Robert Matthew

Cells are the fundamental building blocks of life. Despite their simplicity, cells are extremely versatile, performing a variety of functions including detection, signaling, and repair. While current biomedical devices operate at the organ level, the next generation will operate at the cellular level, combining the nanoscale machinery of cells with the mechanical robustness of synthetic materials in the form of new hybrid devices. This thesis presents advances in four topics concerning the development of nanomedical devices: fabrication, stabilization, control, and operation. First, as feature sizes decrease from the milli- and microscale towards the nanoscale, new fabrication methods must be developed. A new rapid prototyping technique using confocal microscopy was used to produce freely-programmable high-resolution protein patterns of functional motor proteins on thermo-responsive polymer surfaces. Second, hybrid device operation should be temperature-independent, but most biological components have strong responses to temperature fluctuations. To counter operational fluctuations, the temperature-dependent enzymatic activity was characterized for two types of molecular motors with the goal of developing a bionanosystem which is stabilized against temperature fluctuations. Third, replacing electromechanical systems consisting of pumps and batteries with proteins that directly convert chemical potential into mechanical energy increases the efficiency and decreases the size of the bionanodevice, but requires new control methods. An enzymatic network was developed in which fuel was photolytically released to activate molecular shuttles, excess fuel was sequestered using an enzyme, and spatial and temporal control of the system was achieved. Finally, chemically powered bionanodevices will require high-precision nano- and microscale actuators. A two-part hybrid actuator was designed, which consists of a molecular motor-coated synthetic macroscale forcer and a microtubule-based stator. Methods to create and characterize the stator were developed, which can be used to optimize the force generation of the device.

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

Pham, Grace H.; Rana, Ambar S. J. B.; Korkmaz, E. Nihal

Ubiquitin (Ub) chains regulate a wide range of biological processes, and Ub chain connectivity is a critical determinant of the many regulatory roles that this post-translational modification plays in cells. To understand how distinct Ub chains orchestrate different biochemical events, we and other investigators have developed enzymatic and non-enzymatic methods to synthesize Ub chains of well-defined length and connectivity. A number of chemical approaches have been used to generate Ub oligomers connected by non-native linkages; however, few studies have examined the extent to which non-native linkages recapitulate the structural and functional properties associated with native isopeptide bonds. Here, we comparemore » the structure and function of Ub dimers bearing native and non-native linkages. Using small-angle X-ray scattering (SAXS) analysis, we show that scattering profiles for the two types of dimers are similar. Moreover, using an experimental structural library and atomistic simulations to fit the experimental SAXS profiles, we find that the two types of Ub dimers can be matched to analogous structures. An important application of non-native Ub oligomers is to probe the activity and selectivity of deubiquitinases. Through steady-state kinetic analyses, we demonstrate that different families of deubiquitinases hydrolyze native and non-native isopeptide linkages with comparable efficiency and selectivity. Considering the significant challenges associated with building topologically diverse native Ub chains, our results illustrate that chains harboring non-native linkages can serve as surrogate substrates for explorations of Ub function.« less

The Impact of Marine Enzymatic Activity on Sea Spray Aerosol Properties

NASA Astrophysics Data System (ADS)

Ryder, O. S.; Michaud, J. M.; Sauer, J. S.; Lee, C.; Förster, J. D.; Pöhlker, C.; Andreae, M. O.; Prather, K. A.

2016-12-01

The composition of sea spray aerosol (SSA) and the relationship between its organic fraction and biological ocean conditions is not well understood, resulting in considerable disagreement in the literature linking biological markers to SSA chemical composition. Recent work suggests that enzymatic activity in seawater may play a key role in dictating aerosol composition by changing the organic pool from which SSA is formed. Here we investigate the role of enzymatic activity on SSA spatial chemical composition, aerosol phase and morphological microstructure. In these experiments, SSA was generated using a novel mini-Marine Aerosol Reference Tank system. SSA collected onto substrates was generated from artificial salt water that had been doped with either 1) unsaturated triglycerides or 2) diatom cellular lysate, both followed by lipase. Results from analysis including morphological studies via atomic force microscopy, and chemical composition investigations both under dry and RH conditions via STXM-NEXAFS are presented.

Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease

PubMed Central

Uderhardt, Stefan; Ackermann, Jochen A.; Fillep, Tobias; Hammond, Victoria J.; Willeit, Johann; Stark, Konstantin; Rossaint, Jan; Schubert, Irene; Mielenz, Dirk; Dietel, Barbara; Raaz-Schrauder, Dorette; Ay, Cihan; Thaler, Johannes; Heim, Christian; Collins, Peter W.; Schabbauer, Gernot; Mackman, Nigel; Voehringer, David; Nadler, Jerry L.; Lee, James J.; Massberg, Steffen; Rauh, Manfred; O’Donnell, Valerie B.

2017-01-01

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase–derived hydroxyeicosatetraenoic acid–phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease. PMID:28566277

Biotransformation of β-keto nitriles to chiral (S)-β-amino acids using nitrilase and ω-transaminase.

PubMed

Mathew, Sam; Nadarajan, Saravanan Prabhu; Sundaramoorthy, Uthayasuriya; Jeon, Hyunwoo; Chung, Taeowan; Yun, Hyungdon

2017-04-01

To enzymatically synthesize enantiomerically pure β-amino acids from β-keto nitriles using nitrilase and ω-transaminase. An enzyme cascade system was designed where in β-keto nitriles are initially hydrolyzed to β-keto acids using nitrilase from Bradyrhizobium japonicum and subsequently β-keto acids were converted to β-amino acids using ω-transaminases. Five different ω-transaminases were tested for this cascade reaction, To enhance the yields of β-amino acids, the concentrations of nitrilase and amino donor were optimized. Using this enzymatic reaction, enantiomerically pure (S)-β-amino acids (ee > 99%) were generated. As nitrilase is the bottleneck in this reaction, molecular docking analysis was carried out to depict the poor affinity of nitrilase towards β-keto acids. A novel enzymatic route to generate enantiomerically pure aromatic (S)-β-amino acids from β-keto nitriles is demonstrated for the first time.

Modeling reactive nitrogen in North America: recent developments, observational needs and future directions

EPA Science Inventory

Nitrogen is an essential building block of all proteins and thus an essential nutrient for all life. The bulk of nitrogen in the environment is tightly bound as non-reactive N₂. Reactive nitrogen, which is naturally produced via enzymatic reactions, forest ...

Enzymatic Transition States, Transition-State Analogs, Dynamics, Thermodynamics, and Lifetimes

PubMed Central

Schramm, Vern L.

2017-01-01

Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein’s dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states. PMID:21675920

Comparative Proteomic Analyses of Human Adipose Extracellular Matrices Decellularized Using Alternative Procedures.

PubMed

Thomas-Porch, Caasy; Li, Jie; Zanata, Fabiana; Martin, Elizabeth C; Pashos, Nicholas; Genemaras, Kaylynn; Poche, J Nicholas; Totaro, Nicholas P; Bratton, Melyssa R; Gaupp, Dina; Frazier, Trivia; Wu, Xiying; Ferreira, Lydia Masako; Tian, Weidong; Wang, Guangdi; Bunnell, Bruce A; Flynn, Lauren; Hayes, Daniel; Gimble, Jeffrey M

2018-04-25

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for DNA depletion, ECM composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy (SEM). Mass spectrometry (MS) based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP + transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

PubMed

Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

2016-07-09

In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

SINEs as driving forces in genome evolution.

PubMed

Schmitz, J

2012-01-01

SINEs are short interspersed elements derived from cellular RNAs that repetitively retropose via RNA intermediates and integrate more or less randomly back into the genome. SINEs propagate almost entirely vertically within their host cells and, once established in the germline, are passed on from generation to generation. As non-autonomous elements, their reverse transcription (from RNA to cDNA) and genomic integration depends on the activity of the enzymatic machinery of autonomous retrotransposons, such as long interspersed elements (LINEs). SINEs are widely distributed in eukaryotes, but are especially effectively propagated in mammalian species. For example, more than a million Alu-SINE copies populate the human genome (approximately 13% of genomic space), and few master copies of them are still active. In the organisms where they occur, SINEs are a challenge to genomic integrity, but in the long term also can serve as beneficial building blocks for evolution, contributing to phenotypic heterogeneity and modifying gene regulatory networks. They substantially expand the genomic space and introduce structural variation to the genome. SINEs have the potential to mutate genes, to alter gene expression, and to generate new parts of genes. A balanced distribution and controlled activity of such properties is crucial to maintaining the organism's dynamic and thriving evolution. Copyright © 2012 S. Karger AG, Basel.

Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

PubMed

Xie, Yuliang; Ahmed, Daniel; Lapsley, Michael Ian; Lin, Sz-Chin Steven; Nawaz, Ahmad Ahsan; Wang, Lin; Huang, Tony Jun

2012-09-04

In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K(m) and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K(m) and k(cat) can then be obtained from the reaction rate curves for different concentrations of substrate while holding the enzyme concentration constant. We studied the enzymatic reaction for β-galactosidase and its substrate (resorufin-β-D-galactopyranoside) and found K(m) and k(cat) to be 333 ± 130 μM and 64 ± 8 s(-1), respectively, which are in agreement with published data. Our approach is valuable for studying the kinetics of high-speed enzymatic reactions and other chemical reactions.

Biomimetic cell wall model studies to identify new lignin bioengineering targets for improving biomass susceptibility to pretreatment and enzymatic saccharification

USDA-ARS?s Scientific Manuscript database

Increasingly, bioengineering of lignin to contain atypical building blocks from other metabolic pathways is being pursued to custom-design lignin that is easier to remove by chemical pretreatments and less inhibitory toward polysaccharide saccharification. Because plants produce such a diverse array...

A micromotor based on polymer single crystals and nanoparticles: toward functional versatility.

PubMed

Liu, Mei; Liu, Limei; Gao, Wenlong; Su, Miaoda; Ge, Ya; Shi, Lili; Zhang, Hui; Dong, Bin; Li, Christopher Y

2014-08-07

We report a multifunctional micromotor fabricated by the self-assembly technique using multifunctional materials, i.e. polymer single crystals and nanoparticles, as basic building blocks. Not only can this micromotor achieve autonomous and directed movement, it also possesses unprecedented functions, including enzymatic degradation-induced micromotor disassembly, sustained release and molecular detection.

Programmable bioelectronics in a stimuli-encoded 3D graphene interface

NASA Astrophysics Data System (ADS)

Parlak, Onur; Beyazit, Selim; Tse-Sum-Bui, Bernadette; Haupt, Karsten; Turner, Anthony P. F.; Tiwari, Ashutosh

2016-05-01

The ability to program and mimic the dynamic microenvironment of living organisms is a crucial step towards the engineering of advanced bioelectronics. Here, we report for the first time a design for programmable bioelectronics, with `built-in' switchable and tunable bio-catalytic performance that responds simultaneously to appropriate stimuli. The designed bio-electrodes comprise light and temperature responsive compartments, which allow the building of Boolean logic gates (i.e. ``OR'' and ``AND'') based on enzymatic communications to deliver logic operations.The ability to program and mimic the dynamic microenvironment of living organisms is a crucial step towards the engineering of advanced bioelectronics. Here, we report for the first time a design for programmable bioelectronics, with `built-in' switchable and tunable bio-catalytic performance that responds simultaneously to appropriate stimuli. The designed bio-electrodes comprise light and temperature responsive compartments, which allow the building of Boolean logic gates (i.e. ``OR'' and ``AND'') based on enzymatic communications to deliver logic operations. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr02355j

Effects of Extrusion Pretreatment Parameters on Sweet Sorghum Bagasse Enzymatic Hydrolysis and Its Subsequent Conversion into Bioethanol

PubMed Central

Heredia-Olea, Erick; Pérez-Carrillo, Esther; Serna-Saldívar, Sergio O.

2015-01-01

Second-generation bioethanol production from sweet sorghum bagasse first extruded at different conditions and then treated with cell wall degrading enzymes and fermented with I. orientalis was determined. The twin extruder parameters tested were barrel temperature, screws speed, and feedstock moisture content using surface response methodology. The best extrusion conditions were 100°C, 200 rpm, and 30% conditioning moisture content. This nonchemical and continuous pretreatment did not generate inhibitory compounds. The extruded feedstocks were saccharified varying the biocatalysis time and solids loading. The best conditions were 20% solids loading and 72 h of enzymatic treatment. These particular conditions converted 70% of the total fibrous carbohydrates into total fermentable C5 and C6 sugars. The extruded enzymatically hydrolyzed sweet sorghum bagasse was fermented with the strain I. orientalis at 12% solids obtaining a yield of 198.1 mL of ethanol per kilogram of bagasse (dw). PMID:25866776

Electricity generation from rapeseed straw hydrolysates using microbial fuel cells.

PubMed

Jablonska, Milena A; Rybarczyk, Maria K; Lieder, Marek

2016-05-01

Rapeseed straw is an attractive fuel material for microbial fuel cells (MFCs) due to its high content of carbohydrates (more than 60% carbohydrates). This study has demonstrated that reducing sugars can be efficiently extracted from raw rapeseed straw by combination of hydrothermal pretreatment and enzymatic hydrolysis followed by utilization as a fuel in two-chamber MFCs for electrical power generation. The most efficient method of saccharification of this lignocellulosic biomass (17%) turned out hydrothermal pretreatment followed by enzymatic hydrolysis. Electricity was produced using hydrolysate concentrations up to 150 mg/dm(3). The power density reached 54 mW/m(2), while CEs ranged from 60% to 10%, corresponding to the initial reducing sugar concentrations of 10-150 mg/dm(3). The COD degradation rates based on charge calculation increased from 0.445 g COD/m(2)/d for the hydrolysate obtained with the microwave treatment to 0.602 g COD/m(2)/d for the most efficient combination of hydrothermal treatment followed by enzymatic hydrolysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preliminary studies on the activities of spin traps as scavengers of free radicals

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

Ogunbiyi, P.O.; Washington, I.

1991-03-15

The spin trapping agents, N-t-Butyl-a-phenyl-nitrone (PBN) and 5,5-Dimethyl-1-pyroline-N-oxide (DMPO) have been used to investigate the primary free radicals involved in various tissue injuries. Also, PBN and DMPO can provide some protection against free radical-induced lung injuries. However, their therapeutic potentials as free radical scavengers remained unexamined. In this study, the effects of PBN and DMPO on guinea pig lung microsomal lipid peroxidation were investigated using thiobarbituric acid-reactive substance assay. Superoxide anions (O{sup 2}{minus}) were generated in an enzymatic and a non-enzymatic system. PBN and DMPO each, significantly inhibited NADPH-stimulated lipid peroxidation irrespective of the presence of Fe{sup 3+}. Cytochrome cmore » reduction by the enzymatic and nitro blue tetrazolium reduction by the non-enzymatic O{sup 2}{minus} generating systems were both inhibited by PBN and DMPO as well as superoxide dismutase and dimethyl sulfoxide when compared with the controls. The spin traps exhibited lower potencies in these systems than the reference compounds, SOD and DMSO, which are well established as O{sup 2}{minus} and hydroxyl radical scavengers respectively. Results demonstrate the free radical scavenging properties of PBN and DMPO. This is an indication of their possible usefulness as antioxidants.« less

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

PubMed Central

Berron, Brad J; Johnson, Leah M; Ba, Xiao; McCall, Joshua D; Alvey, Nicholas J; Anseth, Kristi S; Bowman, Christopher N

2011-01-01

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc. PMID:21337335

Characterization of Volatile Flavor Compounds in Chinese Rice Wine Fermented from Enzymatic Extruded Rice.

PubMed

Xu, Enbo; Long, Jie; Wu, Zhengzong; Li, Hongyan; Wang, Fang; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

2015-07-01

Enzymatic extrusion, instead of traditional steam cooking, to treat rice is an efficient and alternative pretreatment for Chinese rice wine fermentation. In order to determine the formation of volatiles in enzymatic extrusion-processed rice wine (EE), and to confirm its characteristic flavor compounds, headspace solid-phase micro-extraction followed by GC-MS was used. A total of 66 volatile compounds were identified in EE. During fermentation, most volatiles generated from enzymatic extruded rice had the similar trends with those from steam-cooked rice, but the differences in the concentration of volatiles indicated a changed balance of flavors release caused by enzymatic extrusion. Besides, the concentrations and sorts of volatiles in EEs fermented from different rice particle sizes, were not dramatically different. By principal component analysis, EE could be distinctly separated from other traditional Chinese rice wines according to its characteristic volatiles, namely, 2-heptanol, 1-octen-3-ol, ethyl 4-hydroxybenzoate, methylpentyl 2-propenoate, γ-hexalactone, and 4-vinylguaiacol. Enzymatic extrusion liquefaction has been a popular thermal treatment for cereals, and gradually being applied in fermentation and liquor-making industry all over the world. The characterization of volatile flavor compounds in Chinese rice wine processed by enzymatic extrusion liquefaction pretreatment, might be made use not only for a better understanding of this new-type rice wine, but for the further utilization of enzymatic extrusion in other wine or alcohol production as well. © 2015 Institute of Food Technologists®

Carborane-linked 2'-deoxyuridine 5'-O-triphosphate as building block for polymerase synthesis of carborane-modified DNA.

PubMed

Balintová, Jana; Simonova, Anna; Białek-Pietras, Magdalena; Olejniczak, Agnieszka; Lesnikowski, Zbigniew J; Hocek, Michal

2017-11-01

5-[(p-Carborane-2-yl)ethynyl]-2'-deoxyuridine 5'-O-triphosphate was synthesized and used as a good substrate in enzymatic construction of carborane-modified DNA or oligonucleotides containing up to 21 carborane moieties in primer extension reactions by DNA polymerases. Copyright © 2017 Elsevier Ltd. All rights reserved.

A mathematical model for the generation and control of a pH gradient in an immobilized enzyme system involving acid generation.

PubMed

Chen, G; Fournier, R L; Varanasi, S

1998-02-20

An optimal pH control technique has been developed for multistep enzymatic synthesis reactions where the optimal pH differs by several units for each step. This technique separates an acidic environment from a basic environment by the hydrolysis of urea within a thin layer of immobilized urease. With this technique, a two-step enzymatic reaction can take place simultaneously, in proximity to each other, and at their respective optimal pH. Because a reaction system involving an acid generation represents a more challenging test of this pH control technique, a number of factors that affect the generation of such a pH gradient are considered in this study. The mathematical model proposed is based on several simplifying assumptions and represents a first attempt to provide an analysis of this complex problem. The results show that, by choosing appropriate parameters, the pH control technique still can generate the desired pH gradient even if there is an acid-generating reaction in the system. Copyright 1998 John Wiley & Sons, Inc.

A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

PubMed

Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

2018-02-01

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzyme-Initiated Quinone-Chitosan Conjugation Chemistry: Toward A General in Situ Strategy for High-Throughput Photoelectrochemical Enzymatic Bioanalysis.

PubMed

Wang, Guang-Li; Yuan, Fang; Gu, Tiantian; Dong, Yuming; Wang, Qian; Zhao, Wei-Wei

2018-02-06

Herein we report a general and novel strategy for high-throughput photoelectrochemical (PEC) enzymatic bioanalysis on the basis of enzyme-initiated quinone-chitosan conjugation chemistry (QCCC). Specifically, the strategy was illustrated by using a model quinones-generating oxidase of tyrosinase (Tyr) to catalytically produce 1,2-bezoquinone or its derivative, which can easily and selectively be conjugated onto the surface of the chitosan deposited PbS/NiO/FTO photocathode via the QCCC. Upon illumination, the covalently attached quinones could act as electron acceptors of PbS quantum dots (QDs), improving the photocurrent generation and thus allowing the elegant probing of Tyr activity. Enzyme cascades, such as alkaline phosphatase (ALP)/Tyr and β-galactosidase (Gal)/Tyr, were further introduced into the system for the successful probing of the corresponding targets. This work features not only the first use of QCCC in PEC bioanalysis but also the separation of enzymatic reaction from the photoelectrode as well as the direct signal recording in a split-type protocol, which enables quite convenient and high-throughput detection as compared to previous formats. More importantly, by using numerous other oxidoreductases that involve quinones as reactants/products, this protocol could serve as a common basis for the development of a new class of QCCC-based PEC enzymatic bioanalysis and further extended for general enzyme-labeled PEC bioanalysis of versatile targets.

Utility of antioxidants during assisted reproductive techniques: an evidence based review.

PubMed

Agarwal, Ashok; Durairajanayagam, Damayanthi; du Plessis, Stefan S

2014-11-24

Assisted reproductive technology (ART) is a common treatment of choice for many couples facing infertility issues, be it due to male or female factor, or idiopathic. Employment of ART techniques, however, come with its own challenges as the in vitro environment is not nearly as ideal as the in vivo environment, where reactive oxygen species (ROS) build-up leading to oxidative stress is kept in check by the endogenous antioxidants system. While physiological amounts of ROS are necessary for normal reproductive function in vivo, in vitro manipulation of gametes and embryos exposes these cells to excessive ROS production either by endogenous or exogenous environmental factors. In this review, we discuss the sources of ROS in an in vitro clinical setting and the influence of oxidative stress on gamete/embryo quality and the outcome of IVF/ICSI. Sources of ROS and different strategies of overcoming the excessive generation of ROS in vitro are also highlighted. Endogenously, the gametes and the developing embryo become sources of ROS. Multiple exogenous factors act as potential sources of ROS, including exposure to visible light, composition of culture media, pH and temperature, oxygen concentration, centrifugation during spermatozoa preparation, ART technique involving handling of gamete/embryo and cryopreservation technique (freeze/thawing process). Finally, the use of antioxidants as agents to minimize ROS generation in the in vitro environment and as oral therapy is highlighted. Both enzymatic and non-enzymatic antioxidants are discussed and the outcome of studies using these antioxidants as oral therapy in the male or female or its use in vitro in media is presented. While results of studies using certain antioxidant agents are promising, the current body of evidence as a whole suggests the need for further well-designed and larger scale randomized controlled studies, as well as research to minimize oxidative stress conditions in the clinical ART setting.

Cholesterol and related sterols autoxidation.

PubMed

Zerbinati, Chiara; Iuliano, Luigi

2017-10-01

Cholesterol is a unique lipid molecule providing the building block for membranes, hormones, vitamin D and bile acid synthesis. Metabolism of cholesterol involves several enzymes acting on the sterol nucleus or the isooctyl tail. In the recent years, research interest has been focused on oxysterols, cholesterol derivatives generated by the addition of oxygen to the cholesterol backbone. Oxysterols can be produced enzymatically or by autoxidation. Autoxidation of cholesterol proceeds through type I or type II mechanisms. Type I autoxidation is initiated by free radical species, such as those arising from the superoxide/hydrogen peroxide/hydroxyl radical system. Type II autoxidation occurs stoichiometrically by non-radical highly reactive oxygen species such as singlet oxygen, HOCl, and ozone. The vulnerability of cholesterol towards high reactive species has raised considerable interest for mechanistic studies and for the potential biological activity of oxysterols, as well as for the use of oxysterols as biomarkers for the non-invasive study of oxidative stress in vivo. Copyright © 2017. Published by Elsevier Inc.

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz)

PubMed Central

2014-01-01

Background Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Results Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H2O2, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H2O2 synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. Conclusions These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress. PMID:25091029

Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz).

PubMed

Xu, Jia; Yang, Jun; Duan, Xiaoguang; Jiang, Yueming; Zhang, Peng

2014-08-05

Cassava (Manihot esculenta Crantz) is a tropical root crop, and is therefore, extremely sensitive to low temperature; its antioxidative response is pivotal for its survival under stress. Timely turnover of reactive oxygen species (ROS) in plant cells generated by chilling-induced oxidative damages, and scavenging can be achieved by non-enzymatic and enzymatic reactions in order to maintain ROS homeostasis. Transgenic cassava plants that co-express cytosolic superoxide dismutase (SOD), MeCu/ZnSOD, and ascorbate peroxidase (APX), MeAPX2, were produced and tested for tolerance against oxidative and chilling stresses. The up-regulation of MeCu/ZnSOD and MeAPX2 expression was confirmed by the quantitative reverse transcriptase-polymerase chain reaction, and enzymatic activity analyses in the leaves of transgenic cassava plant lines with a single-transgene integration site. Upon exposure to ROS-generating agents, 100 μM ROS-generating reagent methyl viologen and 0.5 M H₂O₂, higher levels of enzymatic activities of SOD and APX were detected in transgenic plants than the wild type. Consequently, the oxidative stress parameters, such as lipid peroxidation, chlorophyll degradation and H₂O₂ synthesis, were lower in the transgenic lines than the wild type. Tolerance to chilling stress at 4°C for 2 d was greater in transgenic cassava, as observed by the higher levels of SOD, catalase, and ascorbate-glutathione cycle enzymes (e.g., APX, monodehydroascorbate reductase, dehydroascorbate reducatase and glutathione reductase) and lower levels of malondialdehyde content. These results suggest that the expression of native cytosolic SOD and APX simultaneously activated the antioxidative defense mechanisms via cyclic ROS scavenging, thereby improving its tolerance to cold stress.

Influence of nanosecond pulsed plasma on the non-enzymatic pathway for the generation of nitric oxide from L-arginine and the modification of graphite oxide to increase the solar cell efficiency.

PubMed

Attri, Pankaj; Park, Ji Hoon; Gaur, Jitender; Kumar, Naresh; Park, Dae Hoon; Jeon, Su Nam; Park, Bong Sang; Chand, Suresh; Uhm, Han Sup; Choi, Eun Ha

2014-09-14

In this work, we demonstrated the action of nanosecond pulsed plasma (NPP) on the generation of nitric oxide (NO) from the non-enzymatic pathway and on the modification of graphite oxide (GO) sheets to increase polymer solar cells (PSCs) efficiency. NO is an important signal and an effector molecule in animals, which is generated from the enzyme-catalyzed oxidation of L-arginine to NO and L-citrulline. Hence, L-arginine is an important biological precursor for NO formation. Therefore, we developed a new non-enzymatic pathway for the formation of NO and L-citrulline using NPP and characterized the pathway using NO detection kit, NMR, liquid chromatography/capillary electrophoresis-mass spectrometry (LC/CE-MS) for both quantitative and qualitative bioanalysis. We then synthesized and modified the functional groups of GO using NPP, and it was characterised by X-ray photoelectron spectroscopy (XPS), confocal Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) imaging, cathodoluminescence (CL) and work function using γ-FIB. Further, we also tested the power conversion efficiency of the PSCs devices with modified GO that is similar to the one obtained with poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) as HTL. This work is perceived to have great implications for inexpensive and efficient methodology for NO generation and modification of GO, which are applicable in materials from nanomaterials to biomolecules.

Assignment of EC Numbers to Enzymatic Reactions with Reaction Difference Fingerprints

PubMed Central

Hu, Qian-Nan; Zhu, Hui; Li, Xiaobing; Zhang, Manman; Deng, Zhe; Yang, Xiaoyan; Deng, Zixin

2012-01-01

The EC numbers represent enzymes and enzyme genes (genomic information), but they are also utilized as identifiers of enzymatic reactions (chemical information). In the present work (ECAssigner), our newly proposed reaction difference fingerprints (RDF) are applied to assign EC numbers to enzymatic reactions. The fingerprints of reactant molecules minus the fingerprints of product molecules will generate reaction difference fingerprints, which are then used to calculate reaction Euclidean distance, a reaction similarity measurement, of two reactions. The EC number of the most similar training reaction will be assigned to an input reaction. For 5120 balanced enzymatic reactions, the RDF with a fingerprint length at 3 obtained at the sub-subclass, subclass, and main class level with cross-validation accuracies of 83.1%, 86.7%, and 92.6% respectively. Compared with three published methods, ECAssigner is the first fully automatic server for EC number assignment. The EC assignment system (ECAssigner) is freely available via: http://cadd.whu.edu.cn/ecassigner/. PMID:23285222

Enzymatic transformation of nonfood biomass to starch

PubMed Central

You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

2013-01-01

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

PubMed

Sporck, Daniele; Reinoso, Felipe A M; Rencoret, Jorge; Gutiérrez, Ana; Del Rio, José C; Ferraz, André; Milagres, Adriane M F

2017-01-01

New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O -methyl-glucuronic acid and p -coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

Rapid analysis of composition and reactivity in cellulosic biomass feedstocks with near-infrared spectroscopy.

PubMed

Payne, Courtney E; Wolfrum, Edward J

2015-01-01

Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. Our objective was to use near-infrared (NIR) spectroscopy and partial least squares (PLS) multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. Major feedstocks included in the calibration models are corn stover, sorghum, switchgrass, perennial cool season grasses, rice straw, and miscanthus. We present individual model statistics to demonstrate model performance and validation samples to more accurately measure predictive quality of the models. The PLS-2 model for composition predicts glucan, xylan, lignin, and ash (wt%) with uncertainties similar to primary measurement methods. A PLS-2 model was developed to predict glucose and xylose release following pretreatment and enzymatic hydrolysis. An additional PLS-2 model was developed to predict glucan and xylan yield. PLS-1 models were developed to predict the sum of glucose/glucan and xylose/xylan for release and yield (grams per gram). The release and yield models have higher uncertainties than the primary methods used to develop the models. It is possible to build effective multispecies feedstock models for composition, as well as carbohydrate release and yield. The model for composition is useful for predicting glucan, xylan, lignin, and ash with good uncertainties. The release and yield models have higher uncertainties; however, these models are useful for rapidly screening sample populations to identify unusual samples.

Biocalcite, a multifunctional inorganic polymer: Building block for calcareous sponge spicules and bioseed for the synthesis of calcium phosphate-based bone

PubMed Central

Schröder, Heinz C; Müller, Werner E G

2014-01-01

Summary Calcium carbonate is the material that builds up the spicules of the calcareous sponges. Recent results revealed that the calcium carbonate/biocalcite-based spicular skeleton of these animals is formed through an enzymatic mechanism, such as the skeleton of the siliceous sponges, evolutionarily the oldest animals that consist of biosilica. The enzyme that mediates the calcium carbonate deposition has been identified as a carbonic anhydrase (CA) and has been cloned from the calcareous sponge species Sycon raphanus. Calcium carbonate deposits are also found in vertebrate bones besides the main constituent, calcium phosphate/hydroxyapatite (HA). Evidence has been presented that during the initial phase of HA synthesis poorly crystalline carbonated apatite is deposited. Recent data summarized here indicate that during early bone formation calcium carbonate deposits enzymatically formed by CA, act as potential bioseeds for the precipitation of calcium phosphate mineral onto bone-forming osteoblasts. Two different calcium carbonate phases have been found during CA-driven enzymatic calcium carbonate deposition in in vitro assays: calcite crystals and round-shaped vaterite deposits. The CA provides a new target of potential anabolic agents for treatment of bone diseases; a first CA activator stimulating the CA-driven calcium carbonate deposition has been identified. In addition, the CA-driven calcium carbonate crystal formation can be frozen at the vaterite state in the presence of silintaphin-2, an aspartic acid/glutamic acid-rich sponge-specific protein. The discovery that calcium carbonate crystals act as bioseeds in human bone formation may allow the development of novel biomimetic scaffolds for bone tissue engineering. Na-alginate hydrogels, enriched with biosilica, have recently been demonstrated as a suitable matrix to embed bone forming cells for rapid prototyping bioprinting/3D cell printing applications. PMID:24991497

Biocalcite, a multifunctional inorganic polymer: Building block for calcareous sponge spicules and bioseed for the synthesis of calcium phosphate-based bone.

PubMed

Wang, Xiaohong; Schröder, Heinz C; Müller, Werner E G

2014-01-01

Calcium carbonate is the material that builds up the spicules of the calcareous sponges. Recent results revealed that the calcium carbonate/biocalcite-based spicular skeleton of these animals is formed through an enzymatic mechanism, such as the skeleton of the siliceous sponges, evolutionarily the oldest animals that consist of biosilica. The enzyme that mediates the calcium carbonate deposition has been identified as a carbonic anhydrase (CA) and has been cloned from the calcareous sponge species Sycon raphanus. Calcium carbonate deposits are also found in vertebrate bones besides the main constituent, calcium phosphate/hydroxyapatite (HA). Evidence has been presented that during the initial phase of HA synthesis poorly crystalline carbonated apatite is deposited. Recent data summarized here indicate that during early bone formation calcium carbonate deposits enzymatically formed by CA, act as potential bioseeds for the precipitation of calcium phosphate mineral onto bone-forming osteoblasts. Two different calcium carbonate phases have been found during CA-driven enzymatic calcium carbonate deposition in in vitro assays: calcite crystals and round-shaped vaterite deposits. The CA provides a new target of potential anabolic agents for treatment of bone diseases; a first CA activator stimulating the CA-driven calcium carbonate deposition has been identified. In addition, the CA-driven calcium carbonate crystal formation can be frozen at the vaterite state in the presence of silintaphin-2, an aspartic acid/glutamic acid-rich sponge-specific protein. The discovery that calcium carbonate crystals act as bioseeds in human bone formation may allow the development of novel biomimetic scaffolds for bone tissue engineering. Na-alginate hydrogels, enriched with biosilica, have recently been demonstrated as a suitable matrix to embed bone forming cells for rapid prototyping bioprinting/3D cell printing applications.

Natural and engineered biosynthesis of nucleoside antibiotics in Actinomycetes.

PubMed

Chen, Wenqing; Qi, Jianzhao; Wu, Pan; Wan, Dan; Liu, Jin; Feng, Xuan; Deng, Zixin

2016-03-01

Nucleoside antibiotics constitute an important family of microbial natural products bearing diverse bioactivities and unusual structural features. Their biosynthetic logics are unique with involvement of complex multi-enzymatic reactions leading to the intricate molecules from simple building blocks. Understanding how nature builds this family of antibiotics in post-genomic era sets the stage for rational enhancement of their production, and also paves the way for targeted persuasion of the cell factories to make artificial designer nucleoside drugs and leads via synthetic biology approaches. In this review, we discuss the recent progress and perspectives on the natural and engineered biosynthesis of nucleoside antibiotics.

Structural basis of stereospecificity in the bacterial enzymatic cleavage of β-aryl ether bonds in lignin

DOE PAGES

Helmich, Kate E.; Pereira, Jose Henrique; Gall, Daniel L.; ...

2015-12-04

Here, lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, wemore » present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.« less

Functional Cellular Mimics for the Spatiotemporal Control of Multiple Enzymatic Cascade Reactions.

PubMed

Liu, Xiaoling; Formanek, Petr; Voit, Brigitte; Appelhans, Dietmar

2017-12-18

Next-generation therapeutic approaches are expected to rely on the engineering of biomimetic cellular systems that can mimic specific cellular functions. Herein, we demonstrate a highly effective route for constructing structural and functional eukaryotic cell mimics by loading pH-sensitive polymersomes as membrane-associated and free-floating organelle mimics inside the multifunctional cell membrane. Metabolism mimicry has been validated by performing successive enzymatic cascade reactions spatially separated at specific sites of cell mimics in the presence and absence of extracellular organelle mimics. These enzymatic reactions take place in a highly controllable, reproducible, efficient, and successive manner. Our biomimetic approach to material design for establishing functional principles brings considerable enrichment to the fields of biomedicine, biocatalysis, biotechnology, and systems biology. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Networks Approach to Modeling Enzymatic Reactions.

PubMed

Imhof, P

2016-01-01

Modeling enzymatic reactions is a demanding task due to the complexity of the system, the many degrees of freedom involved and the complex, chemical, and conformational transitions associated with the reaction. Consequently, enzymatic reactions are not determined by precisely one reaction pathway. Hence, it is beneficial to obtain a comprehensive picture of possible reaction paths and competing mechanisms. By combining individually generated intermediate states and chemical transition steps a network of such pathways can be constructed. Transition networks are a discretized representation of a potential energy landscape consisting of a multitude of reaction pathways connecting the end states of the reaction. The graph structure of the network allows an easy identification of the energetically most favorable pathways as well as a number of alternative routes. © 2016 Elsevier Inc. All rights reserved.

Industrial enzymatic production of cephalosporin-based beta-lactams.

PubMed

Barber, Michael S; Giesecke, Ulrich; Reichert, Arno; Minas, Wolfgang

2004-01-01

Cephalosporins are chemically closely related to penicillins both work by inhibiting the cell wall synthesis of bacteria. The first generation cephalosporins entered the market in 1964. Second and third generation cephalosporins were subsequently developed that were more powerful than the original products. Fourth generation cephalosporins are now reaching the market. Each newer generation of cephalosporins has greater Gram-negative antimicrobial properties than the preceding generation. Conversely, the 'older' generations of cephalosporins have greater Gram-positive (Staphylococcus and Streptococcus) coverage than the 'newer' generations. Frequency of dosing decreases and palatability generally improve with increasing generations. The advent of fourth generation cephalosporins with the launch of cefepime extended the spectrum against Gram-positive organisms without a significant loss of activity towards Gram-negative bacteria. Its greater stability to beta-lactamases increases its efficacy against drug-resistant bacteria. In this review we present the current situation of this mature market. In addition, we present the current state of the technologies employed for the production of cephalosporins, focusing on the new and environmentally safer 'green' routes to the products. Starting with the fermentation and purification of CPC, enzymatic conversion in conjunction with aqueous chemistry will lead to some key intermediates such as 7-ACA, TDA and TTA, which then can be converted into the active pharmaceutical ingredient (API), again applying biocatalytic technologies and aqueous chemistry. Examples for the costing of selected products are provided as well.

Second generation bioethanol production from Saccharum spontaneum L. ssp. aegyptiacum (Willd.) Hack.

Treesearch

Danilo Scordia; Salvatore L. Consentino; Thomas W. Jeffries

2010-01-01

Saccharum (Saccharum spontaneum L. ssp. aegyptiacum (Willd.) Hack.), is a rapidly growing, wide ranging high-yield perennial, suitable for second generation bioethanol production. This study evaluated oxalic acid as a pretreatment for bioconversion. Overall sugar yields, sugar degradation products, enzymatic glucan hydrolysis and ethanol production were studied as...

Removal of p-alkylphenols from aqueous solutions by combined use of mushroom tyrosinase and chitosan beads.

PubMed

Yamada, Kazunori; Inoue, Tomoaki; Akiba, Yuji; Kashiwada, Ayumi; Matsuda, Kiyomi; Hirata, Mitsuo

2006-10-01

Enzymatic removal of p-alkylphenols from aqueous solutions was investigated through the two-step approach, the quinone conversion of p-alkylphenols with mushroom tyrosinase (EC 1.14.18.1) and the subsequent adsorption of quinone derivatives enzymatically generated on chitosan beads at pH 7.0 and 45 degrees C as the optimum conditions. This technique is quite effective for removal of various p-alkylphenols from an aqueous solution. The % removal values of 97-100% were obtained for p-n-alkylphenols with carbon chain lengths of 5 to 9. In addition, removal of other p-alkylphenols was enhanced by increasing either the tyrosinase concentration or the amount of added chitosan beads, and their % removal values reached >93 except for 4-tert-pentylphenol. This technique was also applicable to remove 4-n-octylphenol (4NOP) and 4-n-nonylphenol (4NNP) as suspected endocrine disrupting chemicals. The reaction of quinone derivatives enzymatically generated with the chitosan's amino groups was confirmed by the appearance of peaks for UV-visible spectrum measurements of the chitosan films incubated in the p-alkylphenol and tyrosinase mixture solutions. In addition, 4-tert-pentylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide.

Multidimensional Self-Assembled Structures of Alkylated Cellulose Oligomers Synthesized via in Vitro Enzymatic Reactions.

PubMed

Yataka, Yusuke; Sawada, Toshiki; Serizawa, Takeshi

2016-10-04

The self-assembly of biomolecules into highly ordered nano-to-macroscale structures is essential in the construction of biological tissues and organs. A variety of biomolecular assemblies composed of nucleic acids, peptides, and lipids have been used as molecular building units for self-assembled materials. However, crystalline polysaccharides have rarely been utilized in self-assembled materials. In this study, we describe multidimensional self-assembled structures of alkylated cellulose oligomers synthesized via in vitro enzymatic reactions. We found that the alkyl chain length drastically affected the assembled morphologies and allomorphs of cellulose moieties. The modulation of the intermolecular interactions of cellulose oligomers by alkyl substituents was highly effective at controlling their assembly into multidimensional structures. This study proposes a new potential of crystalline oligosaccharides for structural components of molecular assemblies with controlled morphologies and crystal structures.

PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes.

PubMed

Li, Qing; Subbulakshmi, Venkita; Oldfield, Claudine M; Aamir, Rozina; Weyman, Crystal M; Wolfman, Alan; Cathcart, Martha K

2007-02-01

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCalpha and cPLA(2) in O(2)(-) generation are solely due to their seminal role in generating arachidonic acid.

Reactive oxygen species, essential molecules, during plant-pathogen interactions.

PubMed

Camejo, Daymi; Guzmán-Cedeño, Ángel; Moreno, Alexander

2016-06-01

Reactive oxygen species (ROS) are continually generated as a consequence of the normal metabolism in aerobic organisms. Accumulation and release of ROS into cell take place in response to a wide variety of adverse environmental conditions including salt, temperature, cold stresses and pathogen attack, among others. In plants, peroxidases class III, NADPH oxidase (NOX) locates in cell wall and plasma membrane, respectively, may be mainly enzymatic systems involving ROS generation. It is well documented that ROS play a dual role into cells, acting as important signal transduction molecules and as toxic molecules with strong oxidant power, however some aspects related to its function during plant-pathogen interactions remain unclear. This review focuses on the principal enzymatic systems involving ROS generation addressing the role of ROS as signal molecules during plant-pathogen interactions. We described how the chloroplasts, mitochondria and peroxisomes perceive the external stimuli as pathogen invasion, and trigger resistance response using ROS as signal molecule. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Improved pretreatment of lignocellulosic biomass using enzymatically-generated peracetic acid.

PubMed

Yin, DeLu Tyler; Jing, Qing; AlDajani, Waleed Wafa; Duncan, Shona; Tschirner, Ulrike; Schilling, Jonathan; Kazlauskas, Romas J

2011-04-01

Release of sugars from lignocellulosic biomass is inefficient because lignin, an aromatic polymer, blocks access of enzymes to the sugar polymers. Pretreatments remove lignin and disrupt its structure, thereby enhancing sugar release. In previous work, enzymatically generated peracetic acid was used to pretreat aspen wood. This pretreatment removed 45% of the lignin and the subsequent saccharification released 97% of the sugars remaining after pretreatment. In this paper, the amount of enzyme needed is reduced tenfold using first, an improved enzyme variant that makes twice as much peracetic acid and second, a two-phase reaction to generate the peracetic acid, which allows enzyme reuse. In addition, the eight pretreatment cycles are reduced to only one by increasing the volume of peracetic acid solution and increasing the temperature to 60 °C and the reaction time to 6h. For the pretreatment step, the weight ratio of peracetic acid to wood determines the amount of lignin removed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Utilization of hydrolysate from lignocellulosic biomass pretreatment to generate electricity by enzymatic fuel cell system.

PubMed

Kim, Sung Bong; Kim, Dong Sup; Yang, Ji Hyun; Lee, Junyoung; Kim, Seung Wook

2016-04-01

The waste hydrolysate after dilute acid pretreatment (DAP) of lignocellulosic biomass was utilized to generate electricity using an enzymatic fuel cell (EFC) system. During DAP, the components of biomass containing hemicellulose and other compounds are hydrolyzed, and glucose is solubilized into the dilute acid solution, called as the hydrolysate liquid. Glucose oxidase (GOD) and laccase (Lac) were assembled on the electrode of the anode and cathode, respectively. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were measured, and the maximum power density was found to be 1.254×10(3) μW/cm(2). The results indicate that the hydrolysate from DAP is a reliable electrolyte containing the fuel of EFC. Moreover, the impurities in the hydrolysate such as phenols and furans slightly affected the charge transfer on the surface of the electrode, but did not affect the power generation of the EFC system in principal. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of Protein Targets of 12/15-Lipoxygenase-Derived Lipid Electrophiles in Mouse Peritoneal Macrophages Using Omega-Alkynyl Fatty Acid.

PubMed

Isobe, Yosuke; Kawashima, Yusuke; Ishihara, Tomoaki; Watanabe, Kenji; Ohara, Osamu; Arita, Makoto

2018-04-20

The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of highest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Here, we describe a chemoproteomic method to identify the targets of enzymatically generated LDE. By treating mouse peritoneal macrophages with omega-alkynyl arachidonic acid (aAA), we identified a series of proteins adducted by LDE generated through a 12/15-LOX catalyzed reaction. Pathway analysis revealed a dramatic enrichment of proteins involved in energy metabolism and found that glycolytic flux and mitochondrial respiration were significantly affected by the expression of 12/15-LOX. Our findings thus highlight the utility of chemoproteomics using aAA for identifying intracellular targets of enzymatically generated LDE.

Synthesis of (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid vinyl-ACCA) derivatives: key intermediates for the preparation of inhibitors of the hepatitis C virus NS3 protease.

PubMed

Beaulieu, Pierre L; Gillard, James; Bailey, Murray D; Boucher, Colette; Duceppe, Jean-Simon; Simoneau, Bruno; Wang, Xiao-Jun; Zhang, Li; Grozinger, Karl; Houpis, Ioannis; Farina, Vittorio; Heimroth, Heidi; Krueger, Thomas; Schnaubelt, Jürgen

2005-07-22

(1R,2S)-1-Amino-2-vinylcyclopropanecarboxylic acid (vinyl-ACCA) is a key building block in the synthesis of potent inhibitors of the hepatitis C virus NS3 protease such as BILN 2061, which was recently shown to dramatically reduce viral load after administration to patients infected with HCV genotype 1. We have developed a scalable process that delivers derivatives of this unusual amino acid in >99% ee. The strategy was based on the dialkylation of a glycine Schiff base using trans-1,4-dibromo-2-butene as an electrophile to produce racemic vinyl-ACCA, which was subsequently resolved using a readily available, inexpensive esterase enzyme (Alcalase 2.4L). Factors that affect diastereoselection in the initial dialkylation steps were examined and the conditions optimized to deliver the desired diastereomer selectively. Product inhibition, which was encountered during the enzymatic resolution step, initially resulted in prolonged cycle times. Enrichment of racemic vinyl-ACCA through a chemical resolution via diastereomeric salt formation or the use of forcing conditions in the enzymatic reaction both led to improvements in throughput and the development of a viable process. The chemistry described herein was scaled up to produce multikilogram quantities of this building block.

Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway

NASA Astrophysics Data System (ADS)

Zhu, Zhiguang; Wang, Yiran; Minteer, Shelley D.; Percival Zhang, Y.-H.

Enzymatic fuel cells (EFCs) use a variety of fuels to generate electricity through oxidoreductase enzymes, such as oxidases or dehydrogenases, as catalysts on electrodes. We have developed a novel synthetic enzymatic pathway containing two free enzymes (maltodextrin phosphorylase and phosphoglucomutase) and one immobilized glucose-6-phosphate dehydrogenase that can utilize an oligomeric substrate maltodextrin for producing electrons mediated via a diaphorase and vitamin K 3 electron shuttle system. Three different enzyme immobilization approaches were compared based on electrostatic force entrapment, chemical cross-linking, and cross-linking with the aid of carbon nanotubes. At 10 mM glucose-6-phosphate (G6P) as a substrate concentration, the maximum power density of 0.06 mW cm -2 and retaining 42% of power output after 11 days were obtained through the method of chemical cross-linking with carbon nanotubes, approximately 6-fold and 3.5-fold better than those of the electrostatic force-based method, respectively. When changed to maltodextrin (degree of polymerization = 19) as the substrate, the EFC achieved a maximum power density of 0.085 mW cm -2. With the advantages of stable, low cost, high energy density, non-inhibitor to enzymes, and environmental friendly, maltodextrin is suggested to be an ideal fuel to power enzymatic fuel cells.

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines.

PubMed

Hanavan, Paul D; Borges, Chad R; Katchman, Benjamin A; Faigel, Douglas O; Ho, Thai H; Ma, Chen-Ting; Sergienko, Eduard A; Meurice, Nathalie; Petit, Joachim L; Lake, Douglas F

2015-07-30

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

PubMed

Lin, Hsin Hung; Pan, Yih Jiuan; Hsu, Shen Hsing; Van, Ru Chuan; Hsiao, Yi Yuong; Chen, Jiun Hsien; Pan, Rong Long

2005-10-15

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.

A heating-superfusion platform technology for the investigation of protein function in single cells.

PubMed

Xu, Shijun; Ainla, Alar; Jardemark, Kent; Jesorka, Aldo; Jeffries, Gavin D M

2015-01-06

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.

Building energy analysis tool

DOEpatents

Brackney, Larry; Parker, Andrew; Long, Nicholas; Metzger, Ian; Dean, Jesse; Lisell, Lars

2016-04-12

A building energy analysis system includes a building component library configured to store a plurality of building components, a modeling tool configured to access the building component library and create a building model of a building under analysis using building spatial data and using selected building components of the plurality of building components stored in the building component library, a building analysis engine configured to operate the building model and generate a baseline energy model of the building under analysis and further configured to apply one or more energy conservation measures to the baseline energy model in order to generate one or more corresponding optimized energy models, and a recommendation tool configured to assess the one or more optimized energy models against the baseline energy model and generate recommendations for substitute building components or modifications.

Rapid analysis of composition and reactivity in cellulosic biomass feedstocks with near-infrared spectroscopy

DOE PAGES

Payne, Courtney E.; Wolfrum, Edward J.

2015-03-12

Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. Our objective was to use near-infrared (NIR) spectroscopy and partial least squares (PLS) multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. Major feedstocks included in the calibration models are corn stover, sorghum, switchgrass, perennial cool season grasses, rice straw, and miscanthus. Here are the results: We present individual model statistics tomore » demonstrate model performance and validation samples to more accurately measure predictive quality of the models. The PLS-2 model for composition predicts glucan, xylan, lignin, and ash (wt%) with uncertainties similar to primary measurement methods. A PLS-2 model was developed to predict glucose and xylose release following pretreatment and enzymatic hydrolysis. An additional PLS-2 model was developed to predict glucan and xylan yield. PLS-1 models were developed to predict the sum of glucose/glucan and xylose/xylan for release and yield (grams per gram). The release and yield models have higher uncertainties than the primary methods used to develop the models. In conclusion, it is possible to build effective multispecies feedstock models for composition, as well as carbohydrate release and yield. The model for composition is useful for predicting glucan, xylan, lignin, and ash with good uncertainties. The release and yield models have higher uncertainties; however, these models are useful for rapidly screening sample populations to identify unusual samples.« less

Rapid analysis of composition and reactivity in cellulosic biomass feedstocks with near-infrared spectroscopy

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

Payne, Courtney E.; Wolfrum, Edward J.

Obtaining accurate chemical composition and reactivity (measures of carbohydrate release and yield) information for biomass feedstocks in a timely manner is necessary for the commercialization of biofuels. Our objective was to use near-infrared (NIR) spectroscopy and partial least squares (PLS) multivariate analysis to develop calibration models to predict the feedstock composition and the release and yield of soluble carbohydrates generated by a bench-scale dilute acid pretreatment and enzymatic hydrolysis assay. Major feedstocks included in the calibration models are corn stover, sorghum, switchgrass, perennial cool season grasses, rice straw, and miscanthus. Here are the results: We present individual model statistics tomore » demonstrate model performance and validation samples to more accurately measure predictive quality of the models. The PLS-2 model for composition predicts glucan, xylan, lignin, and ash (wt%) with uncertainties similar to primary measurement methods. A PLS-2 model was developed to predict glucose and xylose release following pretreatment and enzymatic hydrolysis. An additional PLS-2 model was developed to predict glucan and xylan yield. PLS-1 models were developed to predict the sum of glucose/glucan and xylose/xylan for release and yield (grams per gram). The release and yield models have higher uncertainties than the primary methods used to develop the models. In conclusion, it is possible to build effective multispecies feedstock models for composition, as well as carbohydrate release and yield. The model for composition is useful for predicting glucan, xylan, lignin, and ash with good uncertainties. The release and yield models have higher uncertainties; however, these models are useful for rapidly screening sample populations to identify unusual samples.« less

Plant cell walls to ethanol.

USDA-ARS?s Scientific Manuscript database

Conversion of plant cell walls to ethanol constitutes generation 2 bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation, and separation. Ultimately, it is desired to combine as man...

Quenching of graphene quantum dots fluorescence by alkaline phosphatase activity in the presence of hydroquinone diphosphate.

PubMed

Pereira da Silva Neves, Marta Maria; González-García, María Begoña; Pérez-Junquera, Alejandro; Hernández-Santos, David; Fanjul-Bolado, Pablo

2018-05-01

In this work, a turn-off photoluminescent sensing proof-of-concept based on blue luminescent graphene quantum dots (GQDs) as the fluorescent probe was developed. For that purpose, GQDs optical response was related with the catalytic enzymatic activity of alkaline phosphatase (ALP), in the presence of hydroquinone diphosphate (HQDP). The hydrolysis of HQDP by ALP generated hydroquinone (HQ). The oxidation of HQ, enzymatically produced, to p-benzoquinone (BQ) resulted in the quenching of GQDs fluorescence (FL). Therefore, the developed luminescent sensing mechanism allowed the FL quenching with ALP activity to be related and thus quantified the concentration of ALP down to 0.5 nM of enzyme. This innovative design principle appears as a promising tool for the development of enzymatic sensors based on ALP labeling with fluorescent detection or even for direct ALP luminescent quantification in an easy, fast and sensitive manner. Copyright © 2018 John Wiley & Sons, Ltd.

Production and Characterization of F(Ab')2 Fragments Obtained by Enzymatic Digestion from Murine Anti-MRSA PBP2a Monoclonal Antibodies.

PubMed

de Araujo, Anna Erika Vieira; de Souza, Natalia Plinio; de Sousa, Alvaro Paiva Braga; Lara, Flavio Alves; Senna, Jose Procopio Moreno

2018-05-01

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab') 2 antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab') 2 fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab') 2 fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab') 2 affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab') 2 presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab') 2 fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.

A sensitive immunosensor via in situ enzymatically generating efficient quencher for electrochemiluminescence of iridium complexes doped SiO2 nanoparticles.

PubMed

Liang, Wenbin; Zhuo, Ying; Xiong, Chengyi; Zheng, Yingning; Chai, Yaqin; Yuan, Ruo

2017-08-15

A sensitive electrochemiluminescent (ECL) sandwich immunosensor was proposed herein based on the tris (2-phenylpyridine) iridium [Ir(ppy) 3 ] doped silica nanoparticles (SiO 2 @Ir) with improved ECL emission as signal probes and glucose oxidase (GOD)-based in situ enzymatic reaction to generate H 2 O 2 for efficiently quenching the ECL emission of SiO 2 @Ir. Typically, the SiO 2 @Ir not only increased the loading amount of Ir(ppy) 3 as ECL indicators with high ECL emission, but also improved their water-solubility, which efficiently enhanced the ECL emission. Furthermore, by the efficient quench effect of H 2 O 2 from in situ glucose oxidase (GOD)-based enzymatic reaction on the ECL emission of SiO 2 @Ir, a signal-off ECL immunsensor could be established for sensitive assay. With N-terminal of the prohormone brain natriuretic peptide (BNPT) as a model, the proposed ECL assay performed high sensitivity and low detection limit. Importantly, the proposed sensitive ECL strategy was not only suitable for the detection of BNPT for acute myocardial infarction, but also revealed a new avenue for early diagnosis of various diseases via proteins, nucleotide sequence, microRNA and cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Cannabidiol-2',6'-dimethyl ether as an effective protector of 15-lipoxygenase-mediated low-density lipoprotein oxidation in vitro.

PubMed

Takeda, Shuso; Hirayama, Akari; Urata, Shino; Mano, Nobutaka; Fukagawa, Keiko; Imamura, Midori; Irii, Ayumi; Kitajima, Satomi; Masuyama, Tomoko; Nomiyama, Mai; Tatei, Sachiko; Tomita, Saari; Kudo, Taichi; Noguchi, Momoko; Yamaguchi, Yasuhiro; Okamoto, Yoshiko; Amamoto, Toshiaki; Fukunishi, Yoshifumi; Watanabe, Kazuhito; Omiecinski, Curtis John; Aramaki, Hironori

2011-01-01

15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein (ox-LDL), a major causal factor for atherosclerosis. Both enzymatic (15-LOX) and non-enzymatic (Cu(2+)) mechanisms have been proposed for the production of ox-LDL. We have recently reported that cannabidiol-2',6'-dimethyl ether (CBDD) is a selective and potent inhibitor of 15-LOX-catalyzed linoleic acid oxygenation (Takeda et al., Drug Metab. Dispos., 37, 1733-1737 (2009)). In the LDL, linoleic acid is present as cholesteryl linoleate, the major fatty acid esterified to cholesterol, and is susceptible to oxidative modification by 15-LOX or Cu(2+). In this investigation, we examined the efficacy of CBDD on i) 15-LOX-catalyzed oxygenation of cholesteryl linoleate, and ii) ox-LDL formation catalyzed by 15-LOX versus Cu(2+)-mediated non-enzymatic generation of this important mediator. The results obtained demonstrate that CBDD is a potent and selective inhibitor of ox-LDL formation generated by the 15-LOX pathway. These studies establish CBDD as both an important experimental tool for characterizing 15-LOX-mediated ox-LDL formation, and as a potentially useful therapeutic agent for treatment of atherosclerosis.

Cannabidiol-2′,6′-dimethyl Ether as an Effective Protector of 15-Lipoxygenase-Mediated Low-Density Lipoprotein Oxidation in Vitro

PubMed Central

Takeda, Shuso; Hirayama, Akari; Urata, Shino; Mano, Nobutaka; Fukagawa, Keiko; Imamura, Midori; Irii, Ayumi; Kitajima, Satomi; Masuyama, Tomoko; Nomiyama, Mai; Tatei, Sachiko; Tomita, Saari; Kudo, Taichi; Noguchi, Momoko; Yamaguchi, Yasuhiro; Okamoto, Yoshiko; Amamoto, Toshiaki; Fukunishi, Yoshifumi; Watanabe, Kazuhito; Omiecinski, Curtis John; Aramaki, Hironori

2014-01-01

15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein (ox-LDL), a major causal factor for atherosclerosis. Both enzymatic (15-LOX) and non-enzymatic (Cu2+) mechanisms have been proposed for the production of ox-LDL. We have recently reported that cannabidiol-2′,6′-dimethyl ether (CBDD) is a selective and potent inhibitor of 15-LOX-catalyzed linoleic acid oxygenation (Takeda et al., Drug Metab. Dispos., 37, 1733–1737 (2009)). In the LDL, linoleic acid is present as cholesteryl linoleate, the major fatty acid esterified to cholesterol, and is susceptible to oxidative modification by 15-LOX or Cu2+. In this investigation, we examined the efficacy of CBDD on i) 15-LOX-catalyzed oxygenation of cholesteryl linoleate, and ii) ox-LDL formation catalyzed by 15-LOX versus Cu2+-mediated non-enzymatic generation of this important mediator. The results obtained demonstrate that CBDD is a potent and selective inhibitor of ox-LDL formation generated by the 15-LOX pathway. These studies establish CBDD as both an important experimental tool for characterizing 15-LOX-mediated ox-LDL formation, and as a potentially useful therapeutic agent for treatment of atherosclerosis. PMID:21804214

Discovery, Characterization, and Analogue Synthesis of Bohemamine Dimers Generated by Non-enzymatic Biosynthesis.

PubMed

Fu, Peng; Legako, Aaron; La, Scott; MacMillan, John B

2016-03-01

Dibohemamines A-C (5-7), three new dimeric bohemamine analogues dimerized through a methylene group, were isolated from a marine-derived Streptomyces spinoverrucosus. The structures determined by spectroscopic analysis were confirmed through the semi-synthetic derivatization of monomeric bohemamines and formaldehyde. These reactions, which could occur under mild conditions, together with the detection of formaldehyde in the culture, revealed that this dimerization is a non-enzymatic process. In addition to the unique dimerization of the dibohemamines, dibohemamines B and C were found to have nm cytotoxicity against the non-small cell-lung cancer cell line A549. In view of the potent cytotoxicity of compounds 6 and 7, a small library of bohemamine analogues was generated for biological evaluation by utilizing a series of aryl and alkyl aldehydes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATLAS of Biochemistry: A Repository of All Possible Biochemical Reactions for Synthetic Biology and Metabolic Engineering Studies.

PubMed

Hadadi, Noushin; Hafner, Jasmin; Shajkofci, Adrian; Zisaki, Aikaterini; Hatzimanikatis, Vassily

2016-10-21

Because the complexity of metabolism cannot be intuitively understood or analyzed, computational methods are indispensable for studying biochemistry and deepening our understanding of cellular metabolism to promote new discoveries. We used the computational framework BNICE.ch along with cheminformatic tools to assemble the whole theoretical reactome from the known metabolome through expansion of the known biochemistry presented in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We constructed the ATLAS of Biochemistry, a database of all theoretical biochemical reactions based on known biochemical principles and compounds. ATLAS includes more than 130 000 hypothetical enzymatic reactions that connect two or more KEGG metabolites through novel enzymatic reactions that have never been reported to occur in living organisms. Moreover, ATLAS reactions integrate 42% of KEGG metabolites that are not currently present in any KEGG reaction into one or more novel enzymatic reactions. The generated repository of information is organized in a Web-based database ( http://lcsb-databases.epfl.ch/atlas/ ) that allows the user to search for all possible routes from any substrate compound to any product. The resulting pathways involve known and novel enzymatic steps that may indicate unidentified enzymatic activities and provide potential targets for protein engineering. Our approach of introducing novel biochemistry into pathway design and associated databases will be important for synthetic biology and metabolic engineering.

Production of biofuels and biochemicals by in vitro synthetic biosystems: Opportunities and challenges.

PubMed

Zhang, Yi-Heng Percival

2015-11-15

The largest obstacle to the cost-competitive production of low-value and high-impact biofuels and biochemicals (called biocommodities) is high production costs catalyzed by microbes due to their inherent weaknesses, such as low product yield, slow reaction rate, high separation cost, intolerance to toxic products, and so on. This predominant whole-cell platform suffers from a mismatch between the primary goal of living microbes - cell proliferation and the desired biomanufacturing goal - desired products (not cell mass most times). In vitro synthetic biosystems consist of numerous enzymes as building bricks, enzyme complexes as building modules, and/or (biomimetic) coenzymes, which are assembled into synthetic enzymatic pathways for implementing complicated bioreactions. They emerge as an alternative solution for accomplishing a desired biotransformation without concerns of cell proliferation, complicated cellular regulation, and side-product formation. In addition to the most important advantage - high product yield, in vitro synthetic biosystems feature several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this perspective review, the general design rules of in vitro synthetic pathways are presented with eight supporting examples: hydrogen, n-butanol, isobutanol, electricity, starch, lactate,1,3-propanediol, and poly-3-hydroxylbutyrate. Also, a detailed economic analysis for enzymatic hydrogen production from carbohydrates is presented to illustrate some advantages of this system and the remaining challenges. Great market potentials will motivate worldwide efforts from multiple disciplines (i.e., chemistry, biology and engineering) to address the remaining obstacles pertaining to cost and stability of enzymes and coenzymes, standardized building parts and modules, biomimetic coenzymes, biosystem optimization, and scale-up, soon. Copyright © 2014 Elsevier Inc. All rights reserved.

Synthetic generation of influenza vaccine viruses for rapid response to pandemics.

PubMed

Dormitzer, Philip R; Suphaphiphat, Pirada; Gibson, Daniel G; Wentworth, David E; Stockwell, Timothy B; Algire, Mikkel A; Alperovich, Nina; Barro, Mario; Brown, David M; Craig, Stewart; Dattilo, Brian M; Denisova, Evgeniya A; De Souza, Ivna; Eickmann, Markus; Dugan, Vivien G; Ferrari, Annette; Gomila, Raul C; Han, Liqun; Judge, Casey; Mane, Sarthak; Matrosovich, Mikhail; Merryman, Chuck; Palladino, Giuseppe; Palmer, Gene A; Spencer, Terika; Strecker, Thomas; Trusheim, Heidi; Uhlendorff, Jennifer; Wen, Yingxia; Yee, Anthony C; Zaveri, Jayshree; Zhou, Bin; Becker, Stephan; Donabedian, Armen; Mason, Peter W; Glass, John I; Rappuoli, Rino; Venter, J Craig

2013-05-15

During the 2009 H1N1 influenza pandemic, vaccines for the virus became available in large quantities only after human infections peaked. To accelerate vaccine availability for future pandemics, we developed a synthetic approach that very rapidly generated vaccine viruses from sequence data. Beginning with hemagglutinin (HA) and neuraminidase (NA) gene sequences, we combined an enzymatic, cell-free gene assembly technique with enzymatic error correction to allow rapid, accurate gene synthesis. We then used these synthetic HA and NA genes to transfect Madin-Darby canine kidney (MDCK) cells that were qualified for vaccine manufacture with viral RNA expression constructs encoding HA and NA and plasmid DNAs encoding viral backbone genes. Viruses for use in vaccines were rescued from these MDCK cells. We performed this rescue with improved vaccine virus backbones, increasing the yield of the essential vaccine antigen, HA. Generation of synthetic vaccine seeds, together with more efficient vaccine release assays, would accelerate responses to influenza pandemics through a system of instantaneous electronic data exchange followed by real-time, geographically dispersed vaccine production.

A Cell surface β-Hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma

PubMed Central

Aihara, Arihiro; Huang, Chiung-Kuei; Olsen, Mark J.; Lin, Qiushi; Chung, Waihong; Tang, Qi; Dong, Xiaoqun; Wands, Jack R.

2014-01-01

Hepatocellular carcinoma (HCC) has a poor prognosis due to widespread intrahepatic and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(Asparaginyl)-β-hydroxylase (ASPH) is a cell surface enzyme that generates enhanced cell motility, migration, invasion and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer assisted drug design. Candidate compounds were tested for inhibition of β-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biologic effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor MO-I-1100 was selected since it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion and anchorage independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. Conclusions These studies suggest that the enzymatic activity of ASPH was important for hepatic oncogenesis. Reduced β-hydroxylase activity generated by the SMI MO-I-1100 led to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease. PMID:24954865

Oxidative stress induced by cadmium in the plasma, erythrocytes and lymphocytes of rats: Attenuation by grape seed proanthocyanidins.

PubMed

Nazima, B; Manoharan, V; Miltonprabu, S

2016-04-01

The present study has been designed to investigate the ameliorative effect of grape seed proanthocyanidins (GSP) on cadmium (Cd)-induced oxidative damage in rat erythrocytes. Twenty four male Wistar rats were divided into four groups: control, GSP-treated group (100 mg kg(-1) body weight (BW)), Cd-treated group (cadmium chloride, 5 mg kg(-1) BW), and GSP + Cd-treated group in which GSP was orally pre-administered 90 min before Cd intoxication for 4 weeks. At the end of the experimental period, blood samples were collected by cardiac puncture and were processed for various biochemical estimations. The extent of oxidative damage in isolated rat erythrocyte membrane was assessed by measuring lipid peroxidation, enzymatic and non-enzymatic content, calcium ion (Ca(2+))/magnesium ion (Mg(2+))-ATPase and sodium ion (Na(+))/potassium ion (K(+))-ATPase activities, free iron, calcium, hydrogen peroxide (H2O2) concentration, and osmotic fragility. Our results unveiled that Cd intoxication significantly increased the erythrocyte lipid peroxidation markers and decreased the activity of enzymatic and non-enzymatic markers in erythrocytes. Conversely, GSP pretreatment significantly prevented the decrease in the activities of antioxidant enzymes and membrane-bound ATPases. GSP also restored the levels of iron, calcium, and H2O2 in Cd-treated rats. Conformational changes in erythrocytes of various groups were also determined using morphological and ultrastructural electron microscopic analysis. The findings of our study clearly revealed that GSP affords superior protection against Cd-induced reactive oxygen species generation, lipid peroxidation, and free radical generation in Cd-treated rats, which presumably reflects the ability of this flavonoid to protect erythrocytes and lymphocytes of rats from the toxic effects of Cd. © The Author(s) 2015.

Cultured astrocytes do not release adenosine during hypoxic conditions

PubMed Central

Fujita, Takumi; Williams, Erika K; Jensen, Tina K; Smith, Nathan A; Takano, Takahiro; Tieu, Kim; Nedergaard, Maiken

2012-01-01

Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine from cultured astrocytes. Similar results were obtained using a chemiluminescent enzymatic assay. Moreover, since the chemiluminescent enzymatic assay relies on hydrogen peroxide generation, release of free-radical scavengers from hypoxic cells can interfere with the assay. Accordingly, adenosine added to samples collected from hypoxic cultures could not be detected using the chemiluminescent enzymatic assay. Furthermore, addition of free-radical scavengers sharply reduced the sensitivity of adenosine detection. Conversely, use of a single-step assay inflated measured values due to the inability of the assay to distinguish adenosine and its metabolite inosine. These results show that cultured astrocytes do not release adenosine during mild hypoxia, an observation consistent with their high resistance to hypoxia. PMID:21989480

A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

PubMed Central

Guo, Hou-Fu; Cho, Eun Jeong; Devkota, Ashwini K.; Chen, Yulong; Russell, William; Phillips, George N.; Yamauchi, Mitsuo; Dalby, Kevin; Kurie, Jonathan M.

2017-01-01

Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated previously using E coli– or insect-based expression systems was either insoluble or enzymatically unstable, and LH2 enzymatic activity assays have measured radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems. PMID:28216326

Enzymatic Specific Production and Chemical Functionalization of Phenylpropanone Platform Monomers from Lignin

PubMed Central

Hasegawa, Ryoichi; Kurosawa, Kanako; Maeda, Allyn H.; Koizumi, Toshio; Nishimura, Hiroshi; Okada, Hitomi; Qu, Chen; Saito, Kaori; Watanabe, Takashi; Hatada, Yuji

2016-01-01

Abstract Enzymatic catalysis is an ecofriendly strategy for the production of high‐value low‐molecular‐weight aromatic compounds from lignin. Although well‐definable aromatic monomers have been obtained from synthetic lignin‐model dimers, enzymatic‐selective synthesis of platform monomers from natural lignin has not been accomplished. In this study, we successfully achieved highly specific synthesis of aromatic monomers with a phenylpropane structure directly from natural lignin using a cascade reaction of β‐O‐4‐cleaving bacterial enzymes in one pot. Guaiacylhydroxylpropanone (GHP) and the GHP/syringylhydroxylpropanone (SHP) mixture are exclusive monomers from lignin isolated from softwood (Cryptomeria japonica) and hardwood (Eucalyptus globulus). The intermediate products in the enzymatic reactions show the capacity to accommodate highly heterologous substrates at the substrate‐binding sites of the enzymes. To demonstrate the applicability of GHP as a platform chemical for bio‐based industries, we chemically generate value‐added GHP derivatives for bio‐based polymers. Together with these chemical conversions for the valorization of lignin‐derived phenylpropanone monomers, the specific and enzymatic production of the monomers directly from natural lignin is expected to provide a new stream in “white biotechnology” for sustainable biorefineries. PMID:27878983

Kinetic modelling of non-enzymatic browning and changes of physio-chemical parameters of peach juice during storage.

PubMed

Lyu, Jian; Liu, Xuan; Bi, Jinfeng; Wu, Xinye; Zhou, Linyan; Ruan, Weihong; Zhou, Mo; Jiao, Yi

2018-03-01

Kinetics of non-enzymatic browning and loss of free amino acids during different storage temperature (4, 25, 37 °C) were investigated. Changes of browning degree ( A 420 ), color parameters, Vitamin C ( V c ), free amino acids and 5-hydroxymethylfurfural (5-HMF) were analyzed to evaluate the non-enzymatic browning reactions, which were significantly affected by storage temperature. The lower temperature (4 °C) decreased the loss of V c and the generation of 5-HMF, but induce the highest loss of serine. At the end of storage, loss of serine, alanine and aspartic acid were mainly lost. Results showed that zero-order kinetic model ( R 2  > 0.859), the first-order model ( R 2  > 0.926) and the combined kinetic model ( R 2  > 0.916) were the most appropriate to describe the changes of a * and b * values, the degradation of V c and the changes of A 420 , L * and 5-HMF during different storage temperatures. These kinetic models can be applied for predicting and minimizing the non-enzymatic browning of fresh peach juice during storage.

Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes

USDA-ARS?s Scientific Manuscript database

Second generation feedstock, especially nonfood lignocellulosic biomass, has been seen as a potential source for biofuel production. Cost intensive pretreatment operations, including physical, chemical, biological, and slow enzymatic hydrolysis, make the overall process of lignocellulosic conversio...

75 FR 63404 - Fossil Fuel-Generated Energy Consumption Reduction for New Federal Buildings and Major...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-15

... [Docket No. EERE-2010-BT-STD-0031] RIN 1904-AB96 Fossil Fuel-Generated Energy Consumption Reduction for... of fossil fuel-generated energy consumption in new Federal buildings and Federal buildings undergoing... full fossil fuel-generated energy consumption reduction level is technically impracticable in light of...

Automatic 3d Building Model Generations with Airborne LiDAR Data

NASA Astrophysics Data System (ADS)

Yastikli, N.; Cetin, Z.

2017-11-01

LiDAR systems become more and more popular because of the potential use for obtaining the point clouds of vegetation and man-made objects on the earth surface in an accurate and quick way. Nowadays, these airborne systems have been frequently used in wide range of applications such as DEM/DSM generation, topographic mapping, object extraction, vegetation mapping, 3 dimensional (3D) modelling and simulation, change detection, engineering works, revision of maps, coastal management and bathymetry. The 3D building model generation is the one of the most prominent applications of LiDAR system, which has the major importance for urban planning, illegal construction monitoring, 3D city modelling, environmental simulation, tourism, security, telecommunication and mobile navigation etc. The manual or semi-automatic 3D building model generation is costly and very time-consuming process for these applications. Thus, an approach for automatic 3D building model generation is needed in a simple and quick way for many studies which includes building modelling. In this study, automatic 3D building models generation is aimed with airborne LiDAR data. An approach is proposed for automatic 3D building models generation including the automatic point based classification of raw LiDAR point cloud. The proposed point based classification includes the hierarchical rules, for the automatic production of 3D building models. The detailed analyses for the parameters which used in hierarchical rules have been performed to improve classification results using different test areas identified in the study area. The proposed approach have been tested in the study area which has partly open areas, forest areas and many types of the buildings, in Zekeriyakoy, Istanbul using the TerraScan module of TerraSolid. The 3D building model was generated automatically using the results of the automatic point based classification. The obtained results of this research on study area verified that automatic 3D building models can be generated successfully using raw LiDAR point cloud data.

Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening

PubMed Central

Lane, Andrew B.; Strzelecka, Magdalena; Ettinger, Andreas; Grenfell, Andrew W.; Wittmann, Torsten; Heald, Rebecca

2015-01-01

Summary CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency. PMID:26212133

Enzymatic added extraction and clarification of fruit juices-A review.

PubMed

Sharma, Harsh P; Patel, Hiral; Sugandha

2017-04-13

Enzymatic treatment for juice extraction is most commonly used now a days. The enzymatic process is claimed to offer a number of advantages over mechanical-thermal comminution of several fruit pulps. Enzymes are an integral component of modern fruit juice manufacturing and are highly suitable for optimizing processes. Their main purposes are: increase extraction of juice from raw material, increase processing efficiency (pressing, solid settling or removal), and generate a final product that is clear and visually attractive. Juice extraction can be done by using various mechanical processes, which may be achieved through diffusion extraction, decanter centrifuge, screw type juice extractor, fruit pulper and by different types of presses. Enzymatic treatment prior to mechanical extraction significantly improves juice recovery compared to any other extraction process. Enzymatic hydrolysis of the cell walls increases the extraction yield, reducing sugars, soluble dry matter content and galacturonic acid content and titrable acidity of the products. Enzymatic degradation of the biomaterial depends upon the type of enzyme, incubation time, incubation temperature, enzyme concentration, agitation, pH and use of different enzyme combinations. We can conclude from the technical literature that use of the enzymes i.e. cellulases, pectinases, amylases and combination of these enzymes can give better juice yield with superior quality of the fruit juice. Pectinase enzyme can give maximum juice yield i.e. 92.4% at 360 minutes incubation time, 37°C incubation temperature and 5 mg/100 g of enzyme concentration. Whereas the combination of two enzymes i.e. pectin methyl esterase (PME) and polygalacturonase (PG) at 120 minutes of incubation time, 50°C of incubation temperature and 0.05 mg/100 gm of enzymatic concentration can give the maximum yield of 96.8% for plum fruits. This paper discusses the use of enzymes in fruit juice production focusing on the juice recovery, clarity and effect of the particular enzyme on the biochemical properties of the fruit juices.

75 FR 66008 - Fossil Fuel-Generated Energy Consumption Reduction for New Federal Buildings and Major...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-27

... Fossil Fuel-Generated Energy Consumption Reduction for New Federal Buildings and Major Renovations of Federal Buildings; Correction AGENCY: Office of Energy Efficiency and Renewable Energy, Department of... the fossil fuel- generated energy consumption [[Page 66009

Mycotoxins: A fungal genomics perspective

USDA-ARS?s Scientific Manuscript database

The chemical and enzymatic diversity in the fungal kingdom is staggering. Large-scale fungal genome sequencing projects are generating a massive catalog of secondary metabolite biosynthetic genes and pathways. Fungal natural products are a boon and bane to man as valuable pharmaceuticals and harmful...

Saccharification of biomass using whole solid-state fermentation medium to avoid additional separation steps.

PubMed

Pirota, Rosangela D P B; Baleeiro, Flávio C F; Farinas, Cristiane S

2013-01-01

The enzymatic hydrolysis of steam-exploded sugarcane bagasse (SESB) was investigated using enzymatic extracts (EE) and whole fermentation media (WM), produced in-house, from Aspergillus niger 3T5B8 and Trichoderma reesei Rut-C30 cultivated on wheat bran under solid-state fermentation (SSF). A detailed and quantitative comparison of the different hydrolysis conditions tested was carried out using the Chrastil approach for modeling enzymatic reactions by fitting the experimental data of total reducing sugar (TRS) released according to hydrolysis time. Conversion of SESB using A. niger enzymatic complex were up to 3.2-fold higher (in terms of TRS) than T. reesei at similar enzyme loadings, which could be correlated to the higher β-glucosidase levels (up to 35-fold higher) of A. niger enzymatic complex. Conversion yields after 72 h exceeded 40% in terms of TRS when the WM was supplemented with a low dosage of a commercial enzyme preparation. When the combination of WM (from either T. reesei or A. niger) and commercial cellulase was used, the dosage of the commercial enzyme could be reduced by half, while still providing a hydrolysis that was up to 36% more efficient. Furthermore, SESB hydrolysis using either EE or WM resulted in similar yields, indicating that the enzyme extraction/filtration steps could be eliminated from the overall process. This procedure is highly advantageous in terms of reduced enzyme and process costs, and also avoids the generation of unnecessary effluent streams. Thus, the enzymatic conversion of SESB using the WM from SSF is cost-effective and compatible with the biorefinery concept. © 2013 American Institute of Chemical Engineers.

Age effect in generating mental images of buildings but not common objects.

PubMed

Piccardi, L; Nori, R; Palermo, L; Guariglia, C; Giusberti, F

2015-08-18

Imagining a familiar environment is different from imagining an environmental map and clinical evidence demonstrated the existence of double dissociations in brain-damaged patients due to the contents of mental images. Here, we assessed a large sample of young and old participants by considering their ability to generate different kinds of mental images, namely, buildings or common objects. As buildings are environmental stimuli that have an important role in human navigation, we expected that elderly participants would have greater difficulty in generating images of buildings than common objects. We found that young and older participants differed in generating both buildings and common objects. For young participants there were no differences between buildings and common objects, but older participants found easier to generate common objects than buildings. Buildings are a special type of visual stimuli because in urban environments they are commonly used as landmarks for navigational purposes. Considering that topographical orientation is one of the abilities mostly affected in normal and pathological aging, the present data throw some light on the impaired processes underlying human navigation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Programmable bioelectronics in a stimuli-encoded 3D graphene interface.

PubMed

Parlak, Onur; Beyazit, Selim; Tse-Sum-Bui, Bernadette; Haupt, Karsten; Turner, Anthony P F; Tiwari, Ashutosh

2016-05-21

The ability to program and mimic the dynamic microenvironment of living organisms is a crucial step towards the engineering of advanced bioelectronics. Here, we report for the first time a design for programmable bioelectronics, with 'built-in' switchable and tunable bio-catalytic performance that responds simultaneously to appropriate stimuli. The designed bio-electrodes comprise light and temperature responsive compartments, which allow the building of Boolean logic gates (i.e."OR" and "AND") based on enzymatic communications to deliver logic operations.

Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation.

PubMed

Jiang, Yu; Tao, Rongsheng; Shen, Zhengquan; Sun, Liangdong; Zhu, Fuyun; Yang, Sheng

2016-12-01

Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is a pharmaceutical compound often used in food additives and the cosmetics industry. GSH can be produced biologically from L-glutamic acid, L-cysteine, and glycine through an enzymatic process traditionally involving two sequential adenosine triphosphate (ATP)-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS or GSHI, EC 6.3.2.2) and GSH synthetase (GS or GSHII, EC 6.3.2.3). Here, we report the enzymatic production of GSH by recombinant cell-free bifunctional γ-glutamylcysteine synthetase/glutathione synthetase (γ-GCS-GS or GshF) coupled with in vitro acetate kinase-based ATP generation. GSH production by an acetate kinase-integrated Escherichia coli Rosetta(DE3) mutant expressing Streptococcus thermophilus GshF reached 18.3 ± 0.1 g l -1 (59.5 ± 0.3 mM) within 3 h, with a molar yield of 0.75 ± 0.00 mol mol -1 added cysteine and a productivity of 6.1 ± 0.0 g l -1  h -1 . This is the highest GSH titer reported to date. This newly developed biocatalytic process offers a promising approach for meeting the industrial requirements for GSH production.

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

Helmich, Kate E.; Pereira, Jose Henrique; Gall, Daniel L.

Here, lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, wemore » present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.« less

Control of dispatch dynamics for lowering the cost of distributed generation in the built environment

NASA Astrophysics Data System (ADS)

Flores, Robert Joseph

Distributed generation can provide many benefits over traditional central generation such as increased reliability and efficiency while reducing emissions. Despite these potential benefits, distributed generation is generally not purchased unless it reduces energy costs. Economic dispatch strategies can be designed such that distributed generation technologies reduce overall facility energy costs. In this thesis, a microturbine generator is dispatched using different economic control strategies, reducing the cost of energy to the facility. Several industrial and commercial facilities are simulated using acquired electrical, heating, and cooling load data. Industrial and commercial utility rate structures are modeled after Southern California Edison and Southern California Gas Company tariffs and used to find energy costs for the simulated buildings and corresponding microturbine dispatch. Using these control strategies, building models, and utility rate models, a parametric study examining various generator characteristics is performed. An economic assessment of the distributed generation is then performed for both the microturbine generator and parametric study. Without the ability to export electricity to the grid, the economic value of distributed generation is limited to reducing the individual costs that make up the cost of energy for a building. Any economic dispatch strategy must be built to reduce these individual costs. While the ability of distributed generation to reduce cost depends of factors such as electrical efficiency and operations and maintenance cost, the building energy demand being serviced has a strong effect on cost reduction. Buildings with low load factors can accept distributed generation with higher operating costs (low electrical efficiency and/or high operations and maintenance cost) due to the value of demand reduction. As load factor increases, lower operating cost generators are desired due to a larger portion of the building load being met in an effort to reduce demand. In addition, buildings with large thermal demand have access to the least expensive natural gas, lowering the cost of operating distributed generation. Recovery of exhaust heat from DG reduces cost only if the buildings thermal demand coincides with the electrical demand. Capacity limits exist where annual savings from operation of distributed generation decrease if further generation is installed. For low operating cost generators, the approximate limit is the average building load. This limit decreases as operating costs increase. In addition, a high capital cost of distributed generation can be accepted if generator operating costs are low. As generator operating costs increase, capital cost must decrease if a positive economic performance is desired.

Development of Next Generation Energy Audit Protocols for the Rapid and Advanced Analysis of Building Energy Use

NASA Astrophysics Data System (ADS)

Hartley, Christopher Ahlvin

Current building energy auditing techniques are outdated and lack targeted, actionable information. These analyses only use one year's worth of monthly electricity and gas bills to define energy conservation and efficiency measures. These limited data sets cannot provide robust, directed energy reduction recommendations. The need is apparent for an overhaul of existing energy audit protocols to utilize all data that is available from the building's utility provider, installed energy management system (EMS), and sub-metering devices. This thesis analyzed the current state-of-the-art in energy audits, generated a next generation energy audit protocol, and conducted both audits types on four case study buildings to find out what additional information can be obtained from additional data sources and increased data gathering resolutions. Energy data from each case study building were collected using a variety of means including utility meters, whole building energy meters, EMS systems, and sub-metering devices. In addition to conducting an energy analysis for each case study building using the current and next generation energy audit protocols, two building energy models were created using the programs eQuest and EnergyPlus. The current and next generation energy audit protocol results were compared to one another upon completion. The results show that using the current audit protocols, only variations in season are apparent. Results from the developed next generation energy audit protocols show that in addition to seasonal variations, building heating, ventilation and air conditioning (HVAC) schedules, occupancy schedules, baseline and peak energy demand levels, and malfunctioning equipment can be found. This new protocol may also be used to quickly generate accurate building models because of the increased resolution that yields scheduling information. The developed next generation energy auditing protocol is scalable and can work for many building types across the United States, and perhaps the world.

Feasibility of reusing the black liquor for enzymatic hydrolysis and ethanol fermentation.

PubMed

Wang, Wen; Chen, Xiaoyan; Tan, Xuesong; Wang, Qiong; Liu, Yunyun; He, Minchao; Yu, Qiang; Qi, Wei; Luo, Yu; Zhuang, Xinshu; Yuan, Zhenhong

2017-03-01

The black liquor (BL) generated in the alkaline pretreatment process is usually thought as the environmental pollutant. This study found that the pure alkaline lignin hardly inhibited the enzymatic hydrolysis of cellulose (EHC), which led to the investigation on the feasibility of reusing BL as the buffer via pH adjustment for the subsequent enzymatic hydrolysis and fermentation. The pH value of BL was adjusted from 13.23 to 4.80 with acetic acid, and the alkaline lignin was partially precipitated. It deposited on the surface of cellulose and negatively influenced the EHC via blocking the access of cellulase to cellulose and adsorbing cellulase. The supernatant separated from the acidified BL scarcely affected the EHC, but inhibited the ethanol fermentation. The 4-times diluted supernatant and the last-time waste wash water of the alkali-treated sugarcane bagasse didn't inhibit the EHC and ethanol production. This work gives a clue of saving water for alkaline pretreatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Liquid nitrogen pretreatment of eucalyptus sawdust and rice hull for enhanced enzymatic saccharification.

PubMed

Castoldi, Rafael; Correa, Vanesa G; de Morais, Gutierrez Rodrigues; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosely A; Peralta-Muniz Moreira, Regina F; Peralta, Rosane M

2017-01-01

In this work, liquid nitrogen was used for the first time in the pretreatment of plant biomasses for purposes of enzymatic saccharification. After treatment (cryocrushing), the initial rates of the enzymatic hydrolysis of eucalyptus sawdust and rice hull were increased more than ten-fold. Cryocrushing did not modify significantly the contents of cellulose, hemicellulose and lignin in both eucalyptus sawdust and rice hulls. However, substantial disorganization of the lignocellulosic materials in consequence of the pretreatment could be observed by electron microscopy. Cryocrushing was highly efficient in improving the saccharification of the holocellulose component of the plant biomasses (from 4.3% to 54.1% for eucalyptus sawdust and from 3.9% to 40.6% for rice hull). It is important to emphasize that it consists in a simple operation with low requirements of water and chemicals, no corrosion, no release of products such as soluble phenolics, furfural and hydroxymethylfurfural and no waste generation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Second-generation ethanol production from elephant grass at high total solids.

PubMed

Menegol, Daiane; Fontana, Roselei Claudete; Dillon, Aldo José Pinheiro; Camassola, Marli

2016-07-01

The enzymatic hydrolysis of Pennisetum purpureum (elephant grass) was evaluated at high total solid levels (from 4% to 20% (w/v)) in a concomitant ball milling treatment in a rotating hydrolysis reactor (RHR). The greatest glucose yield was 20.17% when 4% (w/v) untreated biomass was employed. When sugars obtained from enzymatic hydrolysis were submitted to fermentation with Saccharomyces cerevisiae, the greatest ethanol yield was 22.61% when 4% (w/v) untreated biomass was employed; however, the highest glucose concentration (12.47g/L) was obtaining using 20% (w/v) solids and highest ethanol concentration (6.1g/L) was obtained using 16% (w/v) solids. When elephant grass was hydrolyzed in the rotating hydrolysis reactor, ethanol production was about double that was produced when the biomass was hydrolyzed in a static reactor (SR). These data indicate that it is possible to produce ethanol from elephant grass when milling treatment and enzymatic hydrolysis are performed at the same time. Copyright © 2016. Published by Elsevier Ltd.

Regioselectivity of enzymatic and photochemical single electron transfer promoted carbon-carbon bond fragmentation reactions of tetrameric lignin model compounds.

PubMed

Cho, Dae Won; Latham, John A; Park, Hea Jung; Yoon, Ung Chan; Langan, Paul; Dunaway-Mariano, Debra; Mariano, Patrick S

2011-04-15

New types of tetrameric lignin model compounds, which contain the common β-O-4 and β-1 structural subunits found in natural lignins, have been prepared and carbon-carbon bond fragmentation reactions of their cation radicals, formed by photochemical (9,10-dicyanoanthracene) and enzymatic (lignin peroxidase) SET-promoted methods, have been explored. The results show that cation radical intermediates generated from the tetrameric model compounds undergo highly regioselective C-C bond cleavage in their β-1 subunits. The outcomes of these processes suggest that, independent of positive charge and odd-electron distributions, cation radicals of lignins formed by SET to excited states of sensitizers or heme-iron centers in enzymes degrade selectively through bond cleavage reactions in β-1 vs β-O-4 moieties. In addition, the findings made in the enzymatic studies demonstrate that the sterically large tetrameric lignin model compounds undergo lignin peroxidase-catalyzed cleavage via a mechanism involving preliminary formation of an enzyme-substrate complex.

Two step esterification-transesterification process of wet greasy sewage sludge for biodiesel production.

PubMed

Urrutia, C; Sangaletti-Gerhard, N; Cea, M; Suazo, A; Aliberti, A; Navia, R

2016-01-01

Sewage sludge generated in municipal wastewater treatment plants was used as a feedstock for biodiesel production via esterification/transesterification in a two-step process. In the first esterification step, greasy and secondary sludge were tested using acid and enzymatic catalysts. The results indicate that both catalysts performed the esterification of free fatty acids (FFA) simultaneously with the transesterification of triacylglycerols (TAG). Acid catalyst demonstrated better performance in FFA esterification compared to TAG transesterification, while enzymatic catalyst showed the ability to first hydrolyze TAG in FFA, which were esterified to methyl esters. In addition, FAME concentration using greasy sludge were higher (63.9% and 58.7%), compared with those of secondary sludge (11% and 16%), using acid and enzymatic catalysts, respectively. Therefore, only greasy sludge was used in the second step of alkaline transesterification. The alkaline transesterification of the previously esterified greasy sludge reached a maximum FAME concentration of 65.4% when using acid catalyst. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dynamic modeling and validation of a lignocellulosic enzymatic hydrolysis process--a demonstration scale study.

PubMed

Prunescu, Remus Mihail; Sin, Gürkan

2013-12-01

The enzymatic hydrolysis process is one of the key steps in second generation biofuel production. After being thermally pretreated, the lignocellulosic material is liquefied by enzymes prior to fermentation. The scope of this paper is to evaluate a dynamic model of the hydrolysis process on a demonstration scale reactor. The following novel features are included: the application of the Convection-Diffusion-Reaction equation to a hydrolysis reactor to assess transport and mixing effects; the extension of a competitive kinetic model with enzymatic pH dependency and hemicellulose hydrolysis; a comprehensive pH model; and viscosity estimations during the course of reaction. The model is evaluated against real data extracted from a demonstration scale biorefinery throughout several days of operation. All measurements are within predictions uncertainty and, therefore, the model constitutes a valuable tool to support process optimization, performance monitoring, diagnosis and process control at full-scale studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enzyme pretreatment of fats, oil and grease from restaurant waste to prolong septic soil treatment system effectiveness.

PubMed

Dong, Younsuk; Safferman, Steven I; Ostahowski, Jeff; Herold, Tom; Panter, Ronald

2017-01-02

When a fast-food restaurant's wastewater containing fats, oil and grease (FOG) is discharged into a collection system, it builds up over time and clogs pipes. Similarly, when such wastewater flows into a septic soil treatment system, it adheres to the surface of inlet pipes, gravel/distribution media and soil, restricting the flow and eventually clogging the septic soil treatment system. In this study, an enzymatic pretreatment system was tested on wastewater from a fast-food restaurant to determine its effectiveness in preventing septic soil treatment system clogging. This system used aeration equipment, baffles and a one-time inoculum that excretes enzymes to reduce the molecular weight and number of double bonds associated with FOG. FOG containing triglycerides having lower molecular weights and fewer double bonds are less sticky. The enzymatic pretreatment system was found to cause these changes as verified by measuring the types of triglycerides (compounds in FOG) using liquid chromatography/mass spectrometry. A unique bench-scale septic soil treatment system (soil trench) was also used. Each contained six soil moisture sensors to enable the determination of moisture saturation trends among the five tested conditions: sanitary wastewater only, a combination of sanitary and kitchen wastewater, enzymatically pretreated sanitary and kitchen wastewater, kitchen wastewater, and enzymatically pretreated kitchen wastewater. For all influent types, a significant amount of FOG and other pollutants were removed, regardless of the initial concentrations. Moisture sensor readings showed differences among the tested conditions, indicating that septic soil treatment system clogging was delayed. Inspection of the influent pipe and gravel at the end of testing verified these differences as did the measurements of volatile solids.

Excited singlet molecular O2 (1Δg) is generated enzymatically from excited carbonyls in the dark

PubMed Central

Mano, Camila M.; Prado, Fernanda M.; Massari, Júlio; Ronsein, Graziella E.; Martinez, Glaucia R.; Miyamoto, Sayuri; Cadet, Jean; Sies, Helmut; Medeiros, Marisa H. G.; Bechara, Etelvino J. H.; Di Mascio, Paolo

2014-01-01

In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 (1Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 (1Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 (1Δg). Indirect analysis of O2 (1Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [18O]-labeled triplet, ground state molecular oxygen [18O2 (3Σg-)], chemical trapping of 18O2 (1Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[18O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 (1Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates 18O2 (1Δg) by triplet-triplet energy transfer to ground state oxygen O2 (3Σg−), and supports the long formulated hypothesis of O2 (1Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light. PMID:25087485

A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.

PubMed

Cahn, Jackson K B; Werlang, Caroline A; Baumschlager, Armin; Brinkmann-Chen, Sabine; Mayo, Stephen L; Arnold, Frances H

2017-02-17

The ability to control enzymatic nicotinamide cofactor utilization is critical for engineering efficient metabolic pathways. However, the complex interactions that determine cofactor-binding preference render this engineering particularly challenging. Physics-based models have been insufficiently accurate and blind directed evolution methods too inefficient to be widely adopted. Building on a comprehensive survey of previous studies and our own prior engineering successes, we present a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity. This heuristic-based approach leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale. We demonstrate the efficacy of this strategy by inverting the cofactor specificity of four structurally diverse NADP-dependent enzymes: glyoxylate reductase, cinnamyl alcohol dehydrogenase, xylose reductase, and iron-containing alcohol dehydrogenase. The analytical components of this approach have been fully automated and are available in the form of an easy-to-use web tool: Cofactor Specificity Reversal-Structural Analysis and Library Design (CSR-SALAD).

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

PubMed

Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

2016-01-01

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Quantitative Analysis of Cellular Metabolic Dissipative, Self-Organized Structures

PubMed Central

de la Fuente, Ildefonso Martínez

2010-01-01

One of the most important goals of the postgenomic era is understanding the metabolic dynamic processes and the functional structures generated by them. Extensive studies during the last three decades have shown that the dissipative self-organization of the functional enzymatic associations, the catalytic reactions produced during the metabolite channeling, the microcompartmentalization of these metabolic processes and the emergence of dissipative networks are the fundamental elements of the dynamical organization of cell metabolism. Here we present an overview of how mathematical models can be used to address the properties of dissipative metabolic structures at different organizational levels, both for individual enzymatic associations and for enzymatic networks. Recent analyses performed with dissipative metabolic networks have shown that unicellular organisms display a singular global enzymatic structure common to all living cellular organisms, which seems to be an intrinsic property of the functional metabolism as a whole. Mathematical models firmly based on experiments and their corresponding computational approaches are needed to fully grasp the molecular mechanisms of metabolic dynamical processes. They are necessary to enable the quantitative and qualitative analysis of the cellular catalytic reactions and also to help comprehend the conditions under which the structural dynamical phenomena and biological rhythms arise. Understanding the molecular mechanisms responsible for the metabolic dissipative structures is crucial for unraveling the dynamics of cellular life. PMID:20957111

Multiplex newborn screening for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases using a digital microfluidic platform.

PubMed

Sista, Ramakrishna S; Wang, Tong; Wu, Ning; Graham, Carrie; Eckhardt, Allen; Winger, Theodore; Srinivasan, Vijay; Bali, Deeksha; Millington, David S; Pamula, Vamsee K

2013-09-23

New therapies for lysosomal storage diseases (LSDs) have generated interest in screening newborns for these conditions. We present performance validation data on a digital microfluidic platform that performs multiplex enzymatic assays for Pompe, Fabry, Hunter, Gaucher, and Hurler diseases. We developed an investigational disposable digital microfluidic cartridge that uses a single dried blood spot (DBS) punch for performing a 5-plex fluorometric enzymatic assay on up to 44 DBS samples. Precision and linearity of the assays were determined by analyzing quality control DBS samples; clinical performance was determined by analyzing 600 presumed normal and known affected samples (12 for Pompe, 7 for Fabry and 10 each for Hunter, Gaucher and Hurler). Overall coefficient of variation (CV) values between cartridges, days, instruments, and operators ranged from 2 to 21%; linearity correlation coefficients were ≥0.98 for all assays. The multiplex enzymatic assay performed from a single DBS punch was able to discriminate presumed normal from known affected samples for 5 LSDs. Digital microfluidic technology shows potential for rapid, high-throughput screening for 5 LSDs in a newborn screening laboratory environment. Sample preparation to enzymatic activity on each cartridge is less than 3h. Copyright © 2013 Elsevier B.V. All rights reserved.

The effect of irradiation temperature on the non-enzymatic browning reaction in cooked rice

NASA Astrophysics Data System (ADS)

Lee, Ju-Woon; Oh, Sang-Hee; Kim, Jae-Hun; Byun, Eui-Hong; Ree Kim, Mee; Baek, Min; Byun, Myung-Woo

2007-05-01

The effect of irradiation temperature on the non-enzymatic browning reaction in a sugar-glycine solution and cooked rice generated by gamma irradiation was evaluated in the present study. When the sugar-glycine solution and cooked rice were irradiated at room temperature, the browning reaction was dramatically increased during the post-irradiation period. In the case of irradiation at below the freezing point, the browning by irradiation was retarded during not only irradiation but also a post-irradiation period. The changes of the sugar profile, such as a sugar loss or reducing power of the irradiated sugar-glycine solution and the electron spin resonance signal intensity of the irradiated cooked rice were also decreased with lower irradiation temperature. The present results may suggest that the production of free radicals and a radiolysis product is inhibited during gamma irradiation in the frozen state and it may prevent the browning reaction generated by gamma irradiation from occurring.

Air bio-battery with a gas/liquid porous diaphragm cell for medical and health care devices.

PubMed

Arakawa, Takahiro; Xie, Rui; Seshima, Fumiya; Toma, Koji; Mitsubayashi, Kohji

2018-04-30

Powering future generations of medical and health care devices mandates the transcutaneous transfer of energy or harvesting energy from the human body fluid. Glucose-driven bio fuel cells (bio-batteries) demonstrate promise as they produce electrical energy from glucose, which is a substrate presents in physiological fluids. Enzymatic biofuel cells can convert chemical energy into electrical energy using enzymes as catalysts. In this study, an air bio-battery was developed for healthcare and medical applications, consisting of a glucose-driven enzymatic biofuel cell using a direct gas-permeable membrane or a gas/liquid porous diaphragm. The power generation characteristics included a maximum current density of 285μA/cm 2 and maximum power density of 70.7μW/cm 2 in the presence of 5mmol/L of glucose in solution. In addition, high-performance, long-term-stabilized power generation was achieved using the gas/liquid porous diaphragm for the reactions between oxygen and enzyme. This system can be powered using 5mmol/L of glucose, the value of which is similar to that of the blood sugar range in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

Building blocks for the solution phase synthesis of oligonucleotides: regioselective hydrolysis of 3',5'-Di-O-levulinylnucleosides using an enzymatic approach.

PubMed

García, Javier; Fernández, Susana; Ferrero, Miguel; Sanghvi, Yogesh S; Gotor, Vicente

2002-06-28

A short and convenient synthesis of 3'- and 5'-O-levulinyl-2'-deoxynucleosides has been developed from the corresponding 3',5'-di-O-levulinyl derivatives by regioselective enzymatic hydrolysis, avoiding several tedious chemical protection/deprotection steps. Thus, Candida antartica lipase B (CAL-B) was found to selectively hydrolyze the 5'-levulinate esters, furnishing 3'-O-levulinyl-2'-deoxynucleosides 3 in >80% isolated yields. On the other hand, immobilized Pseudomonas cepacia lipase (PSL-C) and Candida antarctica lipase A (CAL-A) exhibit the opposite selectivity toward the hydrolysis at the 3'-position, affording 5'-O-levulinyl derivatives 4 in >70% yields. A similar hydrolysis procedure was successfully extended to the synthesis of 3'- and 5'-O-levulinyl-protected 2'-O-alkylribonucleosides 7 and 8. This work demonstrates for the first time application of commercial CAL-B and PSL-C toward regioselective hydrolysis of levulinyl esters with excellent selectivity and yields. It is noteworthy that protected cytidine and adenosine base derivatives were not adequate substrates for the enzymatic hydrolysis with CAL-B, whereas PSL-C was able to accommodate protected bases during selective hydrolysis. In addition, we report an improved synthesis of dilevulinyl esters using a polymer-bound carbodiimide as a replacement for dicyclohexylcarbodiimide (DCC), thus considerably simplifying the workup for esterification reactions.

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis.

PubMed

Sumida, Yosuke; Iwai, Sachio; Nishiya, Yoshiaki; Kumagai, Shinya; Yamada, Toshihide; Azuma, Masayuki

2018-03-01

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Gaussian curvature analysis allows for automatic block placement in multi-block hexahedral meshing.

PubMed

Ramme, Austin J; Shivanna, Kiran H; Magnotta, Vincent A; Grosland, Nicole M

2011-10-01

Musculoskeletal finite element analysis (FEA) has been essential to research in orthopaedic biomechanics. The generation of a volumetric mesh is often the most challenging step in a FEA. Hexahedral meshing tools that are based on a multi-block approach rely on the manual placement of building blocks for their mesh generation scheme. We hypothesise that Gaussian curvature analysis could be used to automatically develop a building block structure for multi-block hexahedral mesh generation. The Automated Building Block Algorithm incorporates principles from differential geometry, combinatorics, statistical analysis and computer science to automatically generate a building block structure to represent a given surface without prior information. We have applied this algorithm to 29 bones of varying geometries and successfully generated a usable mesh in all cases. This work represents a significant advancement in automating the definition of building blocks.

Passive emission colorimetric sensor (PECS) for measuring emission rates of formaldehyde based on an enzymatic reaction and reflectance photometry.

PubMed

Shinohara, Naohide; Kajiwara, Tomohisa; Ohnishi, Masato; Kodama, Kenichi; Yanagisawa, Yukio

2008-06-15

A coin-sized passive emission colorimetric sensor (PECS) based on an enzymatic reaction and a portable reflectance photometry device were developed to determine the emission rates of formaldehyde from building materials and other materials found indoors in only 30 minutes on-site. The color change of the PECS linearly correlated to the concentration of formaldehyde aqueous solutions up to 28 microg/mL. The correlation between the emission rates measured by using the PECS and those measured by using a desiccator method or by using a chamber method was fitted with a linear function and a power function, and the determination coefficients were more than 0.98. The reproducible results indicate that the emission rates could be obtained with the correlation equations from the data measured by using the PECS and the portable reflectance photometry device. Limits of detection (LODs) were 0.051 mg/L for the desiccator method and 3.1 microg/m2/h for the chamber method. Thus, it was confirmed that the emission rates of formaldehyde from the building materials classified as F four-star (< 0.3 mg/L (desiccator method) or < 5.0 microg/m2/h (chamber method)), based on Japanese Industrial Standards (JIS), could be measured with the PECS. The measurement with PECS was confirmed to be precise (RSD < 10%). Other chemicals emitted from indoor materials, such as methanol, ethanol, acetone, toluene, and xylene, interfered little with the measurement of formaldehyde emission rates by using the PECS.

Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening.

PubMed

Lane, Andrew B; Strzelecka, Magdalena; Ettinger, Andreas; Grenfell, Andrew W; Wittmann, Torsten; Heald, Rebecca

2015-08-10

CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency. Copyright © 2015 Elsevier Inc. All rights reserved.

Improved enzymatic hydrolysis of lignocellulosic biomass through pretreatment with plasma electrolysis.

PubMed

Gao, Jing; Chen, Li; Zhang, Jian; Yan, Zongcheng

2014-11-01

A comprehensive research on plasma electrolysis as pretreatment method for water hyacinth (WH) was performed based on lignin content, crystalline structure, surface property, and enzymatic hydrolysis. A large number of active particles, such as HO and H2O2, generated by plasma electrolysis could decompose the lignin of the biomass samples and reduce the crystalline index. An efficient pretreatment process made use of WH pretreated at a load of 48 wt% (0.15-0.18 mm) in FeCl3 solution for 30 min at 450 V. After the pretreatment, the sugar yield of WH was increased by 126.5% as compared with unpretreated samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immobilization Techniques in the Fabrication of Nanomaterial-Based Electrochemical Biosensors: A Review

PubMed Central

Putzbach, William; Ronkainen, Niina J.

2013-01-01

The evolution of 1st to 3rd generation electrochemical biosensors reflects a simplification and enhancement of the transduction pathway. However, in recent years, modification of the transducer with nanomaterials has become increasingly studied and imparts many advantages. The sensitivity and overall performance of enzymatic biosensors has improved tremendously as a result of incorporating nanomaterials in their fabrication. Given the unique and favorable qualities of gold nanoparticles, graphene and carbon nanotubes as applied to electrochemical biosensors, a consolidated survey of the different methods of nanomaterial immobilization on transducer surfaces and enzyme immobilization on these species is beneficial and timely. This review encompasses modification of enzymatic biosensors with gold nanoparticles, carbon nanotubes, and graphene. PMID:23580051

Restricting lignin and enhancing sugar deposition in secondary cell walls enhances monomeric sugar release after low temperature ionic liquid pretreatment

DOE PAGES

Scullin, Chessa; Cruz, Alejandro G.; Chuang, Yi -De; ...

2015-07-04

Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype.

Reactions of inorganic free radicals with liver protecting drugs

NASA Astrophysics Data System (ADS)

György, I.; Blázovics, A.; Fehér, J.; Földiák, G.

Liver protecting drugs, silibinin, a flavonolignane, and the dihydroquinoline derivates, CH 402 and MTDQ-DA, were shown to inhibit processes in which enzymatically or non-enzymatically generated free radicals were involved. Inorganic free radicals (N 3, (SCN) -2, OH, Trp, CO -2, O -2) produced by pulse radiolysis readily react with the compounds, which transform into exceptionally long-lived, unreactive transients. Time evolution of the UV and visible spectra indicate that oxidising radicals form a phenoxyl type radical from silibinin, while OH forms an adduct by attacking, simultaneously, at various sites of the molecule. Superoxide radicals reduce silibinin and oxidise CH 402 and MTDQ-DA. It is concluded that the drugs might exhibit antioxidant behavior in living systems.

Restricting lignin and enhancing sugar deposition in secondary cell walls enhances monomeric sugar release after low temperature ionic liquid pretreatment

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

Scullin, Chessa; Cruz, Alejandro G.; Chuang, Yi -De

Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype.

Immobilization techniques in the fabrication of nanomaterial-based electrochemical biosensors: a review.

PubMed

Putzbach, William; Ronkainen, Niina J

2013-04-11

The evolution of 1st to 3rd generation electrochemical biosensors reflects a simplification and enhancement of the transduction pathway. However, in recent years, modification of the transducer with nanomaterials has become increasingly studied and imparts many advantages. The sensitivity and overall performance of enzymatic biosensors has improved tremendously as a result of incorporating nanomaterials in their fabrication. Given the unique and favorable qualities of gold nanoparticles, graphene and carbon nanotubes as applied to electrochemical biosensors, a consolidated survey of the different methods of nanomaterial immobilization on transducer surfaces and enzyme immobilization on these species is beneficial and timely. This review encompasses modification of enzymatic biosensors with gold nanoparticles, carbon nanotubes, and graphene.

Highly Sensitive Biosensing with Solid-State Nanopores Displaying Enzymatically Reconfigurable Rectification Properties.

PubMed

Pérez-Mitta, Gonzalo; Peinetti, Ana S; Cortez, M Lorena; Toimil-Molares, María Eugenia; Trautmann, Christina; Azzaroni, Omar

2018-05-09

Molecular design of biosensors based on enzymatic processes taking place in nanofluidic elements is receiving increasing attention by the scientific community. In this work, we describe the construction of novel ultrasensitive enzymatic nanopore biosensors employing "reactive signal amplifiers" as key elements coupled to the transduction mechanism. The proposed framework offers innovative design concepts not only to amplify the detected ionic signal and develop ultrasensitive nanopore-based sensors but also to construct nanofluidic diodes displaying specific chemo-reversible rectification properties. The integrated approach is demonstrated by electrostatically assembling poly(allylamine) on the anionic pore walls followed by the assembly of urease. We show that the cationic weak polyelectrolyte acts as a "reactive signal amplifier" in the presence of local pH changes induced by the enzymatic reaction. These bioinduced variations in proton concentration ultimately alter the protonation degree of the polyamine resulting in amplifiable, controlled, and reproducible changes in the surface charge of the pore walls, and consequently on the generated ionic signals. The "iontronic" response of the as-obtained devices is fully reversible, and nanopores are reused and assayed with different urea concentrations, thus ensuring reliable design. The limit of detection (LOD) was 1 nM. To the best of our knowledge, this value is the lowest LOD reported to date for enzymatic urea detection. In this context, we envision that this approach based on the use of "reactive signal amplifiers" into solid-state nanochannels will provide new alternatives for the molecular design of highly sensitive nanopore biosensors as well as (bio)chemically addressable nanofluidic elements.

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

DOE PAGES

Shiga, Tânia M.; Xiao, Weihua; Yang, Haibing; ...

2017-12-27

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates. Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 degrees C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability,more » and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl3-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose. Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.« less

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

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

Shiga, Tânia M.; Xiao, Weihua; Yang, Haibing

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates. Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 degrees C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability,more » and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl3-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose. Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.« less

Development of Automated Procedures to Generate Reference Building Models for ASHRAE Standard 90.1 and India’s Building Energy Code and Implementation in OpenStudio

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

Parker, Andrew; Haves, Philip; Jegi, Subhash

This paper describes a software system for automatically generating a reference (baseline) building energy model from the proposed (as-designed) building energy model. This system is built using the OpenStudio Software Development Kit (SDK) and is designed to operate on building energy models in the OpenStudio file format.

Enzymatic analysis of α-ketoglutaramate—A biomarker for hyperammonemia

PubMed Central

Halámková, Lenka; Mailloux, Shay; Halámek, Jan; Cooper, Arthur J.L.; Katz, Evgeny

2012-01-01

Two enzymatic assays were developed for the analysis of α-ketoglutaramate (KGM)—an important biomarker of hepatic encephalopathy and other hyperammonemic diseases. In both procedures, KGM is first converted to α-ketoglutarate (KTG) via a reaction catalyzed by ω-amidase (AMD). In the first procedure, KTG generated in the AMD reaction initiates a biocatalytic cascade in which the concerted action of alanine transaminase and lactate dehydrogenase results in the oxidation of NADH. In the second procedure, KTG generated from KGM is reductively aminated, with the concomitant oxidation of NADH, in a reaction catalyzed by L-glutamic dehydrogenase. In both assays, the decrease in optical absorbance (λ=340 nm) corresponding to NADH oxidation is used to quantify concentrations of KGM. The two analytical procedures were applied to 50% (v/v) human serum diluted with aqueous solutions containing the assay components and spiked with concentrations of KGM estimated to be present in normal human plasma and in plasma from hyperammonemic patients. Since KTG is the product of AMD-catalyzed hydrolysis of KGM, in a separate study, this compound was used as a surrogate for KGM. Statistical analyses of samples mimicking the concentration of KGM assumed to be present in normal and pathological concentration ranges were performed. Both enzymatic assays for KGM were confirmed to discriminate between the predicted normal and pathophysiological concentrations of the analyte. The present study is the first step toward the development of a clinically useful probe for KGM analysis in biological fluids. PMID:23141304

Chitosan-induced Au/Ag nanoalloy dispersed in IL and application in fabricating an ultrasensitive glucose biosensor based on luminol-H₂O₂-Cu²⁺/IL chemiluminescence system.

PubMed

Chaichi, M J; Alijanpour, S O

2014-11-01

A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated hydrogen peroxide (H₂O₂) was constructed by one covalent immobilization of glucose oxidase (GOD) in glutaraldehyde-functionalized glass cell. In following, chitosan-induced Au/Ag nanoparticles dispersed in ion liquid (IL) were synthesised and immobilized on it. Herein, chitosan molecules acted as both the reducing and stabilizing agent for the preparation of NPs and also, as a coupling agent GOD and Au/Ag alloy NPs. In addition to catalyze luminol CL reaction, these NPs offered excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between GOD and glucose. The used IL in fabrication of biosensor increased its stability. Also, IL alongside Cu(2+) accelerated enzymatic and CL reaction kinetic, and decreased luminol CL reaction optimum pH to 7.5 which would enable sensitive and precision determination of glucose. Under optimum condition, linear response range of glucose was found to be 1.0 × 10(-6)-7.5 × 10(-3)M, and detection limit was 4.0 × 10(-7)M. The CL biosensor exhibited good storage stability, i.e., 90% of its initial response was retained after 2 months storage at pH 7.0. The present CL biosensor has been applied satisfactory to analysis of glucose in real serum and urine samples. Copyright © 2014 Elsevier B.V. All rights reserved.

A direct biocombinatorial strategy toward next generation, mussel-glue inspired saltwater adhesives.

PubMed

Wilke, Patrick; Helfricht, Nicolas; Mark, Andreas; Papastavrou, Georg; Faivre, Damien; Börner, Hans G

2014-09-10

Biological materials exhibit remarkable, purpose-adapted properties that provide a source of inspiration for designing new materials to meet the requirements of future applications. For instance, marine mussels are able to attach to a broad spectrum of hard surfaces under hostile conditions. Controlling wet-adhesion of synthetic macromolecules by analogue processes promises to strongly impact materials sciences by offering advanced coatings, adhesives, and glues. The de novo design of macromolecules to mimic complex aspects of mussel adhesion still constitutes a challenge. Phage display allows material scientists to design specifically interacting molecules with tailored affinity to material surfaces. Here, we report on the integration of enzymatic processing steps into phage display biopanning to expand the biocombinatorial procedure and enable the direct selection of enzymatically activable peptide adhesion domains. Adsorption isotherms and single molecule force spectroscopy show that those de novo peptides mimic complex aspects of bioadhesion, such as enzymatic activation (by tyrosinase), the switchability from weak to strong binders, and adsorption under hostile saltwater conditions. Furthermore, peptide-poly(ethylene oxide) conjugates are synthesized to generate protective coatings, which possess anti-fouling properties and suppress irreversible interactions with blood-plasma protein cocktails. The extended phage display procedure provides a generic way to non-natural peptide adhesion domains, which not only mimic nature but also improve biological sequence sections extractable from mussel-glue proteins. The de novo peptides manage to combine several tasks in a minimal 12-mer sequence and thus pave the way to overcome major challenges of technical wet glues.

Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy.

PubMed

Shih, Chien-Ju; Smith, Emily A

2009-10-27

Raman spectroscopy has been used for the quantitative determination of the conversion efficiency at each step in the production of ethanol from biomass. The method requires little sample preparation; therefore, it is suitable for screening large numbers of biomass samples and reaction conditions in a complex sample matrix. Dilute acid or ammonia-pretreated corn stover was used as a model biomass for these studies. Ammonia pretreatment was suitable for subsequent measurements with Raman spectroscopy, but dilute acid-pretreated corn stover generated a large background signal that surpassed the Raman signal. The background signal is attributed to lignin, which remains in the plant tissue after dilute acid pretreatment. A commercial enzyme mixture was used for the enzymatic hydrolysis of corn stover, and glucose levels were measured with a dispersive 785 nm Raman spectrometer. The glucose detection limit in hydrolysis liquor by Raman spectroscopy was 8 g L(-1). The mean hydrolysis efficiency for three replicate measurements obtained with Raman spectroscopy (86+/-4%) was compared to the result obtained using an enzymatic reaction with UV-vis spectrophotometry detection (78+/-8%). The results indicate good accuracy, as determined using a Student's t-test, and better precision for the Raman spectroscopy measurement relative to the enzymatic detection assay. The detection of glucose in hydrolysis broth by Raman spectroscopy showed no spectral interference, provided the sample was filtered to remove insoluble cellulose prior to analysis. The hydrolysate was further subjected to fermentation to yield ethanol. The detection limit for ethanol in fermentation broth by Raman spectroscopy was found to be 6 g L(-1). Comparison of the fermentation efficiencies measured by Raman spectroscopy (80+/-10%) and gas chromatography-mass spectrometry (87+/-9%) were statistically the same. The work demonstrates the utility of Raman spectroscopy for screening the entire conversion process to generate lignocellulosic ethanol.

Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell.

PubMed

Campbell, Alan S; Murata, Hironobu; Carmali, Sheiliza; Matyjaszewski, Krzysztof; Islam, Mohammad F; Russell, Alan J

2016-12-15

Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a "grafting-from" approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective "wiring" of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm(-2)) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Water Reuse and Pathogen Tracking

EPA Science Inventory

Building product water. By designing our buildings to collect and treat water generated on-site, can be and reused for flushing our toilets and irrigating our landscaping. Several water sources are generated with-in a building including: rainwater, stormwater, graywater, blackwa...

EXPRESSION OF AS3MT ALTERS TRANSCRIPTIONAL PROFILES IN HUMAN UROTHELIAL CELLS EXPOSED TO ARSENITE

EPA Science Inventory

Inorganic arsenic (iAs) is an environmental toxin and human carcinogen. The enzymatic methylation of iAs that is catalyzed by arsenic (+3 oxidation state)-methyltransferase (AS3MT) generates reactive methylated intermediates that contribute to the toxic and carcinogenic effects ...

Quantitative profiling of oxylipins through comprehensive lc-ms/ms analysis: Application in cardiac surgery

USDA-ARS?s Scientific Manuscript database

Oxylipins, including eicosanoids, affect a broad range of biological processes, such as the initiation and resolution of inflammation. These compounds, also referred to as lipid mediators, are (non-) enzymatically generated by oxidation of polyunsaturated fatty acids such as arachidonic acid (AA). A...

RECOMBINANT RAT CYT19, AN ARSENIC METHYLTRANSFERASE, EFFICIENTLY GENERATES TRIMETHYLARSINE OXIDE IN THE ABSENCE OF GLUTATHIONE

EPA Science Inventory

Chronic exposure to inorganic arsenic (iAs), a toxic metalloid sometimes present in drinking water, is associated with increased prevalences of various cancers and other disorders. Humans and many other species enzymatically convert iAs into methylated metabolites. The major meta...

Arsenite-induced ROS/RNS generation causes zinc loss and inhibits the activity of poly(ADP-ribose) polymerase-1.

PubMed

Wang, Feng; Zhou, Xixi; Liu, Wenlan; Sun, Xi; Chen, Chen; Hudson, Laurie G; Jian Liu, Ke

2013-08-01

Arsenic enhances the genotoxicity of other carcinogenic agents such as ultraviolet radiation and benzo[a]pyrene. Recent reports suggest that inhibition of DNA repair is an important aspect of arsenic cocarcinogenesis, and DNA repair proteins such as poly(ADP ribose) polymerase (PARP)-1 are direct molecular targets of arsenic. Although arsenic has been shown to generate reactive oxygen/nitrogen species (ROS/RNS), little is known about the role of arsenic-induced ROS/RNS in the mechanism underlying arsenic inhibition of DNA repair. We report herein that arsenite-generated ROS/RNS inhibits PARP-1 activity in cells. Cellular exposure to arsenite, as well as hydrogen peroxide and NONOate (nitric oxide donor), decreased PARP-1 zinc content, enzymatic activity, and PARP-1 DNA binding. Furthermore, the effects of arsenite on PARP-1 activity, DNA binding, and zinc content were partially reversed by the antioxidant ascorbic acid, catalase, and the NOS inhibitor, aminoguanidine. Most importantly, arsenite incubation with purified PARP-1 protein in vitro did not alter PARP-1 activity or DNA-binding ability, whereas hydrogen peroxide or NONOate retained PARP-1 inhibitory activity. These results strongly suggest that cellular generation of ROS/RNS plays an important role in arsenite inhibition of PARP-1 activity, leading to the loss of PARP-1 DNA-binding ability and enzymatic activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Interleukin-1beta and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation.

PubMed

Mathy-Hartert, M; Hogge, L; Sanchez, C; Deby-Dupont, G; Crielaard, J M; Henrotin, Y

2008-07-01

Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated enzymatic antioxidant system formed principally by superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPX). This work was designed to assess the effects of interleukin (IL)-1beta and IL-6 on the enzymatic activity and gene expression of SODs, CAT and GPX in bovine chondrocytes. Bovine chondrocytes were cultured in monolayer for 4-96 h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100 ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20 microM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10 microM) and MG132 (0.1-10 microM)] were used. SODs, CAT and GPX enzymatic activities were evaluated in cellular extract by using colorimetric enzymatic assays. Mn SODs, Cu/Zn SOD, extracellular SOD (EC SOD), CAT and GPX gene expressions were quantified by real-time and quantitative polymerase chain reaction (PCR). Mn SOD and GPX activities were dose and time-dependently increased by IL-1beta. In parallel, IL-1beta markedly enhanced Mn SOD and GPX gene expressions, but decreased Cu/Zn SOD, EC SOD and CAT gene expressions. Induction of SOD enzymatic activity and Mn SOD mRNA expression were inhibited by NF-kappaB inhibitors but not by MAPK inhibitors. IL-6 effects were similar but weaker than those of IL-1beta. In conclusion, IL-1beta, and to a lesser extend IL-6, dysregulates enzymatic antioxidant defenses in chondrocyte. These changes could lead to a transient accumulation of H(2)O(2) in mitochondria, and consequently to mitochondria damage. These changes contribute to explain the mitochondrial dysfunction observed in osteoarthritis chondrocytes.

Fungal accumulation of metals from building materials during brown rot wood decay.

PubMed

Hastrup, Anne Christine Steenkjær; Jensen, Bo; Jellison, Jody

2014-08-01

This study analyzes the accumulation and translocation of metal ions in wood during the degradation performed by one strain of each of the three brown rot fungi; Serpula lacrymans, Meruliporia incrassata and Coniophora puteana. These fungi species are inhabitants of the built environment where the prevention and understanding of fungal decay is of high priority. This study focuses on the influence of various building materials in relation to fungal growth and metal uptake. Changes in the concentration of iron, manganese, calcium and copper ions in the decayed wood were analyzed by induced coupled plasma spectroscopy and related to wood weight loss and oxalic acid accumulation. Metal transport into the fungal inoculated wood was found to be dependent on the individual strain/species. The S. lacrymans strain caused a significant increase in total iron whereas the concentration of copper ions in the wood appeared decreased after 10 weeks of decay. Wood inoculated with the M. incrassata isolate showed the contrary tendency with high copper accumulation and low iron increase despite similar weight losses for the two strains. However, significantly lower oxalic acid accumulation was recorded in M. incrassata degraded wood. The addition of a building material resulted in increased weight loss in wood degraded by C. puteana in the soil-block test; however, this could not be directly linked specifically to the accumulation of any of the four metals recorded. The accumulation of oxalic acid seemed to influence the iron uptake. The study assessing the influence of the presence of soil and glass in the soil-block test revealed that soil contributed the majority of the metals for uptake by the fungi and contributed to increased weight loss. The varying uptake observed among the three brown rot fungi strains toward the four metals analyzed may be related to the specific non-enzymatic and enzymatic properties including bio-chelators employed by each of the species during wood decay.

75 FR 60102 - South Dakota PrairieWinds Project (DOE/EIS-0418)

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-29

... feature 101 wind turbine generators; 6,000 square-foot operations and maintenance building and fence... generation facility that would feature 101 wind turbine generators; 6,000 square-foot operations and... turbine generators, operations and maintenance building and fence perimeter, underground communication...

Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine.

PubMed

Normark, Monica; Winestrand, Sandra; Lestander, Torbjörn A; Jönsson, Leif J

2014-03-19

Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass.

Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry.

PubMed

Zhou, Xiao-Ming; Shimanovich, Ulyana; Herling, Therese W; Wu, Si; Dobson, Christopher M; Knowles, Tuomas P J; Perrett, Sarah

2015-06-23

Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions.

Hybrid microfluidic fuel cell based on Laccase/C and AuAg/C electrodes.

PubMed

López-González, B; Dector, A; Cuevas-Muñiz, F M; Arjona, N; Cruz-Madrid, C; Arana-Cuenca, A; Guerra-Balcázar, M; Arriaga, L G; Ledesma-García, J

2014-12-15

A hybrid glucose microfluidic fuel cell composed of an enzymatic cathode (Laccase/ABTS/C) and an inorganic anode (AuAg/C) was developed and tested. The enzymatic cathode was prepared by adsorption of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and Laccase on Vulcan XC-72, which act as a redox mediator, enzymatic catalyst and support, respectively. The Laccase/ABTS/C composite was characterised by Fourier Transform Infrared (FTIR) Spectroscopy, streaming current measurements (Zeta potential) and cyclic voltammetry. The AuAg/C anode catalyst was characterised by Transmission electron microscopy (TEM) and cyclic voltammetry. The hybrid microfluidic fuel cell exhibited excellent performance with a maximum power density value (i.e., 0.45 mW cm(-2)) that is the highest reported to date. The cell also exhibited acceptable stability over the course of several days. In addition, a Mexican endemic Laccase was used as the biocathode electrode and evaluated in the hybrid microfluidic fuel cell generating 0.5 mW cm(-2) of maximum power density. Copyright © 2014 Elsevier B.V. All rights reserved.

Highly sensitive on-site detection of glucose in human urine with naked eye based on enzymatic-like reaction mediated etching of gold nanorods.

PubMed

Zhang, Zhiyang; Chen, Zhaopeng; Cheng, Fangbin; Zhang, Yaowen; Chen, Lingxin

2017-03-15

Based on enzymatic-like reaction mediated etching of gold nanorods (GNRs), an ultrasensitive visual method was developed for on-site detection of urine glucose. With the catalysis of MoO 4 2 - , GNRs were efficiently etched by H 2 O 2 which was generated by glucose-glucose oxidase enzymatic reaction. The etching of GNRs lead to a blue-shift of logitudinal localized surface plasmon resonance of GNRs, accompanied by an obvious color change from blue to red. The peak-shift and the color change can be used for detection of glucose by the spectrophotometer and the naked eyes. Under optimal condition, an excellent sensitivity toward glucose is obtained with a detection limit of 0.1μM and a visual detection limit of 3μM in buffer solution. Benefiting from the high sensitivity, the successful colorimetric detection of glucose in original urine samples was achieved, which indicates the practical applicability to the on-site determination of urine glucose. Copyright © 2016 Elsevier B.V. All rights reserved.

Harnessing surface-bound enzymatic reactions to organize microcapsules in solution

PubMed Central

Shklyaev, Oleg E.; Shum, Henry; Sen, Ayusman; Balazs, Anna C.

2016-01-01

By developing new computational models, we examine how enzymatic reactions on an underlying surface can be harnessed to direct the motion and organization of reagent-laden microcapsules in a fluid-filled microchannel. In the presence of appropriate reagents, surface-bound enzymes can act as pumps, which drive large-scale fluid flows. When the reagents diffuse through the capsules’ porous shells, they can react with enzymatic sites on the bottom surface. The ensuing reaction generates fluid density variations, which result in fluid flows. These flows carry the suspended microcapsules and drive them to aggregate into “colonies” on and near the enzyme-covered sites. This aggregation continues until the reagent has been depleted and the convection stops. We show that the shape of the assembled colonies can be tailored by patterning the distribution of enzymes on the surface. This fundamental physicochemical mechanism could have played a role in the self-organization of early biological cells (protocells) and can be used to regulate the autonomous motion and targeted delivery of microcarriers in microfluidic devices. PMID:27034990

Use of a new Trichoderma harzianum strain isolated from the Amazon rainforest with pretreated sugar cane bagasse for on-site cellulase production.

PubMed

Delabona, Priscila da Silva; Farinas, Cristiane Sanchez; da Silva, Mateus Ribeiro; Azzoni, Sindelia Freitas; Pradella, José Geraldo da Cruz

2012-03-01

The on-site production of cellulases is an important strategy for the development of sustainable second-generation ethanol production processes. This study concerns the use of a specific cellulolytic enzyme complex for hydrolysis of pretreated sugar cane bagasse. Glycosyl hydrolases (FPase, xylanase, and β-glucosidase) were produced using a new strain of Trichoderma harzianum, isolated from the Amazon rainforest and cultivated under different conditions. The influence of the carbon source was first investigated using shake-flask cultures. Selected carbon sources were then further studied under different pH conditions using a stirred tank bioreactor. Enzymatic activities up to 121 FPU/g, 8000 IU/g, and 1730 IU/g of delignified steam-exploded bagasse+sucrose were achieved for cellulase, xylanase and β-glucosidase, respectively. This enzymatic complex was used to hydrolyze pretreated sugar cane bagasse. A comparative evaluation, using an enzymatic extract from Trichoderma reesei RUTC30, indicated similar performance of the T. harzianum enzyme complex, being a potential candidate for on-site production of enzymes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

PubMed Central

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; Park, Sunkyu; Kim, Seong H.

2015-01-01

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlate with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component. PMID:26463274

Management of enzyme diversity in high-performance cellulolytic cocktails.

PubMed

Reyes-Sosa, Francisco Manuel; López Morales, Macarena; Platero Gómez, Ana Isabel; Valbuena Crespo, Noelia; Sánchez Zamorano, Laura; Rocha-Martín, Javier; Molina-Heredia, Fernando P; Díez García, Bruno

2017-01-01

Modern biorefineries require enzymatic cocktails of improved efficiency to generate fermentable sugars from lignocellulosic biomass. Cellulolytic fungi, among other microorganisms, have demonstrated the highest potential in terms of enzymatic productivity, complexity and efficiency. On the other hand, under cellulolytic-inducing conditions, they often produce a considerable diversity of carbohydrate-active enzymes which allow them to adapt to changing environmental conditions. However, industrial conditions are fixed and adjusted to the optimum of the whole cocktail, resulting in underperformance of individual enzymes. One of these cellulolytic cocktails from Myceliophthora thermophila has been analyzed here by means of LC-MS/MS. Pure GH6 family members detected have been characterized, confirming previous studies, and added to whole cocktails to compare their contribution in the hydrolysis of industrial substrates. Finally, independent deletions of two GH6 family members, as an example of the enzymatic diversity management, led to the development of a strain producing a more efficient cellulolytic cocktail. These data indicate that the deletion of noncontributive cellulases (here EG VI) can increase the cellulolytic efficiency of the cocktail, validating the management of cellulase diversity as a strategy to obtain improved fungal cellulolytic cocktails.

Releasing N-glycan from peptide N-terminus by N-terminal succinylation assisted enzymatic deglycosylation.

PubMed

Weng, Yejing; Sui, Zhigang; Jiang, Hao; Shan, Yichu; Chen, Lingfan; Zhang, Shen; Zhang, Lihua; Zhang, Yukui

2015-04-22

Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests. Therefore, in this study, we developed a novel strategy to identify PGANs by releasing N-glycans through the N-terminal site-selective succinylation assisted enzymatic deglycosylation. The obtained PGANs information is beneficial to not only achieve the deep coverage analysis of glycoproteomes, but also discover the new biological functions of such modification.

Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels.

PubMed

Zeng, Yining; Zhao, Shuai; Yang, Shihui; Ding, Shi-You

2014-06-01

A biochemical platform holds the most promising route toward lignocellulosic biofuels, in which polysaccharides are hydrolyzed by cellulase enzymes into simple sugars and fermented to ethanol by microbes. However, these polysaccharides are cross-linked in the plant cell walls with the hydrophobic network of lignin that physically impedes enzymatic deconstruction. A thermochemical pretreatment process is often required to remove or delocalize lignin, which may also generate inhibitors that hamper enzymatic hydrolysis and fermentation. Here we review recent advances in understanding lignin structure in the plant cell walls and the negative roles of lignin in the processes of converting biomass to biofuels. Perspectives and future directions to improve the biomass conversion process are also discussed. Copyright © 2013. Published by Elsevier Ltd.

Rooftop Solar Photovoltaic Technical Potential in the United States. A Detailed Assessment

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

Gagnon, Pieter; Margolis, Robert; Melius, Jennifer

2016-01-01

How much energy could be generated if PV modules were installed on all of the suitable roof area in the nation? To answer this question, we first use GIS methods to process a lidar dataset and determine the amount of roof area that is suitable for PV deployment in 128 cities nationwide, containing 23% of U.S. buildings, and provide PV-generation results for a subset of those cities. We then extend the insights from that analysis to the entire continental United States. We develop two statistical models--one for small buildings and one for medium and large buildings--and populate them with geographicmore » variables that correlate with rooftop's suitability for PV. We simulate the productivity of PV installed on the suitable roof area, and present the technical potential of PV on both small buildings and medium/large buildings for every state in the continental US. Within the 128 cities covered by lidar data, 83% of small buildings have a location suitable for a PV installation, but only 26% of the total rooftop area of small buildings is suitable for development. The sheer number of buildings in this class, however, gives small buildings the greatest technical potential. Small building rooftops could accommodate 731 GW of PV capacity and generate 926 TWh/year of PV energy, approximately 65% of rooftop PV's total technical potential. We conclude by summing the PV-generation results for all building sizes and therefore answering our original question, estimating that the total national technical potential of rooftop PV is 1,118 GW of installed capacity and 1,432 TWh of annual energy generation. This equates to 39% of total national electric-sector sales.« less

Rooftop Solar Photovoltaic Technical Potential in the United States

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

Gagnon, Pieter; Margolis, Robert; Melius, Jennifer

2016-01-01

How much energy could we generate if PV modules were installed on all of the suitable roof area in the nation? To answer this question, we first use GIS methods to process a lidar dataset and determine the amount of roof area that is suitable for PV deployment in 128 cities nationwide, containing 23% of U.S. buildings, and provide PV-generation results for a subset of those cities. We then extend the insights from that analysis to the entire continental United States. We develop two statistical models -- one for small buildings and one for medium and large buildings -- andmore » populate them with geographic variables that correlate with rooftop's suitability for PV. We simulate the productivity of PV installed on the suitable roof area, and present the technical potential of PV on both small buildings and medium/large buildings for every state in the continental US. Within the 128 cities covered by lidar data, 83% of small buildings have a location suitable for a PV installation, but only 26% of the total rooftop area of small buildings is suitable for development. The sheer number of buildings in this class, however, gives small buildings the greatest technical potential. Small building rooftops could accommodate 731 GW of PV capacity and generate 926 TWh/year of PV energy, approximately 65% of rooftop PV's total technical potential. We conclude by summing the PV-generation results for all building sizes and therefore answering our original question, estimating that the total national technical potential of rooftop PV is 1,118 GW of installed capacity and 1,432 TWh of annual energy generation. This equates to 39% of total national electric-sector sales.« less

A comprehensive study on urban true orthorectification

USGS Publications Warehouse

Zhou, G.; Chen, W.; Kelmelis, J.A.; Zhang, Dongxiao

2005-01-01

To provide some advanced technical bases (algorithms and procedures) and experience needed for national large-scale digital orthophoto generation and revision of the Standards for National Large-Scale City Digital Orthophoto in the National Digital Orthophoto Program (NDOP), this paper presents a comprehensive study on theories, algorithms, and methods of large-scale urban orthoimage generation. The procedures of orthorectification for digital terrain model (DTM)-based and digital building model (DBM)-based orthoimage generation and their mergence for true orthoimage generation are discussed in detail. A method of compensating for building occlusions using photogrammetric geometry is developed. The data structure needed to model urban buildings for accurately generating urban orthoimages is presented. Shadow detection and removal, the optimization of seamline for automatic mosaic, and the radiometric balance of neighbor images are discussed. Street visibility analysis, including the relationship between flight height, building height, street width, and relative location of the street to the imaging center, is analyzed for complete true orthoimage generation. The experimental results demonstrated that our method can effectively and correctly orthorectify the displacements caused by terrain and buildings in urban large-scale aerial images. ?? 2005 IEEE.

Evaluation of a next generation direct whole blood enzymatic assay for hemoglobin A1c on the ARCHITECT c8000 chemistry system.

PubMed

Teodoro-Morrison, Tracy; Janssen, Marcel J W; Mols, Jasper; Hendrickx, Ben H E; Velmans, Mathieu H; Lotz, Johannes; Lackner, Karl; Lennartz, Lieselotte; Armbruster, David; Maine, Gregory; Yip, Paul M

2015-01-01

The utility of HbA1c for the diagnosis of type 2 diabetes requires an accurate, precise and robust test measurement system. Currently, immunoassay and HPLC are the most popular methods for HbA1c quantification, noting however the limitations associated with some platforms, such as imprecision or interference from common hemoglobin variants. Abbott Diagnostics has introduced a fully automated direct enzymatic method for the quantification of HbA1c from whole blood on the ARCHITECT chemistry system. Here we completed a method evaluation of the ARCHITECT HbA1c enzymatic assay for imprecision, accuracy, method comparison, interference from hemoglobin variants and specimen stability. This was completed at three independent clinical laboratories in North America and Europe. The total imprecision ranged from 0.5% to 2.2% CV with low and high level control materials. Around the diagnostic cut-off of 48 mmol/mol, the total imprecision was 0.6% CV. Mean bias using reference samples from IFCC and CAP ranged from -1.1 to 1.0 mmol/mol. The enzymatic assay also showed excellent agreement with HPLC methods, with slopes of 1.01 and correlation coefficients ranging from 0.984 to 0.996 compared to Menarini Adams HA-8160, Bio-Rad Variant II and Variant II Turbo instruments. Finally, no significant effect was observed for erythrocyte sedimentation or interference from common hemoglobin variants in patient samples containing heterozygous HbS, HbC, HbD, HbE, and up to 10% HbF. The ARCHITECT enzymatic assay for HbA1c is a robust and fully automated method that meets the performance requirements to support the diagnosis of type 2 diabetes.

Protective effects of azelaic acid against high-fat diet-induced oxidative stress in liver, kidney and heart of C57BL/6J mice.

PubMed

Muthulakshmi, Shanmugam; Saravanan, Ramalingam

2013-05-01

Excess fat intake induces hyperinsulinaemia, increases nutrient uptake and lipid accumulation, amplifies ROS generation, establishes oxidative stress and morphological changes leading to tissue injury in the liver, kidney and heart of high-fat diet (HFD)-fed mice. The effect of azelaic acid (AzA), a C9 α,ω-dicarboxylic acid, against HFD-induced oxidative stress was investigated by assaying the activities and levels of antioxidants and oxidative stress markers in the liver, kidney and heart of C57BL/6J mice. Mice were segregated into two groups, one fed standard diet (NC) and the other fed high-fat diet (HFD) for 15 weeks. HFD-fed mice were subjected to intragastric administration of AzA (80 mg/kg BW)/RSG (10 mg/kg BW) during 11-15 weeks. Glucose, insulin, triglycerides, hepatic and nephritic markers were analysed in the plasma and the activity of enzymatic, non-enzymatic antioxidants and lipid peroxidation markers were examined in the plasma/erythrocytes, liver, kidney and heart of normal and experimental mice. We inferred significant decrease in enzymatic and non-enzymatic antioxidants along with significant increase in glucose, insulin, hepatic and nephritic markers, triglycerides and lipid peroxidation markers in HFD-fed mice. Administration of AzA could positively restore the levels of plasma glucose, insulin, triglycerides, hepatic and nephritic markers to near normal. AzA increased the levels of enzymatic and nonenzymatic antioxidants with significant reduction in the levels of lipid peroxidation markers. Histopathological examination of liver, kidney and heart substantiated these results. Hence, we put forward that AzA could counteract the potential injurious effects of HFD-induced oxidative stress in C57BL/6J mice.

Decomposition rate and enzymatic activity of composted municipal waste and poultry manure in the soil in a biofuel crops field.

PubMed

Cordovil, Cláudia Marques-Dos-Santos; de Varennes, Amarilis; Pinto, Renata Machado Dos Santos; Alves, Tiago Filipe; Mendes, Pedro; Sampaio, Sílvio César

2017-05-01

Biofuel crops are gaining importance because of the need to replace non-renewable sources. Also, due to the increasing amounts of wastes generated, there is the need to recycle them to the soil, both to fertilize crops and to improve soil physical properties through organic matter increase and microbiological changes in the rhizosphere. We therefore studied the influence of six biofuel crops (elephant grass, giant cane, sugarcane, blue gum, black cottonwood, willow) on the decomposition rate and enzymatic activity of composted municipal solid waste and poultry manure. Organic amendments were incubated in the field (litterbag method), buried near each plant or bare soil. Biomass decrease and dehydrogenase, urease and acid phosphatase level in amendments was monitored over a 180-day period. Soil under the litterbags was analysed for the same enzymatic activity and organic matter fractions (last sampling). After 365 days, a fractionation of organic matter was carried out in both amendments and soil under the litterbags. For compost, willow and sugarcane generally led to the greatest enzymatic activity, at the end of the experiment. For manure, dehydrogenase activity decreased sharply with time, the smallest value near sugarcane, while phosphatase and urease generally presented the highest values, at the beginning or after 90 days' incubation. Clustering showed that plant species could be grouped based on biomass and enzymes measured over time. Plant species influenced the decomposition rate and enzymatic activities of the organic amendments. Overall, mineralization of both amendments was associated with a greater urease activity in soils. Dehydrogenase activity in manure was closely associated with urease activity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Oxidation Inhibits Iron-Induced Blood Coagulation

PubMed Central

Pretorius, Etheresia; Bester, Janette; Vermeulen, Natasha; Lipinski, Boguslaw

2013-01-01

Blood coagulation under physiological conditions is activated by thrombin, which converts soluble plasma fibrinogen (FBG) into an insoluble clot. The structure of the enzymatically-generated clot is very characteristic being composed of thick fibrin fibers susceptible to the fibrinolytic degradation. However, in chronic degenerative diseases, such as atherosclerosis, diabetes mellitus, cancer, and neurological disorders, fibrin clots are very different forming dense matted deposits (DMD) that are not effectively removed and thus create a condition known as thrombosis. We have recently shown that trivalent iron (ferric ions) generates hydroxyl radicals, which subsequently convert FBG into abnormal fibrin clots in the form of DMDs. A characteristic feature of DMDs is their remarkable and permanent resistance to the enzymatic degradation. Therefore, in order to prevent thrombotic incidences in the degenerative diseases it is essential to inhibit the iron-induced generation of hydroxyl radicals. This can be achieved by the pretreatment with a direct free radical scavenger (e.g. salicylate), and as shown in this paper by the treatment with oxidizing agents such as hydrogen peroxide, methylene blue, and sodium selenite. Although the actual mechanism of this phenomenon is not yet known, it is possible that hydroxyl radicals are neutralized by their conversion to the molecular oxygen and water, thus inhibiting the formation of dense matted fibrin deposits in human blood. PMID:23170793

Destructive effect of non-enzymatic glycation on catalase and remediation via curcumin.

PubMed

Mofidi Najjar, Fayezeh; Taghavi, Fereshteh; Ghadari, Rahim; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

2017-09-15

Non-enzymatic glycation of proteins is a post-translational modification that is produced by a covalent binding between reducing sugars and amino groups of lysine and arginine residues. In this paper the effect of pathological conditions, derived from hyperglycemia on bovine liver catalase (BLC) as a model protein was considered by measuring enzyme activity, reactive oxygen species (ROS) generation, and changes in catalase conformational properties. We observed that in the presence of glucose, the catalase activity gradually decreased. ROS generation was also involved in the glycation process. Thus, decreased BLC activity was partly considered as a result of ROS generation through glycation. However, in the presence of curcumin the amount of ROS was reduced resulting in increased activity of the glycated catalase. The effect of high glucose level and the potential inhibitory effect of curcumin on aggregation and structural changes of catalase were also investigated. Molecular dynamic simulations also showed that interaction of catalase with curcumin resulted in changes in accessible surface area (ASA) and pKa, two effective parameters of glycation, in potential glycation lysine residues. Thus, the decrease in ASA and increase in pKa of important lysine residues were considered as predominant factors in decreased glycation of BLC by curcumin. Copyright © 2017 Elsevier Inc. All rights reserved.

Production of biodiesel by transesterification of corn and soybean oils with ethanol or butanol using resin-bound truncated Candida antarctica lipase B

USDA-ARS?s Scientific Manuscript database

Enzymatic catalysts, such as lipases, have advantages over chemical catalysts for transesterification of triglycerides to produce biodiesel. A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western b...

A liposome-based energy conversion system for accelerating the multi-enzyme reactions.

PubMed

Matsumoto, Ryuhei; Kakuta, Masaya; Sugiyama, Taiki; Goto, Yoshio; Sakai, Hideki; Tokita, Yuichi; Hatazawa, Tsuyonobu; Tsujimura, Seiya; Shirai, Osamu; Kano, Kenji

2010-11-14

We report the first example of a liposome-based energy conversion system that is useful for entrapping enzymes and NAD coenzyme to accelerate multi-step enzymatic reactions. The liposome generates a much higher catalytic current compared with the non-liposome system, which is in good consistency with numerical simulations.

EXPRESSON OF AS3MT ALTERS TRANSCRIPTIONAL PROFILES IN HUMAN UROTHELIAL CELLS EXPOSED TO ARSENITE

EPA Science Inventory

Inorganic arsenic (iAs) is an environmental toxin and human carcinogen. The enzymatic methylation of iAs that is catalyzed by As (3+ oxidation state)-methyltransferase (AS3MT) generates reactive methyl-As intermediates that may contribute to the toxic or carcinogenic effects of i...

Proline dehydrogenase promotes senescence through the generation of reactive oxygen species.

PubMed

Nagano, Taiki; Nakashima, Akio; Onishi, Kengo; Kawai, Kosuke; Awai, Yuto; Kinugasa, Mizuki; Iwasaki, Tetsushi; Kikkawa, Ushio; Kamada, Shinji

2017-04-15

Cellular senescence is a complex stress response characterized by permanent loss of proliferative capacity and is implicated in age-related disorders. Although the transcriptional activity of p53 (encoded by TP53 ) is known to be vital for senescence induction, the downstream effector genes critical for senescence remain unsolved. Recently, we have identified the proline dehydrogenase gene ( PRODH ) to be upregulated specifically in senescent cells in a p53-dependent manner, and the functional relevance of this to senescence is yet to be defined. Here, we conducted functional analyses to explore the relationship between PRODH and the senescence program. We found that genetic and pharmacological inhibition of PRODH suppressed senescent phenotypes induced by DNA damage. Furthermore, ectopic expression of wild-type PRODH, but not enzymatically inactive forms, induced senescence associated with the increase in reactive oxygen species (ROS) and the accumulation of DNA damage. Treatment with N-acetyl-L-cysteine, a ROS scavenger, prevented senescence induced by PRODH overexpression. These results indicate that PRODH plays a causative role in DNA damage-induced senescence through the enzymatic generation of ROS. © 2017. Published by The Company of Biologists Ltd.

A 3D paper-based enzymatic fuel cell for self-powered, low-cost glucose monitoring.

PubMed

Fischer, Christopher; Fraiwan, Arwa; Choi, Seokheun

2016-05-15

In this work, we demonstrate a novel low-cost, self-powered paper-based biosensor for glucose monitoring. The device operating mechanism is based on a glucose/oxygen enzymatic fuel cell using an electrochemical energy conversion as a transducing element for glucose monitoring. The self-powered glucose biosensor features (i) a 3D origami paper-based structure for easy system integration onto paper, (ii) an air-cathode on paper for low-cost production and easy operation, and (iii) a screen printed chitosan/glucose oxidase anode for stable current generation as an analytical signal for glucose monitoring. The sensor showed a linear range of output current at 1-5mM glucose (R(2)=0.996) with a sensitivity of 0.02 µA mM(-1). The advantages offered by such a device, including a low cost, lack of external power sources/sophisticated external transducers, and the capacity to rapidly generate reliable results, are well suited for the clinical and social settings of the developing world. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzymatic Depilation of Animal Hide: Identification of Elastase (LasB) from Pseudomonas aeruginosa MCM B-327 as a Depilating Protease

PubMed Central

Pandeeti, Emmanuel Vijay Paul; Pitchika, Gopi Krishna; Jotshi, Jyotsna; Nilegaonkar, Smita S.; Kanekar, Pradnya P.; Siddavattam, Dayananda

2011-01-01

Conventional leather processing involving depilation of animal hide by lime and sulphide treatment generates considerable amounts of chemical waste causing severe environmental pollution. Enzymatic depilation is an environmentally friendly process and has been considered to be a viable alternative to the chemical depilation process. We isolated an extracellular protease from Pseudomonas aeruginosa strain MCM B-327 with high depilation activity using buffalo hide as a substrate. This 33 kDa protease generated a peptide mass fingerprint and de novo sequence that matched perfectly with LasB (elastase), of Pseudomonas aeruginosa. In support of this data a lasB mutant of MCM B-327 strain lacked depilatory activity and failed to produce LasB. LasB heterologously over-produced and purified from Escherichia coli also exhibited high depilating activity. Moreover, reintroduction of the lasB gene to the P. aeruginosa lasB mutant via a knock-in strategy also successfully restored depilation activity thus confirming the role of LasB as the depilating enzyme. PMID:21347249

Trypanosoma brucei Metacaspase 4 Is a Pseudopeptidase and a Virulence Factor*

PubMed Central

Proto, William R.; Castanys-Munoz, Esther; Black, Alana; Tetley, Laurence; Moss, Catherine X.; Juliano, Luiz; Coombs, Graham H.; Mottram, Jeremy C.

2011-01-01

Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1–MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor. PMID:21949125

Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface.

PubMed

Kamiya, Noriho; Doi, Satoshi; Tominaga, Jo; Ichinose, Hirofumi; Goto, Masahiro

2005-01-01

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.

Multigene panel next generation sequencing in a patient with cherry red macular spot: Identification of two novel mutations in NEU1 gene causing sialidosis type I associated with mild to unspecific biochemical and enzymatic findings.

PubMed

Mütze, Ulrike; Bürger, Friederike; Hoffmann, Jessica; Tegetmeyer, Helmut; Heichel, Jens; Nickel, Petra; Lemke, Johannes R; Syrbe, Steffen; Beblo, Skadi

2017-03-01

Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. Cherry red macular spots were first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations as well as cerebral MRI were unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to < 1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699C > A, p.S233R in exon 4 and c.803A > G; p.Y268C in Exon 5 in NEU1 transcript NM_000434.3), leading to amino acid changes predicted to impair protein function. Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

Bioproduction of Chitooligosaccharides: Present and Perspectives

PubMed Central

Jung, Woo-Jin; Park, Ro-Dong

2014-01-01

Chitin and chitosan oligosaccharides (COS) have been traditionally obtained by chemical digestion with strong acids. In light of the difficulties associated with these traditional production processes, environmentally compatible and reproducible production alternatives are desirable. Unlike chemical digestion, biodegradation of chitin and chitosan by enzymes or microorganisms does not require the use of toxic chemicals or excessive amounts of wastewater. Enzyme preparations with chitinase, chitosanase, and lysozymeare primarily used to hydrolyze chitin and chitosan. Commercial preparations of cellulase, protease, lipase, and pepsin provide another opportunity for oligosaccharide production. In addition to their hydrolytic activities, the transglycosylation activity of chitinolytic enzymes might be exploited for the synthesis of desired chitin oligomers and their derivatives. Chitin deacetylase is also potentially useful for the preparation of oligosaccharides. Recently, direct production of oligosaccharides from chitin and crab shells by a combination of mechanochemical grinding and enzymatic hydrolysis has been reported. Together with these, other emerging technologies such as direct degradation of chitin from crustacean shells and microbial cell walls, enzymatic synthesis of COS from small building blocks, and protein engineering technology for chitin-related enzymes have been discussed as the most significant challenge for industrial application. PMID:25353253

Chemoenzymatic synthesis of statine side chain building blocks and application in the total synthesis of the cholesterol-lowering compound solistatin.

PubMed

Rieder, Oliver; Wolberg, Michael; Foegen, Silke E; Müller, Michael

2017-09-20

The synthesis and enzymatic reduction of several 6-substituted dioxohexanoates are presented. Two-step syntheses of tert-butyl 6-bromo-3,5-dioxohexanoate and the corresponding 6-hydroxy compound have been achieved in 89% and 59% yield, respectively. Regio- and enantioselective reduction of these diketones and of the 6-chloro derivative with alcohol dehydrogenase from Lactobacillus brevis (LBADH) gave the (5S)-5-hydroxy-3-oxo products with enantiomeric excesses of 91%, 98.4%, and >99.5%, respectively. Chain elongation of the reduction products by one carbon via cyanide addition, and by more than one carbon by Julia-Kocienski olefination, gave access to well-established statine side-chain building blocks. Application in the synthesis of the cholesterol-lowering natural compound solistatin is given. Copyright © 2017 Elsevier B.V. All rights reserved.

Reactive Oxygen Species Generated by NADPH Oxidases Promote Radicle Protrusion and Root Elongation during Rice Seed Germination

PubMed Central

Li, Wen-Yan; Chen, Bing-Xian; Chen, Zhong-Jian; Gao, Yin-Tao; Chen, Zhuang; Liu, Jun

2017-01-01

Seed germination is a complicated biological process that requires regulation through various enzymatic and non-enzymatic mechanisms. Although it has been recognized that reactive oxygen species (ROS) regulate radicle emergence and root elongation in a non-enzymatic manner during dicot seed germination, the role of ROS in monocot seed germination remains unknown. NADPH oxidases (NOXs) are the major ROS producers in plants; however, whether and how NOXs regulate rice seed germination through ROS generation remains unclear. Here, we report that diphenyleneiodinium (DPI), a specific NOX inhibitor, potently inhibited embryo and seedling growth—especially that of the radicle and of root elongation—in a dose-dependent manner. Notably, the DPI-mediated inhibition of radicle and root growth could be eliminated by transferring seedlings from DPI to water. Furthermore, ROS production/accumulation during rice seed germination was quantified via histochemistry. Superoxide radicals (O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) accumulated steadily in the coleorhiza, radicle and seedling root of germinating rice seeds. Expression profiles of the nine typical NOX genes were also investigated. According to quantitative PCR, OsNOX5, 7 and 9 were expressed relatively higher. When seeds were incubated in water, OsNOX5 expression progressively increased in the embryo from 12 to 48 h, whereas OsNOX7 and 9 expressions increased from 12 to 24 h and decreased thereafter. As expected, DPI inhibits the expression at predetermined time points for each of these genes. Taken together, these results suggest that ROS produced by NOXs are involved in radicle and root elongation during rice seed germination, and OsNOX5, 7 and 9 could play crucial roles in rice seed germination. These findings will facilitate further studies of the roles of ROS generated by NOXs during seed germination and seedling establishment and also provide valuable information for the regulation of NOX family gene expression in germinating seeds of monocot cereals. PMID:28098759

A Parameterized Model of Amylopectin Synthesis Provides Key Insights into the Synthesis of Granular Starch

PubMed Central

Wu, Alex Chi; Morell, Matthew K.; Gilbert, Robert G.

2013-01-01

A core set of genes involved in starch synthesis has been defined by genetic studies, but the complexity of starch biosynthesis has frustrated attempts to elucidate the precise functional roles of the enzymes encoded. The chain-length distribution (CLD) of amylopectin in cereal endosperm is modeled here on the basis that the CLD is produced by concerted actions of three enzyme types: starch synthases, branching and debranching enzymes, including their respective isoforms. The model, together with fitting to experiment, provides four key insights. (1) To generate crystalline starch, defined restrictions on particular ratios of enzymatic activities apply. (2) An independent confirmation of the conclusion, previously reached solely from genetic studies, of the absolute requirement for debranching enzyme in crystalline amylopectin synthesis. (3) The model provides a mechanistic basis for understanding how successive arrays of crystalline lamellae are formed, based on the identification of two independent types of long amylopectin chains, one type remaining in the amorphous lamella, while the other propagates into, and is integral to the formation of, an adjacent crystalline lamella. (4) The model provides a means by which a small number of key parameters defining the core enzymatic activities can be derived from the amylopectin CLD, providing the basis for focusing studies on the enzymatic requirements for generating starches of a particular structure. The modeling approach provides both a new tool to accelerate efforts to understand granular starch biosynthesis and a basis for focusing efforts to manipulate starch structure and functionality using a series of testable predictions based on a robust mechanistic framework. PMID:23762422

Building Damage Assessment after Earthquake Using Post-Event LiDAR Data

NASA Astrophysics Data System (ADS)

Rastiveis, H.; Eslamizade, F.; Hosseini-Zirdoo, E.

2015-12-01

After an earthquake, damage assessment plays an important role in leading rescue team to help people and decrease the number of mortality. Damage map is a map that demonstrates collapsed buildings with their degree of damage. With this map, finding destructive buildings can be quickly possible. In this paper, we propose an algorithm for automatic damage map generation after an earthquake using post-event LiDAR Data and pre-event vector map. The framework of the proposed approach has four main steps. To find the location of all buildings on LiDAR data, in the first step, LiDAR data and vector map are registered by using a few number of ground control points. Then, building layer, selected from vector map, are mapped on the LiDAR data and all pixels which belong to the buildings are extracted. After that, through a powerful classifier all the extracted pixels are classified into three classes of "debris", "intact building" and "unclassified". Since textural information make better difference between "debris" and "intact building" classes, different textural features are applied during the classification. After that, damage degree for each candidate building is estimated based on the relation between the numbers of pixels labelled as "debris" class to the whole building area. Calculating the damage degree for each candidate building, finally, building damage map is generated. To evaluate the ability proposed method in generating damage map, a data set from Port-au-Prince, Haiti's capital after the 2010 Haiti earthquake was used. In this case, after calculating of all buildings in the test area using the proposed method, the results were compared to the damage degree which estimated through visual interpretation of post-event satellite image. Obtained results were proved the reliability of the proposed method in damage map generation using LiDAR data.

Literature search strategies for conducting knowledge-building and theory-generating qualitative systematic reviews.

PubMed

Finfgeld-Connett, Deborah; Johnson, E Diane

2013-01-01

To report literature search strategies for the purpose of conducting knowledge-building and theory-generating qualitative systematic reviews. Qualitative systematic reviews lie on a continuum from knowledge-building and theory-generating to aggregating and summarizing. Different types of literature searches are needed to optimally support these dissimilar reviews. Articles published between 1989-Autumn 2011. These documents were identified using a hermeneutic approach and multiple literature search strategies. Redundancy is not the sole measure of validity when conducting knowledge-building and theory-generating systematic reviews. When conducting these types of reviews, literature searches should be consistent with the goal of fully explicating concepts and the interrelationships among them. To accomplish this objective, a 'berry picking' approach is recommended along with strategies for overcoming barriers to finding qualitative research reports. To enhance integrity of knowledge-building and theory-generating systematic reviews, reviewers are urged to make literature search processes as transparent as possible, despite their complexity. This includes fully explaining and rationalizing what databases were used and how they were searched. It also means describing how literature tracking was conducted and grey literature was searched. In the end, the decision to cease searching also needs to be fully explained and rationalized. Predetermined linear search strategies are unlikely to generate search results that are adequate for purposes of conducting knowledge-building and theory-generating qualitative systematic reviews. Instead, it is recommended that iterative search strategies take shape as reviews evolve. © 2012 Blackwell Publishing Ltd.

Preparation of immobilized L-prolinamide via enzymatic polymerization of phenolic L-prolinamide and evaluation of its catalytic performance for direct asymmetric aldol reaction.

PubMed

Qu, Chengke; Zhao, Wenshan; Zhang, Lei; Cui, Yuanchen

2014-04-01

Phenolic L-prolinamide was allowed to participate in enzymatic polymerization with horseradish peroxidase as the catalyst, generating immobilized L-prolinamide. The catalytic performance of the resultant polymer-supported L-prolinamide for direct asymmetric aldol reaction between aromatic aldehyde and cyclohexanone was studied. The results show that as prepared L-prolinamide can catalyze the aldol reaction at room temperature in the presence of H2O. Relevant aldol addition products are obtained with good yields (up to 91%), high diastereoselectivities (up to 6:94 dr), and medium enantioselectivities (up to 87% ee). Moreover, the title polymer-supported catalyst can be recovered and reused for at least five cycles while the activity remains almost unchanged. Copyright © 2014 Wiley Periodicals, Inc.

Structure of clusters and building blocks in amylopectin from African rice accessions.

PubMed

Gayin, Joseph; Abdel-Aal, El-Sayed M; Marcone, Massimo; Manful, John; Bertoft, Eric

2016-09-05

Enzymatic hydrolysis in combination with gel-permeation and anion-exchange chromatography techniques were employed to characterise the composition of clusters and building blocks of amylopectin from two African rice (Oryza glaberrima) accessions-IRGC 103759 and TOG 12440. The samples were compared with one Asian rice (Oryza sativa) sample (cv WITA 4) and one O. sativa×O. glaberrima cross (NERICA 4). The average DP of clusters from the African rice accessions (ARAs) was marginally larger (DP=83) than in WITA 4 (DP=81). However, regarding average number of chains, clusters from the ARAs represented both the smallest and largest clusters. Overall, the result suggested that the structure of clusters in TOG 12440 was dense with short chains and high degree of branching, whereas the situation was the opposite in NERICA 4. IRGC 103759 and WITA 4 possessed clusters with intermediate characteristics. The commonest type of building blocks in all samples was group 2 (single branched dextrins) representing 40.3-49.4% of the blocks, while groups 3-6 were found in successively lower numbers. The average number of building blocks in the clusters was significantly larger in NERICA 4 (5.8) and WITA 4 (5.7) than in IRGC 103759 and TOG 12440 (5.1 and 5.3, respectively). Copyright © 2016 Elsevier Ltd. All rights reserved.

[Evaluation of various biochemical parameters in the urine and blood of painters in the carpentry business].

PubMed

Sołtys, M; Jakubowska, W; Lenart, E; Nagadowska, H; Lisiecka, M; Ziemer, I; Koćmierkska-Grodzka, D

1986-01-01

The work has been aimed at investigating the effects of organic solvent-toluene-upon the painters of the Building Joinery Factory. Studies carried out during 1978-1980 showed an increased level of toluene metabolite (hippuric acid) in painters' urine, as compared to the environmental control group. The highest level of hippuric acid in painters' urine was found in the first period (first six months) of their job. This was not related to an increase in toluene vapours at the workplace. In painters' blood some irregular and rather small modifications of enzymatic activity were exhibited.

Automatic building information model query generation

DOE PAGES

Jiang, Yufei; Yu, Nan; Ming, Jiang; ...

2015-12-01

Energy efficient building design and construction calls for extensive collaboration between different subfields of the Architecture, Engineering and Construction (AEC) community. Performing building design and construction engineering raises challenges on data integration and software interoperability. Using Building Information Modeling (BIM) data hub to host and integrate building models is a promising solution to address those challenges, which can ease building design information management. However, the partial model query mechanism of current BIM data hub collaboration model has several limitations, which prevents designers and engineers to take advantage of BIM. To address this problem, we propose a general and effective approachmore » to generate query code based on a Model View Definition (MVD). This approach is demonstrated through a software prototype called QueryGenerator. In conclusion, by demonstrating a case study using multi-zone air flow analysis, we show how our approach and tool can help domain experts to use BIM to drive building design with less labour and lower overhead cost.« less

Automatic building information model query generation

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

Jiang, Yufei; Yu, Nan; Ming, Jiang

Energy efficient building design and construction calls for extensive collaboration between different subfields of the Architecture, Engineering and Construction (AEC) community. Performing building design and construction engineering raises challenges on data integration and software interoperability. Using Building Information Modeling (BIM) data hub to host and integrate building models is a promising solution to address those challenges, which can ease building design information management. However, the partial model query mechanism of current BIM data hub collaboration model has several limitations, which prevents designers and engineers to take advantage of BIM. To address this problem, we propose a general and effective approachmore » to generate query code based on a Model View Definition (MVD). This approach is demonstrated through a software prototype called QueryGenerator. In conclusion, by demonstrating a case study using multi-zone air flow analysis, we show how our approach and tool can help domain experts to use BIM to drive building design with less labour and lower overhead cost.« less

Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions

NASA Astrophysics Data System (ADS)

Stasyuk, Nataliya; Gayda, Galina; Zakalskiy, Andriy; Zakalska, Oksana; Errachid, Abdelhamid; Gonchar, Mykhailo

2018-03-01

A novel enzymatic method of manganese (II) and cobalt (II) ions assay, based on using apo-enzyme of Mn2 +-dependent recombinant arginase I (arginase) and 2,3-butanedione monoxime (DMO) as a chemical reagent is proposed. The principle of the method is the evaluation of the activity of L-arginine-hydrolyzing of arginase holoenzyme after the specific binding of Mn2 + or Co2 + with apo-arginase. Urea, which is the product of enzymatic hydrolysis of L-arginine (Arg), reacts with DMO and the resulted compound is detected by both fluorometry and visual spectrophotometry. Thus, the content of metal ions in the tested samples can be determined by measuring the level of urea generated after enzymatic hydrolysis of Arg by reconstructed arginase holoenzyme in the presence of tested metal ions. The linearity range of the fluorometric apo-arginase-DMO method in the case of Mn2 + assay is from 4 pM to 1.10 nM with a limit of detection of 1 pM Mn2 +, whereas the linearity range of the present method in the case of Co2 + assay is from 8 pM to 45 nM with a limit of detection of 2.5 pM Co2 +. The proposed method being highly sensitive, selective, valid and low-cost, may be useful to monitor Mn2 + and Co2 + content in clinical laboratories, food industry and environmental control service.

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

PubMed Central

Lazar, Iulia M.; Deng, Jingren; Smith, Nicole

2016-01-01

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW < 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 ºC. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively. PMID:27078683

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor.

PubMed

Lazar, Iulia M; Deng, Jingren; Smith, Nicole

2016-04-06

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW < 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 ºC. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

Multi-wavelength dye concentration determination for enzymatic assays: evaluation of chromogenic para-nitrophenol over a wide pH range.

PubMed

Max, Jean-Joseph; Meddeb-Mouelhi, Fatma; Beauregard, Marc; Chapados, Camille

2012-12-01

Enzymatic assays need robust, rapid colorimetric methods that can follow ongoing reactions. For this, we developed a highly accurate, multi-wavelength detection method that could be used for several systems. Here, it was applied to the detection of para-nitrophenol (pNP) in basic and acidic solutions. First, we confirmed by factor analysis that pNP has two forms, with unique spectral characteristics in the 240 to 600 nm range: Phenol in acidic conditions absorbs in the lower range, whereas phenolate in basic conditions absorbs in the higher range. Thereafter, the method was used for the determination of species concentration. For this, the intensity measurements were made at only two wavelengths with a microtiter plate reader. This yielded total dye concentration, species relative abundance, and solution pH value. The method was applied to an enzymatic assay. For this, a chromogenic substrate that generates pNP after hydrolysis catalyzed by a lipase from the fungus Yarrowia lipolytica was used. Over the pH range of 3-11, accurate amounts of acidic and basic pNP were determined at 340 and 405 nm, respectively. This method surpasses the commonly used single-wavelength assay at 405 nm, which does not detect pNP acidic species, leading to activity underestimations. Moreover, alleviation of this pH-related problem by neutralization is not necessary. On the whole, the method developed is readily applicable to rapid high-throughput of enzymatic activity measurements over a wide pH range.

Analysis, pretreatment and enzymatic saccharification of different fractions of Scots pine

PubMed Central

2014-01-01

Background Forestry residues consisting of softwood are a major lignocellulosic resource for production of liquid biofuels. Scots pine, a commercially important forest tree, was fractionated into seven fractions of chips: juvenile heartwood, mature heartwood, juvenile sapwood, mature sapwood, bark, top parts, and knotwood. The different fractions were characterized analytically with regard to chemical composition and susceptibility to dilute-acid pretreatment and enzymatic saccharification. Results All fractions were characterized by a high glucan content (38-43%) and a high content of other carbohydrates (11-14% mannan, 2-4% galactan) that generate easily convertible hexose sugars, and by a low content of inorganic material (0.2-0.9% ash). The lignin content was relatively uniform (27-32%) and the syringyl-guaiacyl ratio of the different fractions were within the range 0.021-0.025. The knotwood had a high content of extractives (9%) compared to the other fractions. The effects of pretreatment and enzymatic saccharification were relatively similar, but without pretreatment the bark fraction was considerably more susceptible to enzymatic saccharification. Conclusions Since sawn timber is a main product from softwood species such as Scots pine, it is an important issue whether different parts of the tree are equally suitable for bioconversion processes. The investigation shows that bioconversion of Scots pine is facilitated by that most of the different fractions exhibit relatively similar properties with regard to chemical composition and susceptibility to techniques used for bioconversion of woody biomass. PMID:24641769

The oxidative stress response of the opportunistic fungal pathogen Candida glabrata.

PubMed

Briones-Martin-Del-Campo, Marcela; Orta-Zavalza, Emmanuel; Juarez-Cepeda, Jacqueline; Gutierrez-Escobedo, Guadalupe; Cañas-Villamar, Israel; Castaño, Irene; De Las Peñas, Alejandro

2014-01-01

Organisms have evolved different strategies to respond to oxidative stress generated as a by-product of aerobic respiration and thus maintain the redox homeostasis within the cell. In particular, fungal pathogens are exposed to reactive oxygen species (ROS) when they interact with the phagocytic cells of the host which are the first line of defense against fungal infections. These pathogens have co-opted the enzymatic (catalases, superoxide dismutases (SODs), and peroxidases) and non-enzymatic (glutathione) mechanisms used to maintain the redox homeostasis within the cell, to resist oxidative stress and ensure survival within the host. Several virulence factors have been related to the response to oxidative stress in pathogenic fungi. The opportunistic fungal pathogen Candida glabrata (C. glabrata) is the second most common cause of candidiasis after Candida albicans (C. albicans). C. glabrata has a well defined oxidative stress response (OSR), which include both enzymatic and non-enzymatic mechanisms. C. glabrata OSR is controlled by the well-conserved transcription factors Yap1, Skn7, Msn2 and Msn4. In this review, we describe the OSR of C. glabrata, what is known about its core elements, its regulation and how C. glabrata interacts with the host. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.

Enzymatically Active Microgels from Self-Assembling Protein Nanofibrils for Microflow Chemistry

PubMed Central

2015-01-01

Amyloid fibrils represent a generic class of protein structure associated with both pathological states and with naturally occurring functional materials. This class of protein nanostructure has recently also emerged as an excellent foundation for sophisticated functional biocompatible materials including scaffolds and carriers for biologically active molecules. Protein-based materials offer the potential advantage that additional functions can be directly incorporated via gene fusion producing a single chimeric polypeptide that will both self-assemble and display the desired activity. To succeed, a chimeric protein system must self-assemble without the need for harsh triggering conditions which would damage the appended functional protein molecule. However, the micrometer to nanoscale patterning and morphological control of protein-based nanomaterials has remained challenging. This study demonstrates a general approach for overcoming these limitations through the microfluidic generation of enzymatically active microgels that are stabilized by amyloid nanofibrils. The use of scaffolds formed from biomaterials that self-assemble under mild conditions enables the formation of catalytic microgels while maintaining the integrity of the encapsulated enzyme. The enzymatically active microgel particles show robust material properties and their porous architecture allows diffusion in and out of reactants and products. In combination with microfluidic droplet trapping approaches, enzymatically active microgels illustrate the potential of self-assembling materials for enzyme immobilization and recycling, and for biological flow-chemistry. These design principles can be adopted to create countless other bioactive amyloid-based materials with diverse functions. PMID:26030507

Microbial Enzymatic Degradation of Biodegradable Plastics.

PubMed

Roohi; Bano, Kulsoom; Kuddus, Mohammed; Zaheer, Mohammed R; Zia, Qamar; Khan, Mohammed F; Ashraf, Ghulam Md; Gupta, Anamika; Aliev, Gjumrakch

2017-01-01

The renewable feedstock derived biodegradable plastics are important in various industries such as packaging, agricultural, paper coating, garbage bags and biomedical implants. The increasing water and waste pollution due to the available decomposition methods of plastic degradation have led to the emergence of biodegradable plastics and biological degradation with microbial (bacteria and fungi) extracellular enzymes. The microbes utilize biodegradable polymers as the substrate under starvation and in unavailability of microbial nutrients. Microbial enzymatic degradation is suitable from bioremediation point of view as no waste accumulation occurs. It is important to understand the microbial interaction and mechanism involved in the enzymatic degradation of biodegradable plastics under the influence of several environmental factors such as applied pH, thermo-stability, substrate molecular weight and/or complexity. To study the surface erosion of polymer film is another approach for hydrolytic degradation characteristion. The degradation of biopolymer is associated with the production of low molecular weight monomer and generation of carbon dioxide, methane and water molecule. This review reported the degradation study of various existing biodegradable plastics along with the potent degrading microbes (bacteria and fungi). Patents available on plastic biodegradation with biotechnological significance is also summarized in this paper. This paper assesses that new disposal technique should be adopted for the degradation of polymers and further research is required for the economical production of biodegradable plastics along with their enzymatic degradation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

30. Launch Area, Generator Building, interior view showing diesel fuel ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

30. Launch Area, Generator Building, interior view showing diesel fuel tank, fuel pump (foreground) and fuel lines leading to power-generating units (removed) VIEW NORTHWEST - NIKE Missile Battery PR-79, Launch Area, East Windsor Road south of State Route 101, Foster, Providence County, RI

New efforts in eastern cottonwood biomass production through breeding and clonal refinement

Treesearch

Jason W. Cromer; Randall J. Rousseau; B. Landis Herrin

2014-01-01

First generation biofuels (also known as traditional biofuels) primarily use corn to produce ethanol. Newer techniques and knowledge are now allowing ethanol production from renewable resources such as trees that have more complex molecular structures that inhibit access to sugars. Ethanol production is through an enzymatic process which uses cellulose, or pyrolosis...

Surface modification of lignocellulosic fibers using high-frequency ultrasound

Treesearch

Jayant B. Gadhe; Ram B. Gupta; Thomas Elder

2005-01-01

Enzymatic and chemical oxidation of fiber surfaces has been reported in the literature as a method for producing medium density fiberboards without using synthetic adhesives. This work focuses on modifying the surface properties of wood fibers by the generation of free radicals using high-frequency ultrasound. A sonochemical reactor operating at 610 kHz is used to...

Evaluation of the Fermentation Potential of Pulp Mill Residue to Produce D(-)-Lactic Acid by Separate Hydrolysis and Fermentation Using Lactobacillus coryniformis subsp. torquens.

PubMed

de Oliveira Moraes, Anelize; Ramirez, Ninoska Isabel Bojorge; Pereira, Nei

2016-12-01

Lactic acid is widely used in chemical, pharmaceutical, cosmetic, and food industries, besides it is the building block to produce polylactic acid, which is a sustainable alternative biopolymer to synthetic plastic due to its biodegradability. Aiming at producing an optically pure isomer, the present work evaluated the potential of pulp mill residue as feedstock to produce D(-)-lactic acid by a strain of the bacterium Lactobacillus coryniformis subsp. torquens using separate hydrolysis and fermentation process. Enzymatic hydrolysis, optimized through response surface methodology for 1 g:4 mL solid/liquid ratio and 24.8 FPU/g cellulose enzyme loading, resulted in 140 g L -1 total reducing sugar and 110 g L -1 glucose after 48 h, leading to 61 % of efficiency. In instrumented bioreactor, 57 g L -1 of D(-)-lactic acid was achieved in 20 h of fermentation, while only 0.5 g L -1 of L(+)-lactic acid was generated. Furthermore, product yield of 0.97 g/g and volumetric productivity of 2.8 g L -1  h -1 were obtained.

Toward a new generation of therapeutics: artificial cell targeted delivery of live cells for therapy.

PubMed

Prakash, Satya; Martoni, Christopher

2006-01-01

Scientific evidence in the prevention and treatment of various disorders is accumulating regarding probiotics. The health benefits supported by adequate clinical data include increased resistance to infectious disease, decreased duration of diarrhea, management of inflammatory bowel disease, reduction of serum cholesterol, prevention of allergy, modulation of cytokine gene expression, and suppression of carcinogen production. Recent ventures in metabolic engineering and heterologous protein expression have enhanced the enzymatic and immunomodulatory effects of probiotics and, with time, may allow more active intervention among critical care patients. In addition, a number of approaches are currently being explored, including the physical and chemical protection of cells, to increase probiotic viability and its health benefits. Traditional immobilization of probiotics in gel matrices, most notably calcium alginate and kappa-carrageenan, has frequently been employed, with noted improvements in viability during freezing and storage. Conflicting reports exist, however, on the protection offered by immobilization from harsh physiologic environments. An alternative approach, microencapsulation in "artificial cells," builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading, and greater control of parameters. This review summarizes the current clinical status of probiotics, examines the promises and challenges of current immobilization technologies, and presents the concept of artificial cells for effective delivery of therapeutic bacterial cells.

Style grammars for interactive visualization of architecture.

PubMed

Aliaga, Daniel G; Rosen, Paul A; Bekins, Daniel R

2007-01-01

Interactive visualization of architecture provides a way to quickly visualize existing or novel buildings and structures. Such applications require both fast rendering and an effortless input regimen for creating and changing architecture using high-level editing operations that automatically fill in the necessary details. Procedural modeling and synthesis is a powerful paradigm that yields high data amplification and can be coupled with fast-rendering techniques to quickly generate plausible details of a scene without much or any user interaction. Previously, forward generating procedural methods have been proposed where a procedure is explicitly created to generate particular content. In this paper, we present our work in inverse procedural modeling of buildings and describe how to use an extracted repertoire of building grammars to facilitate the visualization and quick modification of architectural structures and buildings. We demonstrate an interactive application where the user draws simple building blocks and, using our system, can automatically complete the building "in the style of" other buildings using view-dependent texture mapping or nonphotorealistic rendering techniques. Our system supports an arbitrary number of building grammars created from user subdivided building models and captured photographs. Using only edit, copy, and paste metaphors, the entire building styles can be altered and transferred from one building to another in a few operations, enhancing the ability to modify an existing architectural structure or to visualize a novel building in the style of the others.

U.S. Department of Energy awards $200 million for next-generation

Science.gov Websites

build a powerful new next-gen supercomputer, Aurora. The press conference took place at Chicago tech Congress and industry at the announcement of DOE's award of $200 million to Argonne in order to build a accelerate to next-generation exascale computing. DOE earlier announced a $325 million investment to build

Assessing mechanical deconstruction of softwood cell wall for cellulosic biofuels production

NASA Astrophysics Data System (ADS)

Jiang, Jinxue

Mechanical deconstruction offers a promising strategy to overcome biomass recalcitrance for facilitating enzymatic hydrolysis of pretreated substrates with zero chemicals input and presence of inhibitors. The goal of this dissertation research is to gain a more fundamental understanding on the impact of mechanical pretreatment on generating digestible micronized-wood and how the physicochemical characteristics influence the subsequent enzymatic hydrolysis of micronized wood. The initial moisture content of feedstock was found to be the key factor affecting the development of physical features and enzymatic hydrolysis of micronized wood. Lower moisture content resulted in much rounder particles with lower crystallinity, while higher moisture content resulted in the milled particles with larger aspect ratio and crystallinity. The enzymatic hydrolysis of micronized wood was improved as collectively increasing surface area (i.e., reducing particle size and aspect ratio) and decreasing crystallinity during mechanical milling pretreatment. Energy efficiency analysis demonstrated that low-moisture content feedstock with multi-step milling process would contribute to cost-effectiveness of mechanical pretreatment for achieving more than 70% of total sugars conversion. In the early stage of mechanical pretreatment, the types of cell fractures were distinguished by the initial moisture contents of wood, leading to interwall fracture at the middle lamella region for low moisture content samples and intrawall fracture at the inner cell wall for high moisture content samples. The changes in cell wall fractures also resulted in difference in the distribution of surface chemical composition and energy required for milling process. In an effort to exploit the underlying mechanism associated with the reduced recalcitrance in micronized wood, we reported the increased enzymatic sugar yield and correspondingly structural and accessible properties of micronized feedstock. Electronic microscopy analysis detailed the structural alternation of cell wall during mechanical process, including cell fracture and delamination, ultrastructure disintegration, and cell wall fragments amorphization, as coincident with the particle size reduction. It was confirmed with Simons' staining that longer milling time resulted in increased substrate accessibility and porosity. The changes in cellulose molecular structure with respect to degree of polymerization (DP) and crystallinity index (CrI) also benefited to decreasing recalcitrance and facilitating enzymatic hydrolysis of micronized wood.

Non-invasive determination of glucose directly in raw fruits using a continuous flow system based on microdialysis sampling and amperometric detection at an integrated enzymatic biosensor.

PubMed

Vargas, E; Ruiz, M A; Campuzano, S; Reviejo, A J; Pingarrón, J M

2016-03-31

A non-destructive, rapid and simple to use sensing method for direct determination of glucose in non-processed fruits is described. The strategy involved on-line microdialysis sampling coupled with a continuous flow system with amperometric detection at an enzymatic biosensor. Apart from direct determination of glucose in fruit juices and blended fruits, this work describes for the first time the successful application of an enzymatic biosensor-based electrochemical approach to the non-invasive determination of glucose in raw fruits. The methodology correlates, through previous calibration set-up, the amperometric signal generated from glucose in non-processed fruits with its content in % (w/w). The comparison of the obtained results using the proposed approach in different fruits with those provided by other method involving the same commercial biosensor as amperometric detector in stirred solutions pointed out that there were no significant differences. Moreover, in comparison with other available methodologies, this microdialysis-coupled continuous flow system amperometric biosensor-based procedure features straightforward sample preparation, low cost, reduced assay time (sampling rate of 7 h(-1)) and ease of automation. Copyright © 2016 Elsevier B.V. All rights reserved.

Ligand-Receptor Interaction Modulates the Energy Landscape of Enzyme-Instructed Self-Assembly of Small Molecules.

PubMed

Haburcak, Richard; Shi, Junfeng; Du, Xuewen; Yuan, Dan; Xu, Bing

2016-11-30

The concurrence of enzymatic reaction and ligand-receptor interactions is common for proteins, but rare for small molecules and has yet to be explored. Here we show that ligand-receptor interaction modulates the morphology of molecular assemblies formed by enzyme-instructed assembly of small molecules. While the absence of ligand-receptor interaction allows enzymatic dephosphorylation of a precursor to generate the hydrogelator that self-assembles to form long nanofibers, the presence of the ligand-receptor interaction biases the pathway to form precipitous aggregates containing short nanofibers. While the hydrogelators self-assemble to form nanofibers or nanoribbons that are unable to bind with the ligand (i.e., vancomycin), the addition of surfactant breaks up the assemblies to restore the ligand-receptor interaction. In addition, an excess amount of the ligands can disrupt the nanofibers and result in the precipitates. As the first example of the use of ligand-receptor interaction to modulate the kinetics of enzymatic self-assembly, this work not only provides a solution to evaluate the interaction between aggregates and target molecules but also offers new insight for understanding the emergent behavior of sophisticated molecular systems having multiple and parallel processes.

pH sensing characteristics and biosensing application of solution-gated reduced graphene oxide field-effect transistors.

PubMed

Sohn, Il-Yung; Kim, Duck-Jin; Jung, Jin-Heak; Yoon, Ok Ja; Thanh, Tien Nguyen; Quang, Trung Tran; Lee, Nae-Eung

2013-07-15

Solution-gated reduced graphene oxide field-effect transistors (R-GO FETs) were investigated for pH sensing and biochemical sensing applications. A channel of a networked R-GO film formed by self-assembly was incorporated as a sensing layer into a solution-gated FET structure for pH sensing and the detection of acetylcholine (Ach), which is a neurotransmitter in the nerve system, through enzymatic reactions. The fabricated R-GO FET was sensitive to protons (H(+)) with a pH sensitivity of 29 mV/pH in terms of the shift of the charge neutrality point (CNP), which is attributed to changes in the surface potential caused by the interaction of protons with OH surface functional groups present on the R-GO surface. The R-GO FET immobilized with acetylcholinesterase (AchE) was used to detect Ach in the concentration range of 0.1-10mM by sensing protons generated during the enzymatic reactions. The results indicate that R-GO FETs provide the capability to detect protons, demonstrating their applicability as a biosensing device for enzymatic reactions. Copyright © 2013 Elsevier B.V. All rights reserved.

Industrial-scale steam explosion pretreatment of sugarcane straw for enzymatic hydrolysis of cellulose for production of second generation ethanol and value-added products.

PubMed

Oliveira, Fernando M V; Pinheiro, Irapuan O; Souto-Maior, Ana M; Martin, Carlos; Gonçalves, Adilson R; Rocha, George J M

2013-02-01

Steam explosion at 180, 190 and 200°C for 15min was applied to sugarcane straw in an industrial sugar/ethanol reactor (2.5m(3)). The pretreated straw was delignificated by sodium hydroxide and hydrolyzed with cellulases, or submitted directly to enzymatic hydrolysis after the pretreatment. The pretreatments led to remarkable hemicellulose solubilization, with the maximum (92.7%) for pretreatment performed at 200°C. Alkaline treatment of the pretreated materials led to lignin solubilization of 86.7% at 180°C, and only to 81.3% in the material pretreated at 200°C. All pretreatment conditions led to high hydrolysis conversion of cellulose, with the maximum (80.0%) achieved at 200°C. Delignification increase the enzymatic conversion (from 58.8% in the cellulignin to 85.1% in the delignificated pulp) of the material pretreated at 180°C, but for the material pretreated at 190°C, the improvement was less remarkable, while for the pretreated at 200°C the hydrolysis conversion decreased after the alkaline treatment. Copyright © 2012 Elsevier Ltd. All rights reserved.

Kinetic study of lipase-catalyzed glycerolysis of palm olein using Lipozyme TLIM in solvent-free system

PubMed Central

Phuah, Eng-Tong; Lee, Yee-Ying; Tang, Teck-Kim

2018-01-01

Diacylglycerol (DAG) and monoacylglycerol (MAG) are two natural occurring minor components found in most edible fats and oils. These compounds have gained increasing market demand owing to their unique physicochemical properties. Enzymatic glycerolysis in solvent-free system might be a promising approach in producing DAG and MAG-enriched oil. Understanding on glycerolysis mechanism is therefore of great importance for process simulation and optimization. In this study, a commercial immobilized lipase (Lipozyme TL IM) was used to catalyze the glycerolysis reaction. The kinetics of enzymatic glycerolysis reaction between triacylglycerol (TAG) and glycerol (G) were modeled using rate equation with unsteady-state assumption. Ternary complex, ping-pong bi-bi and complex ping-pong bi-bi models were proposed and compared in this study. The reaction rate constants were determined using non-linear regression and sum of square errors (SSE) were minimized. Present work revealed satisfactory agreement between experimental data and the result generated by complex ping-pong bi-bi model as compared to other models. The proposed kinetic model would facilitate understanding on enzymatic glycerolysis for DAG and MAG production and design optimization of a pilot-scale reactor. PMID:29401481

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

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

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlatemore » with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. It was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.« less

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

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

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlatemore » with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. Moreover, it was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.« less

Progressive structural changes of Avicel, bleached softwood, and bacterial cellulose during enzymatic hydrolysis

DOE PAGES

Kafle, Kabindra; Shin, Heenae; Lee, Christopher M.; ...

2015-10-14

A comprehensive picture of structural changes of cellulosic biomass during enzymatic hydrolysis is essential for a better understanding of enzymatic actions and development of more efficient enzymes. In this study, a suite of analytical techniques including sum frequency generation (SFG) spectroscopy, infrared (IR) spectroscopy, x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) were employed for lignin-free model biomass samples—Avicel, bleached softwood, and bacterial cellulose—to find correlations between the decrease in hydrolysis rate over time and the structural or chemical changes of biomass during the hydrolysis reaction. The results showed that the decrease in hydrolysis rate over time appears to correlatemore » with the irreversible deposition of non-cellulosic species (either reaction side products or denatured enzymes, or both) on the cellulosic substrate surface. The crystallinity, degree of polymerization, and meso-scale packing of cellulose do not seem to positively correlate with the decrease in hydrolysis rate observed for all three substrates tested in this study. Moreover, it was also found that the cellulose Iα component of the bacterial cellulose is preferentially hydrolyzed by the enzyme than the cellulose Iβ component.« less

Extraction of green labeled pectins and pectic oligosaccharides from plant byproducts.

PubMed

Zykwinska, Agata; Boiffard, Marie-Hélène; Kontkanen, Hanna; Buchert, Johanna; Thibault, Jean-François; Bonnin, Estelle

2008-10-08

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.

Impact of primary amine group from aminophospholipids and amino acids on marine phospholipids stability: non-enzymatic browning and lipid oxidation.

PubMed

Lu, F S H; Nielsen, N S; Baron, C P; Diehl, B W K; Jacobsen, C

2013-11-15

The main objective of this study was to investigate the oxidative stability and non-enzymatic browning reactions of marine PL in the presence or in the absence of primary amine group from aminophospholipids and amino acids. Marine phospholipids liposomal dispersions were prepared from two authentic standards (phosphatidylcholine and phosphatidylethanolamine) and two purified PL from marine sources with and without addition of amino acids (leucine, methionine and lysine). Samples were incubated at 60°C for 0, 2, 4 and 6days. Non-enzymatic browning reactions were investigated through measurement of (i) Strecker derived volatiles, (ii) yellowness index (YI), (iii) hydrophobic and (iv) hydrophilic pyrroles content. The oxidative stability of the samples was assessed through measurement of secondary lipid derived volatile oxidation products. The result showed that the presence of PE and amino acids caused the formation of pyrroles, generated Strecker derived volatiles, decreased the YI development and lowered lipid oxidation. The lower degree of lipid oxidation in liposomal dispersions containing amino acids might be attributed to antioxidative properties of pyrroles or amino acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

Enzymatic saccharification of seaweeds into fermentable sugars by xylanase from marine Bacillus sp. strain BT21.

PubMed

Parab, Pankaj; Khandeparker, Rakhee; Amberkar, Ujwala; Khodse, Vishwas

2017-10-01

Enzymatic hydrolysis of seaweed biomass was studied using xylanase produced from marine bacteria Bacillus sp. strain BT21 through solid-state fermentation of wheat bran. Three types of seaweeds, Ahnfeltia plicata , Padina tetrastromatica and Ulva lactuca , were selected as representatives of red, brown, and green seaweeds, respectively. Seaweed biomass was pretreated with hot water. The efficiency of pretreated biomass to release reducing sugar by the action of xylanase as well as the type of monosaccharide released during enzyme saccharification of seaweed biomass was studied. It was seen that pretreated biomass of seaweed A. plicata, U. lactuca , and P. tetrastroma , at 121 °C for 45 min, followed by incubation with 50 IU xylanase released reducing sugars of 233 ± 5.3, 100 ± 6.1 and 73.3 ± 4.1 µg/mg of seaweed biomass, respectively. Gas chromatography analysis illustrated the release of xylose, glucose, and mannose during the treatment process. Hot water pre-treatment process enhanced enzymatic conversion of biomass into sugars. This study revealed the important role of xylanase in saccharification of seaweed, a promising feedstock for third-generation bioethanol production.

Modification of the nanostructure of lignocellulose cell walls via a non-enzymatic lignocellulose deconstruction system in brown rot wood-decay fungi.

PubMed

Goodell, Barry; Zhu, Yuan; Kim, Seong; Kafle, Kabindra; Eastwood, Daniel; Daniel, Geoffrey; Jellison, Jody; Yoshida, Makoto; Groom, Leslie; Pingali, Sai Venkatesh; O'Neill, Hugh

2017-01-01

Wood decayed by brown rot fungi and wood treated with the chelator-mediated Fenton (CMF) reaction, either alone or together with a cellulose enzyme cocktail, was analyzed by small angle neutron scattering (SANS), sum frequency generation (SFG) spectroscopy, Fourier transform infrared (FTIR) analysis, X-ray diffraction (XRD), atomic force microscopy (AFM), and transmission electron microscopy (TEM). Results showed that the CMF mechanism mimicked brown rot fungal attack for both holocellulose and lignin components of the wood. Crystalline cellulose and lignin were both depolymerized by the CMF reaction. Porosity of the softwood cell wall did not increase during CMF treatment, enzymes secreted by the fungi did not penetrate the decayed wood. The enzymes in the cellulose cocktail also did not appear to alter the effects of the CMF-treated wood relative to enhancing cell wall deconstruction. This suggests a rethinking of current brown rot decay models and supports a model where monomeric sugars and oligosaccharides diffuse from the softwood cell walls during non-enzymatic action. In this regard, the CMF mechanism should not be thought of as a "pretreatment" used to permit enzymatic penetration into softwood cell walls, but instead it enhances polysaccharide components diffusing to fungal enzymes located in wood cell lumen environments during decay. SANS and other data are consistent with a model for repolymerization and aggregation of at least some portion of the lignin within the cell wall, and this is supported by AFM and TEM data. The data suggest that new approaches for conversion of wood substrates to platform chemicals in biorefineries could be achieved using the CMF mechanism with >75% solubilization of lignocellulose, but that a more selective suite of enzymes and other downstream treatments may be required to work when using CMF deconstruction technology. Strategies to enhance polysaccharide release from lignocellulose substrates for enhanced enzymatic action and fermentation of the released fraction would also aid in the efficient recovery of the more uniform modified lignin fraction that the CMF reaction generates to enhance biorefinery profitability.

Enzymatic reaction paths as determined by transition path sampling

NASA Astrophysics Data System (ADS)

Masterson, Jean Emily

Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems, we observed changes in the reaction mechanism and altered contributions of the mutated residues to the enzymatic reaction coordinate, but we did not detect a substantial change in the time of barrier crossing. These results confirm the importance of maintaining the dynamics and structural scaffolding of the hhLDH PV in order to facilitate facile barrier passage. We also utilized TPS to investigate the possible role of fast protein dynamics in the enzymatic reaction coordinate of human dihydrofolate reductase (hsDHFR). We found that sub-picosecond dynamics of hsDHFR do contribute to the reaction coordinate, whereas this is not the case in the E. coli version of the enzyme. This result indicates a shift in the DHFR family to a more dynamic version of catalysis. The second inquiry we addressed in this thesis regarding enzymatic barrier passage concerns the variability of paths through reactive phase space for a given enzymatic reaction. We further investigated the hhLDH-catalyzed reaction using a high-perturbation TPS algorithm. Though we saw that alternate reaction paths were possible, the dominant reaction path we observed corresponded to that previously elucidated in prior hhLDH TPS studies. Since the additional reaction paths we observed were likely high-energy, these results indicate that only the dominant reaction path contributes significantly to the overall reaction rate. In conclusion, we show that the enzymes hhLDH and hsDHFR exhibit paths through reactive phase space where fast protein motions are involved in the enzymatic reaction coordinate and exhibit a non-negligible contribution to chemical barrier crossing.

2. Detail of panel in generator room, building 501, looking ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. Detail of panel in generator room, building 501, looking north - Offutt Air Force Base, Strategic Air Command Headquarters & Command Center, Command Center, 901 SAC Boulevard, Bellevue, Sarpy County, NE

Predictive model for CO2 generation and decay in building envelopes

NASA Astrophysics Data System (ADS)

Aglan, Heshmat A.

2003-01-01

Understanding carbon dioxide generation and decay patterns in buildings with high occupancy levels is useful to identify their indoor air quality, air change rates, percent fresh air makeup, occupancy pattern, and how a variable air volume system to off-set undesirable CO2 level can be modulated. A mathematical model governing the generation and decay of CO2 in building envelopes with forced ventilation due to high occupancy is developed. The model has been verified experimentally in a newly constructed energy efficient healthy house. It was shown that the model accurately predicts the CO2 concentration at any time during the generation and decay processes.

TLS for generating multi-LOD of 3D building model

NASA Astrophysics Data System (ADS)

Akmalia, R.; Setan, H.; Majid, Z.; Suwardhi, D.; Chong, A.

2014-02-01

The popularity of Terrestrial Laser Scanners (TLS) to capture three dimensional (3D) objects has been used widely for various applications. Development in 3D models has also led people to visualize the environment in 3D. Visualization of objects in a city environment in 3D can be useful for many applications. However, different applications require different kind of 3D models. Since a building is an important object, CityGML has defined a standard for 3D building models at four different levels of detail (LOD). In this research, the advantages of TLS for capturing buildings and the modelling process of the point cloud can be explored. TLS will be used to capture all the building details to generate multi-LOD. This task, in previous works, involves usually the integration of several sensors. However, in this research, point cloud from TLS will be processed to generate the LOD3 model. LOD2 and LOD1 will then be generalized from the resulting LOD3 model. Result from this research is a guiding process to generate the multi-LOD of 3D building starting from LOD3 using TLS. Lastly, the visualization for multi-LOD model will also be shown.

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

PubMed

Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD + ]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD + and enzymes that use NAD + like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

PubMed

Estabrook, R W; Shet, M S; Faulkner, K; Fisher, C W

1996-11-01

A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries.

PubMed

Cobucci-Ponzano, Beatrice; Strazzulli, Andrea; Iacono, Roberta; Masturzo, Giuseppe; Giglio, Rosa; Rossi, Mosè; Moracci, Marco

2015-10-01

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2h vs 12h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications. Copyright © 2015 Elsevier Inc. All rights reserved.

The CanOE strategy: integrating genomic and metabolic contexts across multiple prokaryote genomes to find candidate genes for orphan enzymes.

PubMed

Smith, Adam Alexander Thil; Belda, Eugeni; Viari, Alain; Medigue, Claudine; Vallenet, David

2012-05-01

Of all biochemically characterized metabolic reactions formalized by the IUBMB, over one out of four have yet to be associated with a nucleic or protein sequence, i.e. are sequence-orphan enzymatic activities. Few bioinformatics annotation tools are able to propose candidate genes for such activities by exploiting context-dependent rather than sequence-dependent data, and none are readily accessible and propose result integration across multiple genomes. Here, we present CanOE (Candidate genes for Orphan Enzymes), a four-step bioinformatics strategy that proposes ranked candidate genes for sequence-orphan enzymatic activities (or orphan enzymes for short). The first step locates "genomic metabolons", i.e. groups of co-localized genes coding proteins catalyzing reactions linked by shared metabolites, in one genome at a time. These metabolons can be particularly helpful for aiding bioanalysts to visualize relevant metabolic data. In the second step, they are used to generate candidate associations between un-annotated genes and gene-less reactions. The third step integrates these gene-reaction associations over several genomes using gene families, and summarizes the strength of family-reaction associations by several scores. In the final step, these scores are used to rank members of gene families which are proposed for metabolic reactions. These associations are of particular interest when the metabolic reaction is a sequence-orphan enzymatic activity. Our strategy found over 60,000 genomic metabolons in more than 1,000 prokaryote organisms from the MicroScope platform, generating candidate genes for many metabolic reactions, of which more than 70 distinct orphan reactions. A computational validation of the approach is discussed. Finally, we present a case study on the anaerobic allantoin degradation pathway in Escherichia coli K-12.

A Sensitive and Versatile Fluorescent Activity Assay for ABHD6.

PubMed

Savinainen, Juha R; Navia-Paldanius, Dina; Laitinen, Jarmo T

2016-01-01

The α/β-hydrolase domain-containing 6 (ABHD6) enzyme is a newly found serine hydrolase whose substrate profile resembles that of monoacylglycerol lipase (MAGL), the major 2-arachidonoyl glycerol (2-AG) hydrolase in the brain. Here, we describe a sensitive fluorescent assay of ABHD6 activity in a 96-well-plate format that allows parallel testing of inhibitor activities of up to 40 compounds in a single assay. The method utilizes lysates of HEK293 cells transiently overexpressing human ABHD6 as the enzymatic source, and kinetically monitors glycerol liberated in the hydrolysis of 1(3)-AG, the preferred arachidonoyl glycerol isomer. Glycerol output is coupled to an enzymatic cascade generating the fluorescent end-product resorufin. The approach has major benefits compared to laborious traditional mass spectrometric methods and liquid scintillation-based assays, or approaches using unnatural substrates.

Biotechnological production of arbutins (α- and β-arbutins), skin-lightening agents, and their derivatives.

PubMed

Seo, Dong-Ho; Jung, Jong-Hyun; Lee, Jae-Eun; Jeon, Eun-Jung; Kim, Wooki; Park, Cheon-Seok

2012-09-01

Arbutins (α- and β-arbutins) are glycosylated hydroquinones that are commercially used in the cosmetic industry. These compounds have an inhibitory function against tyrosinase, a critical enzyme for generating pigments, which leads to the prevention of melanin formation, resulting in a whitening effect on the skin. Although β-arbutin is found in various plants including bearberry, wheat, and pear, α-arbutin and other arbutin derivatives are synthesized by chemical and enzymatic methods. This article presents a mini-review of recent studies on the production of α-arbutin and other α- and β-arbutin derivatives via enzymatic bioconversion methods. In addition, the structures of α- and β-arbutin derivatives and their biological activities are discussed. The catalytic characteristics of various enzymes used in the biosynthesis of arbutin derivatives are also reviewed.

Environmental biocatalysis: from remediation with enzymes to novel green processes.

PubMed

Alcalde, Miguel; Ferrer, Manuel; Plou, Francisco J; Ballesteros, Antonio

2006-06-01

Modern biocatalysis is developing new and precise tools to improve a wide range of production processes, which reduce energy and raw material consumption and generate less waste and toxic side-products. Biocatalysis is also achieving new advances in environmental fields, from enzymatic bioremediation to the synthesis of renewable and clean energies and biochemical cleaning of 'dirty' fossil fuels. Despite the obvious benefits of biocatalysis, the major hurdles hindering the exploitation of the repertoire of enzymatic processes are, in many cases, the high production costs and the low yields obtained. This article will discuss these issues, pinpointing specific new advances in recombinant DNA techniques amenable to future biocatalyst development, in addition to drawing the attention of the biotechnology community to the active pursuit and development of environmental biocatalysis, from remediation with enzymes to novel green processes.

Fractionation of corn fiber treated by soaking in aqueous ammonia (SAA) for isolation of hemicellulose B and production of C5 sugars by enzyme hydrolysis

USDA-ARS?s Scientific Manuscript database

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia (SAA) followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cle...

Two-Dimensional NMR Evidence for Cleavage of Lignin and Xylan Substituents in Wheat Straw Through Hydrothermal Pretreatment and Enzymatic Hydrolysis

Treesearch

Daniel J. Yelle; Prasad Kaparaju; Christopher G. Hunt; Kolby Hirth; Hoon Kim; John Ralph; Claus Felby

2012-01-01

Solution-state two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of plant cell walls is a powerful tool for characterizing changes in cell wall chemistry during the hydrothermal pretreatment process of wheat straw for second-generation bioethanol production. One-bond 13C-1H NMR correlation spectroscopy, via...

Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification

USDA-ARS?s Scientific Manuscript database

A pretreatment strategy for dilute H2SO4 pretreatment of corn stover was developed for the purpose of reducing the generation of inhibitory substances during pretreatment so that a detoxification step is not required prior to fermentation while maximizing the sugar yield. We have optimized dilute su...

Optimization of dilute sulfuric acid pretreatment and enzymatic saccharification of corn stover for efficient ethanol production

USDA-ARS?s Scientific Manuscript database

Dilute acid pretreatment is a promising pretreatment technology for conversion of lignocellulosic biomass to fuel ethanol. Corn stover (supplied by a local farmer) used in this study contained 37.0±0.4% cellulose, 31.3±0.6% hemicelluloses, and 17.8±0.2% lignin. Generation of fermentable sugars from ...

Anaerobic ammonia removal in presence of organic matter: a novel route.

PubMed

Sabumon, P C

2007-10-01

This study describes the feasibility of anaerobic ammonia removal process in presence of organic matter. Different sources of biomass collected from diverse eco-systems containing ammonia and organic matter (OM) were screened for potential anaerobic ammonia removal. Sequential batch studies confirmed the possibility of anaerobic ammonia removal in presence of OM, but ammonia was oxidized anoxically to nitrate (at oxidation reduction potential; ORP=-248+/-25 mV) by an unknown mechanism unlike in the reported anammox process. The oxygen required for oxidation of ammonia might have been generated through catalase enzymatic activity of facultative anaerobes in mixed culture. The oxygen generation possibility by catalase enzyme route was demonstrated. Among the inorganic electron acceptors (NO(2)(-), NO(3)(-) and SO(4)(2-)) studied, NO(2)(-) was found to be most effective in total nitrogen removal. Denitrification by the developed culture was much effective and faster compared to ammonia oxidation. The results of this study show that anaerobic ammonia removal is feasible in presence of OM. The novel nitrogen removal route is hypothesized as enzymatic anoxic oxidation of NH(4)(+) to NO(3)(-), followed by denitrification via autotrophic and/or heterotrophic routes. The results of batch study were confirmed in continuous reactor operation.

Thiomers and thiomer-based nanoparticles in protein and DNA drug delivery.

PubMed

Hauptstein, Sabine; Bernkop-Schnürch, Andreas

2012-09-01

Thanks to advances in biotechnology, more and more highly efficient protein- and DNA-based drugs have been developed. Unfortunately, these kinds of drugs underlie poor non-parental bioavailability. To overcome hindrances like low mucosal permeability and enzymatic degradation polymeric excipients are utilized as drug carrier whereat thiolated excipients showed several promising qualities in comparison to the analogical unmodified polymer. The article deals with the comparatively easy modification of well-established polymers like chitosan or poly(acrylates) to synthesize thiomers. Further, the recently developed "next generation" thiomers e.g. preactivated or S-protected thiomers are introduced. Designative properties like mucoadhesion, uptake and permeation enhancement, efflux pump inhibition and protection against enzymatic degradation will be discussed and differences between first and next generation thiomers will be pointed out. Additionally, nanoparticles prepared with thiomers will be dealt with regarding to protein and DNA drug delivery as thiomers seem to be a promising approach to avoid parenteral application. Properties of thiomers per se and results of in vivo studies carried out so far for peptide and DNA drugs demonstrate their potential as multifunctional excipients. However, further investigations and optimizations have to be done before establishing a carrier system ready for clinical approval.

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

PubMed Central

Rodriguez-Rivera, Veronica; Weidner, John W.; Yost, Michael J.

2016-01-01

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material. PMID:26967145

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels.

PubMed

Rodriguez-Rivera, Veronica; Weidner, John W; Yost, Michael J

2016-02-12

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

Genetic engineering including superseding microinjection: new ways to make GM pigs.

PubMed

Galli, Cesare; Perota, Andrea; Brunetti, Dario; Lagutina, Irina; Lazzari, Giovanna; Lucchini, Franco

2010-01-01

Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review.

PubMed

Tiwari, Rameshwar; Nain, Lata; Labrou, Nikolaos E; Shukla, Pratyoosh

2018-03-01

Second generation biofuel production has been appeared as a sustainable and alternative energy option. The ultimate aim is the development of an industrially feasible and economic conversion process of lignocellulosic biomass into biofuel molecules. Since, cellulose is the most abundant biopolymer and also represented as the photosynthetically fixed form of carbon, the efficient hydrolysis of cellulose is the most important step towards the development of a sustainable biofuel production process. The enzymatic hydrolysis of cellulose by suites of hydrolytic enzymes underlines the importance of cellulase enzyme system in whole hydrolysis process. However, the selection of the suitable cellulolytic enzymes with enhanced activities remains a challenge for the biorefinery industry to obtain efficient enzymatic hydrolysis of biomass. The present review focuses on deciphering the novel and effective cellulases from different environmental niches by unculturable metagenomic approaches. Furthermore, a comprehensive functional aspect of cellulases is also presented and evaluated by assessing the structural and catalytic properties as well as sequence identities and expression patterns. This review summarizes the recent development in metagenomics based approaches for identifying and exploring novel cellulases which open new avenues for their successful application in biorefineries.

18. Control Area, Frequency Changer and Generator Building, interior view ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

18. Control Area, Frequency Changer and Generator Building, interior view of remaining control panels VIEW WEST - NIKE Missile Battery PR-79, Control Area, Tucker Hollow Road south of State Route 101, Foster, Providence County, RI

12. LAUNCH CONTROL SUPPORT BUILDING. INTERIOR OF GENERATOR ROOM. VIEW ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. LAUNCH CONTROL SUPPORT BUILDING. INTERIOR OF GENERATOR ROOM. VIEW TO EAST. - Minuteman III ICBM Launch Control Facility November-1, 1.5 miles North of New Raymer & State Highway 14, New Raymer, Weld County, CO

Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

PubMed

Nakanishi, Simone C; Soares, Lauren B; Biazi, Luiz Eduardo; Nascimento, Viviane M; Costa, Aline C; Rocha, George Jackson M; Ienczak, Jaciane L

2017-10-01

Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130°C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g · L -1  · h -1 (first to third batch) for S. stipitis and from 0.38 to 0.81 g · L -1  · h -1 for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Automatic generation and simulation of urban building energy models based on city datasets for city-scale building retrofit analysis

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

Chen, Yixing; Hong, Tianzhen; Piette, Mary Ann

Buildings in cities consume 30–70% of total primary energy, and improving building energy efficiency is one of the key strategies towards sustainable urbanization. Urban building energy models (UBEM) can support city managers to evaluate and prioritize energy conservation measures (ECMs) for investment and the design of incentive and rebate programs. This paper presents the retrofit analysis feature of City Building Energy Saver (CityBES) to automatically generate and simulate UBEM using EnergyPlus based on cities’ building datasets and user-selected ECMs. CityBES is a new open web-based tool to support city-scale building energy efficiency strategic plans and programs. The technical details ofmore » using CityBES for UBEM generation and simulation are introduced, including the workflow, key assumptions, and major databases. Also presented is a case study that analyzes the potential retrofit energy use and energy cost savings of five individual ECMs and two measure packages for 940 office and retail buildings in six city districts in northeast San Francisco, United States. The results show that: (1) all five measures together can save 23–38% of site energy per building; (2) replacing lighting with light-emitting diode lamps and adding air economizers to existing heating, ventilation and air-conditioning (HVAC) systems are most cost-effective with an average payback of 2.0 and 4.3 years, respectively; and (3) it is not economical to upgrade HVAC systems or replace windows in San Francisco due to the city's mild climate and minimal cooling and heating loads. Furthermore, the CityBES retrofit analysis feature does not require users to have deep knowledge of building systems or technologies for the generation and simulation of building energy models, which helps overcome major technical barriers for city managers and their consultants to adopt UBEM.« less

Automatic generation and simulation of urban building energy models based on city datasets for city-scale building retrofit analysis

DOE PAGES

Chen, Yixing; Hong, Tianzhen; Piette, Mary Ann

2017-08-07

Buildings in cities consume 30–70% of total primary energy, and improving building energy efficiency is one of the key strategies towards sustainable urbanization. Urban building energy models (UBEM) can support city managers to evaluate and prioritize energy conservation measures (ECMs) for investment and the design of incentive and rebate programs. This paper presents the retrofit analysis feature of City Building Energy Saver (CityBES) to automatically generate and simulate UBEM using EnergyPlus based on cities’ building datasets and user-selected ECMs. CityBES is a new open web-based tool to support city-scale building energy efficiency strategic plans and programs. The technical details ofmore » using CityBES for UBEM generation and simulation are introduced, including the workflow, key assumptions, and major databases. Also presented is a case study that analyzes the potential retrofit energy use and energy cost savings of five individual ECMs and two measure packages for 940 office and retail buildings in six city districts in northeast San Francisco, United States. The results show that: (1) all five measures together can save 23–38% of site energy per building; (2) replacing lighting with light-emitting diode lamps and adding air economizers to existing heating, ventilation and air-conditioning (HVAC) systems are most cost-effective with an average payback of 2.0 and 4.3 years, respectively; and (3) it is not economical to upgrade HVAC systems or replace windows in San Francisco due to the city's mild climate and minimal cooling and heating loads. Furthermore, the CityBES retrofit analysis feature does not require users to have deep knowledge of building systems or technologies for the generation and simulation of building energy models, which helps overcome major technical barriers for city managers and their consultants to adopt UBEM.« less

Spring Ankle with Regenerative Kinetics to Build a New Generation of Transtibial Prostheses

DTIC Science & Technology

2008-07-31

form factor that is portable to the wearer. The objective is to build a transtibial prosthesis that will support a Military amputee’s return to...active duty. 15. SUBJECT TERMS Transtibial Prosthesis , regenerative, spring, wearable robot 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...Regenerative Kinetics” to build a new generation of transtibial prostheses Keywords: Transtibial Prosthesis , regenerative, spring, wearable robot

Multigenerational Challenges: Team-Building for Positive Clinical Workforce Outcomes

PubMed

Moore, Jill M; Everly, Marcee; Bauer, Renee

2016-05-31

Patient acuity in hospital settings continues to increase, and there is greater emphasis on patient outcomes. The current nursing workforce is comprised of four distinct generational cohorts that include veterans, baby boomers, millennials, and generation Xers. Each group has unique characteristics that add complexity to the workforce and this can add challenges to providing optimal patient care. Team building is one strategy to increase mutual understanding, communication, and respect, and thus potentially improve patient outcomes. In this article, we first briefly define generational cohorts by characteristics, and discuss differing expectations for work/life balance and potential negative outcomes. Our discussion offers team building strategies for positive outcomes, a case scenario, and concludes with resources for team building and organizational opportunities.

Large-scale building scenes reconstruction from close-range images based on line and plane feature

NASA Astrophysics Data System (ADS)

Ding, Yi; Zhang, Jianqing

2007-11-01

Automatic generate 3D models of buildings and other man-made structures from images has become a topic of increasing importance, those models may be in applications such as virtual reality, entertainment industry and urban planning. In this paper we address the main problems and available solution for the generation of 3D models from terrestrial images. We first generate a coarse planar model of the principal scene planes and then reconstruct windows to refine the building models. There are several points of novelty: first we reconstruct the coarse wire frame model use the line segments matching with epipolar geometry constraint; Secondly, we detect the position of all windows in the image and reconstruct the windows by established corner points correspondences between images, then add the windows to the coarse model to refine the building models. The strategy is illustrated on image triple of college building.

Co3O4 based non-enzymatic glucose sensor with high sensitivity and reliable stability derived from hollow hierarchical architecture.

PubMed

Tian, Liangliang; He, Gege; Cai, Yanhua; Wu, Shenping; Su, Yongyao; Yan, Hengqing; Yang, Cong; Chen, Yanling; Li, Lu

2018-02-16

Inspired by kinetics, the design of hollow hierarchical electrocatalysts through large-scale integration of building blocks is recognized as an effective approach to the achievement of superior electrocatalytic performance. In this work, a hollow, hierarchical Co 3 O 4 architecture (Co 3 O 4 HHA) was constructed using a coordinated etching and precipitation (CEP) method followed by calcination. The resulting Co 3 O 4 HHA electrode exhibited excellent electrocatalytic activity in terms of high sensitivity (839.3 μA mM -1 cm -2 ) and reliable stability in glucose detection. The high sensitivity could be attributed to the large specific surface area (SSA), ample unimpeded penetration diffusion paths and high electron transfer rate originating from the unique two-dimensional (2D) sheet-like character and hollow porous architecture. The hollow hierarchical structure also affords sufficient interspace for accommodation of volume change and structural strain, resulting in enhanced stability. The results indicate that Co 3 O 4 HHA could have potential for application in the design of non-enzymatic glucose sensors, and that the construction of hollow hierarchical architecture provides an efficient way to design highly active, stable electrocatalysts.

Co3O4 based non-enzymatic glucose sensor with high sensitivity and reliable stability derived from hollow hierarchical architecture

NASA Astrophysics Data System (ADS)

Tian, Liangliang; He, Gege; Cai, Yanhua; Wu, Shenping; Su, Yongyao; Yan, Hengqing; Yang, Cong; Chen, Yanling; Li, Lu

2018-02-01

Inspired by kinetics, the design of hollow hierarchical electrocatalysts through large-scale integration of building blocks is recognized as an effective approach to the achievement of superior electrocatalytic performance. In this work, a hollow, hierarchical Co3O4 architecture (Co3O4 HHA) was constructed using a coordinated etching and precipitation (CEP) method followed by calcination. The resulting Co3O4 HHA electrode exhibited excellent electrocatalytic activity in terms of high sensitivity (839.3 μA mM-1 cm-2) and reliable stability in glucose detection. The high sensitivity could be attributed to the large specific surface area (SSA), ample unimpeded penetration diffusion paths and high electron transfer rate originating from the unique two-dimensional (2D) sheet-like character and hollow porous architecture. The hollow hierarchical structure also affords sufficient interspace for accommodation of volume change and structural strain, resulting in enhanced stability. The results indicate that Co3O4 HHA could have potential for application in the design of non-enzymatic glucose sensors, and that the construction of hollow hierarchical architecture provides an efficient way to design highly active, stable electrocatalysts.

[Enzymatic degradation of organophosphorus insecticide chlorpyrifos by fungus WZ-I].

PubMed

Xie, Hui; Zhu, Lu-sheng; Wang, Jun; Wang, Xiu-guo; Liu, Wei; Qian, Bo; Wang, Qian

2005-11-01

Degradation characteristics of chlorpyrifos insecticides was determined by the crude enzyme extracted from the isolated strain WZ-I ( Fusarium LK. ex Fx). The best separating condition and the degrading characteristic of chlorpyrifos were studied. Rate of degradation for chlorpyrifos by its intracellular enzyme, extracellular enzyme and cell fragment was 60.8%, 11.3% and 48%, respectively. The degrading enzyme was extracted after this fungus was incubated for 8 generations in the condition of noninducement, and its enzymic activity lost less, the results show that this enzyme is an intracellular and connatural enzyme. The solubility protein of the crude enzyme was determined with Albumin (bovine serum) as standard protein and the solubility protein of the crude enzyme was 3.36 mg x mL(-1). The pH optimum for crude enzyme was 6.8 for enzymatic degradation of chlorpyrifos, and it had comparatively high activity in the range of pH 6.0 - 9.0. The optimum temperature for enzymatic activity was at 40 degrees C, it still had comparatively high activity in the range of temperature 20-50 degrees C, the activity of enzyme rapidly reduced at 55 degrees C, its activity was 41% of the maximal activity. The crude enzyme showed Km value for chlorpyrifos of 1.049 26 mmol x L(-1), and the maximal enzymatic degradation rate was 0.253 5 micromol x (mg x min)(-1). Additional experimental evidence suggests that the enzyme had the stability of endure for temperature and pH, the crude enzyme of fungus WZ-I could effectively degrade chlorpyrifos.

Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS.

PubMed

Muneeruddin, K; Bobst, C E; Frenkel, R; Houde, D; Turyan, I; Sosic, Z; Kaltashov, I A

2017-01-16

Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon β-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.

DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L -1) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration

DOE PAGES

Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W.; ...

2016-04-01

Distilling and purifying ethanol and other products from second generation lignocellulosic biorefineries adds significant capital and operating costs to biofuel production. The energy usage associated with distillation negatively affects plant gate costs and causes environmental and life-cycle impacts, and the lower titers in fermentation caused by lower sugar concentrations from pretreatment and enzymatic hydrolysis increase energy and water usage and ethanol production costs. In addition, lower ethanol titers increase the volumes required for enzymatic hydrolysis and fermentation vessels increase capital expenditure (CAPEX). Therefore, increasing biofuel titers has been a research focus in renewable biofuel production for several decades. In thismore » work, we achieved approximately 230 g L -1 of monomeric sugars after high solid enzymatic hydrolysis using deacetylation and mechanical refining (DMR) processed corn stover substrates produced at the 100 kg per day scale. The high sugar concentrations and low chemical inhibitor concentrations achieved by the DMR process allowed fermentation to ethanol with titers as high as 86 g L -1, which translates into approximately 10.9% v/v ethanol. To our knowledge, this is the first time that titers greater than 10% v/v ethanol in fermentations derived from corn stover without any sugar concentration or purification steps have been reported. As a result, the potential cost savings from high sugar and ethanol titers achieved by the DMR process are also reported using TEA analysis.« less

Over production of fermentable sugar for bioethanol production from carbohydrate-rich Malaysian food waste via sequential acid-enzymatic hydrolysis pretreatment.

PubMed

Hafid, Halimatun Saadiah; Nor 'Aini, Abdul Rahman; Mokhtar, Mohd Noriznan; Talib, Ahmad Tarmezee; Baharuddin, Azhari Samsu; Umi Kalsom, Md Shah

2017-09-01

In Malaysia, the amount of food waste produced is estimated at approximately 70% of total municipal solid waste generated and characterised by high amount of carbohydrate polymers such as starch, cellulose, and sugars. Considering the beneficial organic fraction contained, its utilization as an alternative substrate specifically for bioethanol production has receiving more attention. However, the sustainable production of bioethanol from food waste is linked to the efficient pretreatment needed for higher production of fermentable sugar prior to fermentation. In this work, a modified sequential acid-enzymatic hydrolysis process has been developed to produce high concentration of fermentable sugars; glucose, sucrose, fructose and maltose. The process started with hydrothermal and dilute acid pretreatment by hydrochloric acid (HCl) and sulphuric acid (H 2 SO 4 ) which aim to degrade larger molecules of polysaccharide before accessible for further steps of enzymatic hydrolysis by glucoamylase. A kinetic model is proposed to perform an optimal hydrolysis for obtaining high fermentable sugars. The results suggested that a significant increase in fermentable sugar production (2.04-folds) with conversion efficiency of 86.8% was observed via sequential acid-enzymatic pretreatment as compared to dilute acid pretreatment (∼42.4% conversion efficiency). The bioethanol production by Saccharomyces cerevisiae utilizing fermentable sugar obtained shows ethanol yield of 0.42g/g with conversion efficiency of 85.38% based on the theoretical yield was achieved. The finding indicates that food waste can be considered as a promising substrate for bioethanol production. Copyright © 2017. Published by Elsevier Ltd.

DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L -1) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration

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

Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W.

Distilling and purifying ethanol and other products from second generation lignocellulosic biorefineries adds significant capital and operating costs to biofuel production. The energy usage associated with distillation negatively affects plant gate costs and causes environmental and life-cycle impacts, and the lower titers in fermentation caused by lower sugar concentrations from pretreatment and enzymatic hydrolysis increase energy and water usage and ethanol production costs. In addition, lower ethanol titers increase the volumes required for enzymatic hydrolysis and fermentation vessels increase capital expenditure (CAPEX). Therefore, increasing biofuel titers has been a research focus in renewable biofuel production for several decades. In thismore » work, we achieved approximately 230 g L -1 of monomeric sugars after high solid enzymatic hydrolysis using deacetylation and mechanical refining (DMR) processed corn stover substrates produced at the 100 kg per day scale. The high sugar concentrations and low chemical inhibitor concentrations achieved by the DMR process allowed fermentation to ethanol with titers as high as 86 g L -1, which translates into approximately 10.9% v/v ethanol. To our knowledge, this is the first time that titers greater than 10% v/v ethanol in fermentations derived from corn stover without any sugar concentration or purification steps have been reported. As a result, the potential cost savings from high sugar and ethanol titers achieved by the DMR process are also reported using TEA analysis.« less

Pretreatment of Dried Distiller Grains with Solubles by Soaking in Aqueous Ammonia and Subsequent Enzymatic/Dilute Acid Hydrolysis to Produce Fermentable Sugars.

PubMed

Nghiem, Nhuan P; Montanti, Justin; Kim, Tae Hyun

2016-05-01

Dried distillers grains with solubles (DDGS), a co-product of corn ethanol production in the dry-grind process, was pretreated by soaking in aqueous ammonia (SAA) using a 15 % w/w NH4OH solution at a solid/liquid ratio of 1:10. The effect of pretreatment on subsequent enzymatic hydrolysis was studied at two temperatures (40 and 60 °C) and four reaction times (6, 12, 24, and 48 h). Highest glucose yield of 91 % theoretical was obtained for the DDGS pretreated at 60 °C and 24 h. The solubilized hemicellulose in the liquid fraction was further hydrolyzed with dilute H2SO4 to generate fermentable monomeric sugars. The conditions of acid hydrolysis included 1 and 4 wt% acid, 60 and 120 °C, and 0.5 and 1 h. Highest yields of xylose and arabinose were obtained at 4 wt% acid, 120 °C, and 1 h. The fermentability of the hydrolysate obtained by enzymatic hydrolysis of the SAA-pretreated DDGS was demonstrated in ethanol fermentation by Saccharomyces cerevisiae. The fermentability of the hydrolysate obtained by consecutive enzymatic and dilute acid hydrolysis was demonstrated using a succinic acid-producing microorganism, strain Escherichia coli AFP184. Under the fermentation conditions, complete utilization of glucose and arabinose was observed, whereas only 47 % of xylose was used. The succinic acid yield was 0.60 g/g total sugar consumed.

1. MISSILE TEST AND ASSEMBLY BUILDING, FRONT, LOOKING SOUTH. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. MISSILE TEST AND ASSEMBLY BUILDING, FRONT, LOOKING SOUTH. - NIKE Missile Base SL-40, Missile Test & Assembly Building, South end of launch area, northeast of Generator Building No. 3, Hecker, Monroe County, IL

D Building Reconstruction by Multiview Images and the Integrated Application with Augmented Reality

NASA Astrophysics Data System (ADS)

Hwang, Jin-Tsong; Chu, Ting-Chen

2016-10-01

This study presents an approach wherein photographs with a high degree of overlap are clicked using a digital camera and used to generate three-dimensional (3D) point clouds via feature point extraction and matching. To reconstruct a building model, an unmanned aerial vehicle (UAV) is used to click photographs from vertical shooting angles above the building. Multiview images are taken from the ground to eliminate the shielding effect on UAV images caused by trees. Point clouds from the UAV and multiview images are generated via Pix4Dmapper. By merging two sets of point clouds via tie points, the complete building model is reconstructed. The 3D models are reconstructed using AutoCAD 2016 to generate vectors from the point clouds; SketchUp Make 2016 is used to rebuild a complete building model with textures. To apply 3D building models in urban planning and design, a modern approach is to rebuild the digital models; however, replacing the landscape design and building distribution in real time is difficult as the frequency of building replacement increases. One potential solution to these problems is augmented reality (AR). Using Unity3D and Vuforia to design and implement the smartphone application service, a markerless AR of the building model can be built. This study is aimed at providing technical and design skills related to urban planning, urban designing, and building information retrieval using AR.

WASTE HANDLING BUILDING ELECTRICAL SYSTEM DESCRIPTION DOCUMENT

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

S.C. Khamamkar

2000-06-23

The Waste Handling Building Electrical System performs the function of receiving, distributing, transforming, monitoring, and controlling AC and DC power to all waste handling building electrical loads. The system distributes normal electrical power to support all loads that are within the Waste Handling Building (WHB). The system also generates and distributes emergency power to support designated emergency loads within the WHB within specified time limits. The system provides the capability to transfer between normal and emergency power. The system provides emergency power via independent and physically separated distribution feeds from the normal supply. The designated emergency electrical equipment will bemore » designed to operate during and after design basis events (DBEs). The system also provides lighting, grounding, and lightning protection for the Waste Handling Building. The system is located in the Waste Handling Building System. The system consists of a diesel generator, power distribution cables, transformers, switch gear, motor controllers, power panel boards, lighting panel boards, lighting equipment, lightning protection equipment, control cabling, and grounding system. Emergency power is generated with a diesel generator located in a QL-2 structure and connected to the QL-2 bus. The Waste Handling Building Electrical System distributes and controls primary power to acceptable industry standards, and with a dependability compatible with waste handling building reliability objectives for non-safety electrical loads. It also generates and distributes emergency power to the designated emergency loads. The Waste Handling Building Electrical System receives power from the Site Electrical Power System. The primary material handling power interfaces include the Carrier/Cask Handling System, Canister Transfer System, Assembly Transfer System, Waste Package Remediation System, and Disposal Container Handling Systems. The system interfaces with the MGR Operations Monitoring and Control System for supervisory monitoring and control signals. The system interfaces with all facility support loads such as heating, ventilation, and air conditioning, office, fire protection, monitoring and control, safeguards and security, and communications subsystems.« less

4. MISSILE TEST AND ASSEMBLY BUILDING, LEFT SIDE, LOOKING NORTH. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

4. MISSILE TEST AND ASSEMBLY BUILDING, LEFT SIDE, LOOKING NORTH. - NIKE Missile Base SL-40, Missile Test & Assembly Building, South end of launch area, northeast of Generator Building No. 3, Hecker, Monroe County, IL

2. MISSILE TEST AND ASSEMBLY BUILDING, RIGHT SIDE, LOOKING WEST. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. MISSILE TEST AND ASSEMBLY BUILDING, RIGHT SIDE, LOOKING WEST. - NIKE Missile Base SL-40, Missile Test & Assembly Building, South end of launch area, northeast of Generator Building No. 3, Hecker, Monroe County, IL

3. MISSILE TEST AND ASSEMBLY BUILDING, REAR SIDE, LOOKING NORTH. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. MISSILE TEST AND ASSEMBLY BUILDING, REAR SIDE, LOOKING NORTH. - NIKE Missile Base SL-40, Missile Test & Assembly Building, South end of launch area, northeast of Generator Building No. 3, Hecker, Monroe County, IL

Automatic Large-Scalae 3d Building Shape Refinement Using Conditional Generative Adversarial Networks

NASA Astrophysics Data System (ADS)

Bittner, K.; d'Angelo, P.; Körner, M.; Reinartz, P.

2018-05-01

Three-dimensional building reconstruction from remote sensing imagery is one of the most difficult and important 3D modeling problems for complex urban environments. The main data sources provided the digital representation of the Earths surface and related natural, cultural, and man-made objects of the urban areas in remote sensing are the digital surface models (DSMs). The DSMs can be obtained either by light detection and ranging (LIDAR), SAR interferometry or from stereo images. Our approach relies on automatic global 3D building shape refinement from stereo DSMs using deep learning techniques. This refinement is necessary as the DSMs, which are extracted from image matching point clouds, suffer from occlusions, outliers, and noise. Though most previous works have shown promising results for building modeling, this topic remains an open research area. We present a new methodology which not only generates images with continuous values representing the elevation models but, at the same time, enhances the 3D object shapes, buildings in our case. Mainly, we train a conditional generative adversarial network (cGAN) to generate accurate LIDAR-like DSM height images from the noisy stereo DSM input. The obtained results demonstrate the strong potential of creating large areas remote sensing depth images where the buildings exhibit better-quality shapes and roof forms.

Building a New Generation of Learning: Conversations to Catalyze Our Construction

ERIC Educational Resources Information Center

Milliron, Mark David; Plinske, Kathleen; Noonan-Terry, Coral

2008-01-01

Rather than focus primarily on the next generation of learners, the authors argue we are best served to focus on building out our on-ground and online infrastructures for a new generation of learning--blending multiple learning modes, technologies, and techniques over the course of the next 15-20 years to serve the diverse array of students from…

Building carbon–carbon bonds using a biocatalytic methanol condensation cycle

PubMed Central

Bogorad, Igor W.; Chen, Chang-Ting; Theisen, Matthew K.; Wu, Tung-Yun; Schlenz, Alicia R.; Lam, Albert T.; Liao, James C.

2014-01-01

Methanol is an important intermediate in the utilization of natural gas for synthesizing other feedstock chemicals. Typically, chemical approaches for building C–C bonds from methanol require high temperature and pressure. Biological conversion of methanol to longer carbon chain compounds is feasible; however, the natural biological pathways for methanol utilization involve carbon dioxide loss or ATP expenditure. Here we demonstrated a biocatalytic pathway, termed the methanol condensation cycle (MCC), by combining the nonoxidative glycolysis with the ribulose monophosphate pathway to convert methanol to higher-chain alcohols or other acetyl-CoA derivatives using enzymatic reactions in a carbon-conserved and ATP-independent system. We investigated the robustness of MCC and identified operational regions. We confirmed that the pathway forms a catalytic cycle through 13C-carbon labeling. With a cell-free system, we demonstrated the conversion of methanol to ethanol or n-butanol. The high carbon efficiency and low operating temperature are attractive for transforming natural gas-derived methanol to longer-chain liquid fuels and other chemical derivatives. PMID:25355907

The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes

Treesearch

Dimitrios Floudas; Manfred Binder; Robert Riely; Kerrie Barry; Robert A. Blanchette; Bernard Henrissat; Angel T. Martínez; Robert Otillar; Joseph W. Spatafora; Jagjit S. Yadav; Andrea Aerts; Isabelle Benoit; Alex Boyd; Alexis Carlson; Alex Copeland; Pedro M. Coutinho; Ronald P. deVries; Patricia Ferreira; Keisha Findley; Brian Foster; Jill Gaskell; Dylan Glotzer; Pawe³ Górecki; Joseph Heitman; Cedar Hesse; Chiaki Hori; Kiyohiko Igarashi; Joel A. Jurgens; Nathan Kallen; Phil Kersten; Annegret Kohler; Ursula Kües; T. K. ArunKumar; Alan Kuo; Kurt LaButti; Luis F. Larrondo; Erika Lindquist; Albee Ling; Vincent Lombard; Susan Lucas; Taina Lundell; Rachael Martin; David J. McLaughlin; Ingo Morgenstern; Emanuelle Morin; Claude Murat; Laszlo G. Nagy; Matt Nolan; Robin A. Ohm; Aleksandrina Patyshakuliyeva; Antonis Rokas; Francisco J. Ruiz-Dueñas; Grzegorz Sabat; Asaf Salamov; Masahiro Samejima; Jeremy Schmutz; Jason C. Slot; Franz St. John; Jan Stenlid; Hui Sun; Sheng Sun; Khajamohiddin Syed; Adrian Tsang; Ad Wiebenga; Darcy Young; Antonio Pisabarro; Daniel C. Eastwood; Francis Martin; Dan Cullen; Igor V. Grigoriev; David S. Hibbett

2012-01-01

Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non–lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study)...

5. MISSILE TEST AND ASSEMBLY BUILDING, FRONT AND RIGHT SIDES, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

5. MISSILE TEST AND ASSEMBLY BUILDING, FRONT AND RIGHT SIDES, LOOKING SOUTHEAST. - NIKE Missile Base SL-40, Missile Test & Assembly Building, South end of launch area, northeast of Generator Building No. 3, Hecker, Monroe County, IL

6. MISSILE TEST AND ASSEMBLY BUILDING, REAR AND LEFT SIDES, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

6. MISSILE TEST AND ASSEMBLY BUILDING, REAR AND LEFT SIDES, LOOKING NORTHWEST. - NIKE Missile Base SL-40, Missile Test & Assembly Building, South end of launch area, northeast of Generator Building No. 3, Hecker, Monroe County, IL

Interdependency of Reactive Oxygen Species generating and scavenging system in salt sensitive and salt tolerant cultivars of rice.

PubMed

Kaur, Navdeep; Dhawan, Manish; Sharma, Isha; Pati, Pratap Kumar

2016-06-10

Salinity stress is a major constrain in the global rice production and hence serious efforts are being undertaken towards deciphering its remedial strategies. The comparative analysis of differential response of salt sensitive and salt tolerant lines is a judicious approach to obtain essential clues towards understanding the acquisition of salinity tolerance in rice plants. However, adaptation to salt stress is a fairly complex process and operates through different mechanisms. Among various mechanisms involved, the reactive oxygen species mediated salinity tolerance is believed to be critical as it evokes cascade of responses related to stress tolerance. In this background, the present paper for the first time evaluates the ROS generating and the scavenging system in tandem in both salt sensitive and salt tolerant cultivars of rice for getting better insight into salinity stress adaptation. Comparative analysis of ROS indicates the higher level of hydrogen peroxide (H2O2) and lower level of superoxide ions (O(2-)) in the salt tolerant as compared to salt sensitive cultivars. Specific activity of ROS generating enzyme, NADPH oxidase was also found to be more in the tolerant cultivars. Further, activities of various enzymes involved in enzymatic and non enzymatic antioxidant defence system were mostly higher in tolerant cultivars. The transcript level analysis of antioxidant enzymes were in alignment with the enzymatic activity. Other stress markers like proline were observed to be higher in tolerant varieties whereas, the level of malondialdehyde (MDA) equivalents and chlorophyll content were estimated to be more in sensitive. The present study showed significant differences in the level of ROS production and antioxidant enzymes activities among sensitive and tolerant cultivars, suggesting their possible role in providing natural salt tolerance to selected cultivars of rice. Our study demonstrates that the cellular machinery for ROS production and scavenging system works in an interdependent manner to offer better salt stress adaptation in rice. The present work further highlights that the elevated level of H2O2 which is considered as a key determinant for conferring salt stress tolerance to rice might have originated through an alternative route of photocatalytic activity of chlorophyll.

Influence of Laccase and Tyrosinase on the Antioxidant Capacity of Selected Phenolic Compounds on Human Cell Lines.

PubMed

Riebel, Matthias; Sabel, Andrea; Claus, Harald; Fronk, Petra; Xia, Ning; Li, Huige; König, Helmut; Decker, Heinz

2015-09-18

Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO), including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been proved in physiological test systems. Furthermore, only a few data exist on the antioxidant capacities of the enzymatic oxidation products of phenolic compounds generated by PPO. We report here first results about the antioxidant effects of phenolic substances, before and after oxidation by fungal model tyrosinase and laccase. In general, the common chemical 2,2-diphenyl-1-picrylhydrazyl assay and the biological tests using two different types of cell cultures (monocytes and endothelial cells) delivered similar results. The phenols tested showed significant differences with respect to their antioxidant activity in all test systems. Their antioxidant capacities after enzymatic conversion decreased or increased depending on the individual PPO used.

Physicochemical structural changes of poplar and switchgrass during biomass pretreatment and enzymatic hydrolysis

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

Meng, Xianzhi; Sun, Qining; Kosa, Matyas

Converting lignocellulosics to simple sugars for second generation bioethanol is complicated due to biomass recalcitrance, and it requires a pretreatment stage prior to enzymatic hydrolysis. In this study, native, pretreated (acid and alkaline) and partially hydrolyzed poplar and switchgrass were characterized by using Simons’ staining for cellulose accessibility, GPC for degree of polymerization (DP), and FTIR for chemical structure of plant cell wall. The susceptibility of the pretreated biomass to enzymatic hydrolysis could not be easily predicted from differences in cellulose DP and accessibility. During hydrolysis, the most significant DP reduction occurred at the very beginning of hydrolysis, and themore » DP began to decrease at a significantly slower rate after this initial period, suggesting an existence of a synergistic action of endo- and exoglucanases that contribute to the occurrence of a “peeling off” mechanism. Cellulose accessibility was found to be increased at the beginning of hydrolysis, after reaching a maximum value then started to decrease. In conclusion, the fresh enzyme restart hydrolysis experiment along with the accessibility data indicated that the factors associated with the nature of enzyme such as irreversible nonspecific binding of cellulases by lignin and steric hindrance of enzymes should be responsible for the gradual slowing down of the reaction rate.« less

Physicochemical structural changes of poplar and switchgrass during biomass pretreatment and enzymatic hydrolysis

DOE PAGES

Meng, Xianzhi; Sun, Qining; Kosa, Matyas; ...

2016-07-27

Converting lignocellulosics to simple sugars for second generation bioethanol is complicated due to biomass recalcitrance, and it requires a pretreatment stage prior to enzymatic hydrolysis. In this study, native, pretreated (acid and alkaline) and partially hydrolyzed poplar and switchgrass were characterized by using Simons’ staining for cellulose accessibility, GPC for degree of polymerization (DP), and FTIR for chemical structure of plant cell wall. The susceptibility of the pretreated biomass to enzymatic hydrolysis could not be easily predicted from differences in cellulose DP and accessibility. During hydrolysis, the most significant DP reduction occurred at the very beginning of hydrolysis, and themore » DP began to decrease at a significantly slower rate after this initial period, suggesting an existence of a synergistic action of endo- and exoglucanases that contribute to the occurrence of a “peeling off” mechanism. Cellulose accessibility was found to be increased at the beginning of hydrolysis, after reaching a maximum value then started to decrease. In conclusion, the fresh enzyme restart hydrolysis experiment along with the accessibility data indicated that the factors associated with the nature of enzyme such as irreversible nonspecific binding of cellulases by lignin and steric hindrance of enzymes should be responsible for the gradual slowing down of the reaction rate.« less

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NASA Technical Reports Server (NTRS)

Chueh, Pin-Ju; Kim, Chinpal; Cho, NaMi; Morre, Dorothy M.; Morre, D. James

2002-01-01

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

Fluorescent pyrimidine ribonucleotide: synthesis, enzymatic incorporation and utilization

PubMed Central

Srivatsan, Seergazhi G.

2008-01-01

Fluorescent nucleobase analogs that respond to changes in their microenvironment are valuable for studying RNA structure, dynamics and recognition. The most commonly used fluorescent ribonucleoside is 2-aminopurine, a highly responsive purine analog. Responsive isosteric fluorescent pyrimidine analogs are, however, rare. Appending 5-membered aromatic heterocycles at the 5-position on a pyrimidine core has recently been found to provide a family of responsive fluorescent nucleoside analogs with emission in the visible range. To explore the potential utility of this chromophore for studying RNA–ligand interactions, an efficient incorporation method is necessary. Here we describe the synthesis of the furan-containing ribonucleoside and its triphosphate, as well as their basic photophysical characteristics. We demonstrate that T7 RNA polymerase accepts this fluorescent ribonucleoside triphosphate as a substrate in in vitro transcription reactions and very efficiently incorporates it into RNA oligonucleotides, generating fluorescent constructs. Furthermore, we utilize this triphosphate for the enzymatic preparation of a fluorescent bacterial A-site, an RNA construct of potential therapeutic utility. We show that the binding of this RNA target to aminoglycoside antibiotics, its cognate ligands, can be effectively monitored by fluorescence spectroscopy. These observations are significant since isosteric emissive U derivatives are scarce and the trivial synthesis and effective enzymatic incorporation of the furan-containing U triphosphate make it accessible to the biophysical community. PMID:17256858

Electrochemical Reduction of Carbon Dioxide to Methanol by Direct Injection of Electrons into Immobilized Enzymes on a Modified Electrode.

PubMed

Schlager, Stefanie; Dumitru, Liviu Mihai; Haberbauer, Marianne; Fuchsbauer, Anita; Neugebauer, Helmut; Hiemetsberger, Daniela; Wagner, Annika; Portenkirchner, Engelbert; Sariciftci, Niyazi Serdar

2016-03-21

We present results for direct bio-electrocatalytic reduction of CO2 to C1 products using electrodes with immobilized enzymes. Enzymatic reduction reactions are well known from biological systems where CO2 is selectively reduced to formate, formaldehyde, or methanol at room temperature and ambient pressure. In the past, the use of such enzymatic reductions for CO2 was limited due to the necessity of a sacrificial co-enzyme, such as nicotinamide adenine dinucleotide (NADH), to supply electrons and the hydrogen equivalent. The method reported here in this paper operates without the co-enzyme NADH by directly injecting electrons from electrodes into immobilized enzymes. We demonstrate the immobilization of formate, formaldehyde, and alcohol dehydrogenases on one-and-the-same electrode for direct CO2 reduction. Carbon felt is used as working electrode material. An alginate-silicate hybrid gel matrix is used for the immobilization of the enzymes on the electrode. Generation of methanol is observed for the six-electron reduction with Faradaic efficiencies of around 10%. This method of immobilization of enzymes on electrodes offers the opportunity for electrochemical application of enzymatic electrodes to many reactions in which a substitution of the expensive sacrificial co-enzyme NADH is desired. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tempol Supplementation Restores Diaphragm Force and Metabolic Enzyme Activities in mdx Mice

PubMed Central

Burns, David P.; Ali, Izza; Rieux, Clement; Healy, James; Jasionek, Greg; O’Halloran, Ken D.

2017-01-01

Duchenne muscular dystrophy (DMD) is characterized by striated muscle weakness, cardiomyopathy, and respiratory failure. Since oxidative stress is recognized as a secondary pathology in DMD, the efficacy of antioxidant intervention, using the superoxide scavenger tempol, was examined on functional and biochemical status of dystrophin-deficient diaphragm muscle. Diaphragm muscle function was assessed, ex vivo, in adult male wild-type and dystrophin-deficient mdx mice, with and without a 14-day antioxidant intervention. The enzymatic activities of muscle citrate synthase, phosphofructokinase, and lactate dehydrogenase were assessed using spectrophotometric assays. Dystrophic diaphragm displayed mechanical dysfunction and altered biochemical status. Chronic tempol supplementation in the drinking water increased diaphragm functional capacity and citrate synthase and lactate dehydrogenase enzymatic activities, restoring all values to wild-type levels. Chronic supplementation with tempol recovers force-generating capacity and metabolic enzyme activity in mdx diaphragm. These findings may have relevance in the search for therapeutic strategies in neuromuscular disease. PMID:29210997

Activation of dioxygen by copper metalloproteins and insights from model complexes

PubMed Central

Quist, David A.; Diaz, Daniel E.; Liu, Jeffrey J.; Karlin, Kenneth D.

2017-01-01

Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing met-alloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O2-activation in copper proteins are addressed. PMID:27921179

Biological Fuel Cells and Membranes.

PubMed

Ghassemi, Zahra; Slaughter, Gymama

2017-01-17

Biofuel cells have been widely used to generate bioelectricity. Early biofuel cells employ a semi-permeable membrane to separate the anodic and cathodic compartments. The impact of different membrane materials and compositions has also been explored. Some membrane materials are employed strictly as membrane separators, while some have gained significant attention in the immobilization of enzymes or microorganisms within or behind the membrane at the electrode surface. The membrane material affects the transfer rate of the chemical species (e.g., fuel, oxygen molecules, and products) involved in the chemical reaction, which in turn has an impact on the performance of the biofuel cell. For enzymatic biofuel cells, Nafion, modified Nafion, and chitosan membranes have been used widely and continue to hold great promise in the long-term stability of enzymes and microorganisms encapsulated within them. This article provides a review of the most widely used membrane materials in the development of enzymatic and microbial biofuel cells.

Fungal biodegradation and enzymatic modification of lignin

PubMed Central

Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

2010-01-01

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed. PMID:21968746

The potential value of the seaweed Ceylon moss (Gelidium amansii) as an alternative bioenergy resource.

PubMed

Wi, Seung Gon; Kim, Hyun Joo; Mahadevan, Shobana Arumugam; Yang, Duck-Joo; Bae, Hyeun-Jong

2009-12-01

Sea weed (Ceylon moss) possesses comparable bioenergy production potential to that of land plants. Ceylon moss has high content of carbohydrates, typically galactose (23%) and glucose (20%). We have explored the possibility of sodium chlorite in Ceylon moss pretreatment that can ultimately increase the efficiency of enzymatic saccharification. In an acidic medium, chlorite generates ClO(2) molecules that transform lignin into soluble compounds without any significant loss of carbohydrate content and this procedure is widely used as an analytical method for holocellulose determination. Sodium chlorite-pretreated samples resulted in glucose yield up to 70% with contrast of only 5% was obtained from non-pretreated samples. The efficiency of enzymatic hydrolysis is significantly improved by sodium chlorite pretreatment, and thus sodium chlorite pretreatment is potentially a very useful tool in the utilisation of Ceylon moss biomass for ethanol production or bioenergy purposes.

Isolation and chemoenzymatic treatment of glycoalkaloids from green, sprouting and rotting Solanum tuberosum potatoes for solanidine recovery.

PubMed

Koffi, Grokoré Yvonne; Remaud-Simeon, Magali; Due, Ahipo Edmond; Combes, Didier

2017-04-01

The estimation of glycoalkaloids in the flesh of different types of decayed potatoes was evaluated. The results showed that turned green and also sprouting or rotting potato flesh contain high amounts of toxic solanine and chaconine, exceeding by 2-5-fold the recommended limit, and ranging from 2578±86mg/kg to 5063±230mg/kg of dry weight potato flesh. For safety consideration, these decayed potatoes should be systematically set aside. To avoid a net economic loss and encourage the removal of this hazardous food, a recycling process was investigated to generate added-value compounds from the toxic glycoalkaloids. A simple chemo-enzymatic protocol comprising a partial acidic hydrolysis followed by an enzymatic treatment with the β-glycosidase from Periplaneta americana allowed the efficient conversion of α-chaconine to solanidine. Copyright © 2016 Elsevier Ltd. All rights reserved.

Integrating the protein and metabolic engineering toolkits for next-generation chemical biosynthesis.

PubMed

Pirie, Christopher M; De Mey, Marjan; Jones Prather, Kristala L; Ajikumar, Parayil Kumaran

2013-04-19

Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.

Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants.

PubMed

Kühnel, Lisa C; Nestl, Bettina M; Hauer, Bernhard

2017-11-16

Squalene-hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl-farnesyl ether product from farnesol. Furthermore, the addition of small-chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and, thus, to significant alteration of the product pattern relative to that obtained with the wild type. It is proposed that the mutation of leucine at position 607 may facilitate premature quenching of the intermediate by small alcohol nucleophiles. This mutagenesis-based study opens the field for further intermolecular bond-forming reactions and the generation of non-natural products. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modification of enzymes by use of high-pressure homogenization.

PubMed

Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

2018-07-01

High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biological Fuel Cells and Membranes

PubMed Central

Ghassemi, Zahra; Slaughter, Gymama

2017-01-01

Biofuel cells have been widely used to generate bioelectricity. Early biofuel cells employ a semi-permeable membrane to separate the anodic and cathodic compartments. The impact of different membrane materials and compositions has also been explored. Some membrane materials are employed strictly as membrane separators, while some have gained significant attention in the immobilization of enzymes or microorganisms within or behind the membrane at the electrode surface. The membrane material affects the transfer rate of the chemical species (e.g., fuel, oxygen molecules, and products) involved in the chemical reaction, which in turn has an impact on the performance of the biofuel cell. For enzymatic biofuel cells, Nafion, modified Nafion, and chitosan membranes have been used widely and continue to hold great promise in the long-term stability of enzymes and microorganisms encapsulated within them. This article provides a review of the most widely used membrane materials in the development of enzymatic and microbial biofuel cells. PMID:28106711

Activation of dioxygen by copper metalloproteins and insights from model complexes.

PubMed

Quist, David A; Diaz, Daniel E; Liu, Jeffrey J; Karlin, Kenneth D

2017-04-01

Nature uses dioxygen as a key oxidant in the transformation of biomolecules. Among the enzymes that are utilized for these reactions are copper-containing metalloenzymes, which are responsible for important biological functions such as the regulation of neurotransmitters, dioxygen transport, and cellular respiration. Enzymatic and model system studies work in tandem in order to gain an understanding of the fundamental reductive activation of dioxygen by copper complexes. This review covers the most recent advancements in the structures, spectroscopy, and reaction mechanisms for dioxygen-activating copper proteins and relevant synthetic models thereof. An emphasis has also been placed on cofactor biogenesis, a fundamentally important process whereby biomolecules are post-translationally modified by the pro-enzyme active site to generate cofactors which are essential for the catalytic enzymatic reaction. Significant questions remaining in copper-ion-mediated O 2 -activation in copper proteins are addressed.

Design of an embedded inverse-feedforward biomolecular tracking controller for enzymatic reaction processes.

PubMed

Foo, Mathias; Kim, Jongrae; Sawlekar, Rucha; Bates, Declan G

2017-04-06

Feedback control is widely used in chemical engineering to improve the performance and robustness of chemical processes. Feedback controllers require a 'subtractor' that is able to compute the error between the process output and the reference signal. In the case of embedded biomolecular control circuits, subtractors designed using standard chemical reaction network theory can only realise one-sided subtraction, rendering standard controller design approaches inadequate. Here, we show how a biomolecular controller that allows tracking of required changes in the outputs of enzymatic reaction processes can be designed and implemented within the framework of chemical reaction network theory. The controller architecture employs an inversion-based feedforward controller that compensates for the limitations of the one-sided subtractor that generates the error signals for a feedback controller. The proposed approach requires significantly fewer chemical reactions to implement than alternative designs, and should have wide applicability throughout the fields of synthetic biology and biological engineering.

Non-enzymatic cyclic oxygenated metabolites of adrenic, docosahexaenoic, eicosapentaenoic and α-linolenic acids; bioactivities and potential use as biomarkers.

PubMed

Galano, Jean-Marie; Lee, Jetty Chung-Yung; Gladine, Cecile; Comte, Blandine; Le Guennec, Jean-Yves; Oger, Camille; Durand, Thierry

2015-04-01

Cyclic oxygenated metabolites are formed in vivo through non-enzymatic free radical reaction of n-6 and n-3 polyunsaturated fatty acids (PUFAs) such as arachidonic (ARA C20:4 n-6), adrenic (AdA 22:4 n-6), α-linolenic (ALA 18:3 n-3), eicosapentaenoic (EPA 20:5 n-3) and docosahexaenoic (DHA 22:6 n-3) acids. These cyclic compounds are known as isoprostanes, neuroprostanes, dihomo-isoprostanes and phytoprostanes. Evidence has emerged for their use as biomarkers of oxidative stress and, more recently, the n-3PUFA-derived compounds have been shown to mediate bioactivities as secondary messengers. Accordingly, this review will focus on the cyclic oxygenated metabolites generated from AdA, ALA, EPA and DHA. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance". Copyright © 2014 Elsevier B.V. All rights reserved.

Luciferase Protein Complementation Assays for Bioluminescence Imaging of Cells and Mice

PubMed Central

Luker, Gary D.; Luker, Kathryn E.

2015-01-01

Summary Protein fragment complementation assays (PCAs) with luciferase reporters currently are the preferred method for detecting and quantifying protein-protein interactions in living animals. At the most basic level, PCAs involve fusion of two proteins of interest to enzymatically inactive fragments of luciferase. Upon association of the proteins of interest, the luciferase fragments are capable of reconstituting enzymatic activity to generate luminescence in vivo. In addition to bi-molecular luciferase PCAs, unimolecular biosensors for hormones, kinases, and proteases also have been developed using target peptides inserted between inactive luciferase fragments. Luciferase PCAs offer unprecedented opportunities to quantify dynamics of protein-protein interactions in intact cells and living animals, but successful use of luciferase PCAs in cells and mice involves careful consideration of many technical factors. This chapter discusses the design of luciferase PCAs appropriate for animal imaging, including construction of reporters, incorporation of reporters into cells and mice, imaging techniques, and data analysis. PMID:21153371

A Library of Rad Hard Mixed-Voltage/Mixed-Signal Building Blocks for Integration of Avionics Systems for Deep Space

NASA Technical Reports Server (NTRS)

Mojarradi, M. M.; Blaes, B.; Kolawa, E. A.; Blalock, B. J.; Li, H. W.; Buck, K.; Houge, D.

2001-01-01

To build the sensor intensive system-on-a-chip for the next generation spacecrafts for deep space, Center for Integration of Space Microsystems at JPL (CISM) takes advantage of the lower power rating and inherent radiation resistance of Silicon on Insulator technology (SOI). We are developing a suite of mixed-voltage and mixed-signal building blocks in Honeywell's SOI process that can enable the rapid integration of the next generation avionics systems with lower power rating, higher reliability, longer life, and enhanced radiation tolerance for spacecrafts such as the Europa Orbiter and Europa Lander. The mixed-voltage building blocks are predominantly for design of adaptive power management systems. Their design centers around an LDMOS structure that is being developed by Honeywell, Boeing Corp, and the University of Idaho. The mixed-signal building blocks are designed to meet the low power, extreme radiation requirement of deep space applications. These building blocks are predominantly used to interface analog sensors to the digital CPU of the next generation avionics system on a chip. Additional information is contained in the original extended abstract.

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy

PubMed Central

Kottom, Theodore J.; Hebrink, Deanne M.; Jenson, Paige E.; Ramirez-Prado, Jorge H.

2017-01-01

N-acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)–GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP-GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP-GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc-binding recombinant protein and a novel GlcNAc-binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using high-performance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP-GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms. PMID:27632412

Caribbean yellow band disease compromises the activity of catalase and glutathione S-transferase in the reef-building coral Orbicella faveolata exposed to anthracene.

PubMed

Montilla, Luis Miguel; Ramos, Ruth; García, Elia; Cróquer, Aldo

2016-05-03

Healthy and diseased corals are threatened by different anthropogenic sources, such as pollution, a problem expected to become more severe in the near future. Despite the fact that coastal pollution and coral diseases might represent a serious threat to coral reef health, there is a paucity of controlled experiments showing whether the response of diseased and healthy corals to xenobiotics differs. In this study, we exposed healthy and Caribbean yellow band disease (CYBD)-affected Orbicella faveolata colonies to 3 sublethal concentrations of anthracene to test if enzymatic responses to this hydrocarbon were compromised in CYBD-affected tissues. For this, a 2-factorial fully orthogonal design was used in a controlled laboratory bioassay, using tissue condition (2 levels: apparently healthy and diseased) and pollutant concentration (4 levels: experimental control, 10, 30 and 100 ppb concentration) as fixed factors. A permutation-based ANOVA (PERMANOVA) was used to test the effects of condition and concentration on the specific activity of 3 enzymatic biomarkers: catalase, glutathione S-transferase, and glutathione peroxidase. We found a significant interaction between the concentration of anthracene and the colony condition for catalase (Pseudo-F = 3.84, df = 3, p < 0.05) and glutathione S-transferase (Pseudo-F = 3.29, df = 3, p < 0.05). Moreover, our results indicated that the enzymatic response to anthracene in CYBD-affected tissues was compromised, as the activity of these enzymes decreased 3- to 4-fold compared to healthy tissues. These results suggest that under a potential scenario of increasing hydrocarbon coastal pollution, colonies of O. faveolata affected with CYBD might become more vulnerable to the deleterious effects of chemical pollution.

Combined Heat & Power Using the Infinia Concentrated Solar CHP PowerDish System

DTIC Science & Technology

2013-08-01

been due to a temperature induced reduction in magnetic field strength (inside the generator) that in turn can cause a generator over-stroke which can... Measurements and Sensors 34 Table 4: Results Summary Performance Objectives (same as Table 2) 38 Table 5: Building 9246 Energy Consumption vs...and Building Measurement Instrumentation P&ID 28 Figure 12: Building 9246 Radiant Heating and Hot Water Equipment 29 Figure 13: Coolant Flow

Dietary omega-3 but not omega-6 fatty acids down-regulate maternal dyslipidemia induced oxidative stress: A three generation study in rats.

PubMed

Ramaiyan, Breetha; Bettadahalli, Sadashivaiah; Talahalli, Ramaprasad Ravichandra

2016-09-02

Maternal nutrition modulates fetal metabolic programming and development later. Maternal dyslipidemia effects on oxidative stress (OS) in offsprings and its modulation by dietary fatty acids over generations remains to be elucidated. The objective of present study was to assess the long-term (three generations) effect of omega-3 fatty acids on OS under dyslipidemia. Weanling female Wistar rats were fed with control diet (7% lard), high fat diet (35% lard, HFL), high fat with fish oil (21% fish oil + 14% lard, HFF), high fat with canola oil (21% canola oil + 14% lard, HFC) and high fat with sunflower oil (21% sunflower oil + 14% lard, HFS). Following 60 days feeding, the female rats were mated with sexually matured males (fed normal chow diet) and continued with the above diet regimen during pregnancy and lactation. The pups after lactation were continued with their maternal diet for 60 days and subjected to mating and feeding trial as above for two generations. Serum lipid profiles, OS markers (lipid peroxidation, nitric oxide release and protein carbonyl) and antioxidant defence enzymes (catalase, SOD, glutathione peroxidase and glutathione transferase) were assessed in serum, liver and uterus of rats fed on experimental and control diets for three generations. Feeding HFL diet increased blood lipids, OS and lowered the antioxidant enzymes activity in serum, liver and uterus (p < 0.05). The reduction in the antioxidant enzymes in HFL group were higher in third followed by second generation compared to first generation (p < 0.05). Omega-3 fatty acids prevented the dyslipidemia induced loss of antioxidant enzyme activities in serum, liver and uterus. Our data show for the first time that offsprings born to dyslipidemic mothers' exhibit diminished enzymatic antioxidant defence and its progressive reduction in future generation, and dietary omega-3 fatty acids restore the enzymatic antioxidant defence in offsprings and suppress the markers of OS. Copyright © 2016 Elsevier Inc. All rights reserved.

Cold enzymatic bleaching of fluid whey.

PubMed

Campbell, R E; Drake, M A

2013-01-01

Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically bleached WPC80, lactoperoxidase-bleached WPC80 contained higher relative abundance of 2,3-octadienone, 2-pentyl furan, and hexanal than those bleached with added EP. Bleach times, bleaching efficacy, and flavor results suggest that enzymatic bleaching may be a viable and desirable alternative to HP bleaching of fluid whey or retentate. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

a New Mask for Automatic Building Detection from High Density Point Cloud Data and Multispectral Imagery

NASA Astrophysics Data System (ADS)

Awrangjeb, M.; Siddiqui, F. U.

2017-11-01

In complex urban and residential areas, there are buildings which are not only connected with and/or close to one another but also partially occluded by their surrounding vegetation. Moreover, there may be buildings whose roofs are made of transparent materials. In transparent buildings, there are point returns from both the ground (or materials inside the buildings) and the rooftop. These issues confuse the previously proposed building masks which are generated from either ground points or non-ground points. The normalised digital surface model (nDSM) is generated from the non-ground points and usually it is hard to find individual buildings and trees using the nDSM. In contrast, the primary building mask is produced using the ground points, thereby it misses the transparent rooftops. This paper proposes a new building mask based on the non-ground points. The dominant directions of non-ground lines extracted from the multispectral imagery are estimated. A dummy grid with the target mask resolution is rotated at each dominant direction to obtain the corresponding height values from the non-ground points. Three sub-masks are then generated from the height grid by estimating the gradient function. Two of these sub-masks capture planar surfaces whose height remain constant in along and across the dominant direction, respectively. The third sub-mask contains only the flat surfaces where the height (ideally) remains constant in all directions. All the sub-masks generated in all estimated dominant directions are combined to produce the candidate building mask. Although the application of the gradient function helps in removal of most of the vegetation, the final building mask is obtained through removal of planar vegetation, if any, and tiny isolated false candidates. Experimental results on three Australian data sets show that the proposed method can successfully remove vegetation, thereby separate buildings from occluding vegetation and detect buildings with transparent roof materials. While compared to existing building detection techniques, the proposed technique offers higher objectbased completeness, correctness and quality, specially in complex scenes with aforementioned issues. It is not only capable of detecting transparent buildings, but also small garden sheds which are sometimes as small as 5 m2 in area.

Finding New Water: Development of On-Site Non-Potable Water Reuse Systems

EPA Science Inventory

By designing our buildings to collect and treat water generated on-site, can be and reused for flushing our toilets and irrigating our landscaping. Several water sources are generated with-in a building including: rainwater, stormwater, graywater, blackwater and foundation drain...

Energy Efficient Building Management | Climate Neutral Research Campuses |

Science.gov Websites

NREL Efficient Building Management Energy Efficient Building Management As campuses complete generate the greatest climate impact. Energy efficient management in the existing stock of buildings is the following links go to sections that describe how an energy buildings management and maintenance program may

RCRA, superfund and EPCRA hotline training module. Introduction to: Containment buildings (40 cfr parts 264/265, subpart dd) updated July 1996

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

NONE

1996-07-01

The module outlines the regulatory history and purpose of containment buildings. It disucsses the relationship between LDR and containment buildings. It summarizes the design and operating standard applicable to containment buildings and describes the relationship between generator accumulation standards and containment buildings.

Effect of Gestational Intake of Fisetin (3,3',4',7-Tetrahydroxyflavone) on Developmental Methyl Mercury Neurotoxicity in F1 Generation Rats.

PubMed

Jacob, Sherin; Thangarajan, Sumathi

2017-06-01

Methyl mercury (MeHg) is a developmental neurotoxin that causes irreversible cognitive damage in offspring of gestationally exposed mothers. Currently, no preventive drugs are established against MeHg developmental neurotoxicity. The neuroprotective effect of gestational administration of a flavanoid against in utero toxicity of MeHg is not explored much. Hence, the present study validated the effect of a bioactive flavanoid, fisetin, on MeHg developmental neurotoxicity outcomes in rat offspring at postnatal weaning age. Pregnant Wistar rats were simultaneously given MeHg (1.5 mg/kg b.w.) and two doses of fisetin (10 and 50 mg/kg b.w. in two separate groups) orally from gestational day (GD) 5 till parturition. Accordingly, after parturition, on postnatal day (PND) 24, weaning F 1 generation rats were studied for motor and cognitive behavioural changes. Biochemical and histopathological changes were also studied in the cerebral cortex, cerebellum and hippocampus on PND 25. Administration of fisetin during pregnancy prevented behavioural impairment due to transplacental MeHg exposure in weaning rats. Fisetin decreased the levels of oxidative stress markers, increased enzymatic and non-enzymatic antioxidant levels and increased the activity of membrane-bound ATPases and cholinergic function in F 1 generation rats. In light microscopic studies, fisetin treatment protected the specific offspring brain regions from significant morphological aberrations. Between the two doses of fisetin studied, 10 mg/kg b.w. was found to be more satisfactory and effective than 50 mg/kg b.w. The present study shows that intake of fisetin during pregnancy in rats ameliorated in utero MeHg exposure-induced neurotoxicity outcomes in postnatal weaning F 1 generation rats.

A bacterial model for expression of mutations in the human ornithine transcarbamylase (OTC) gene

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

Tuchman, M.; McCann, M.T.; Qureshi, A.A.

1994-09-01

OTC is a mitochondrial enzyme catalyzing the formation of citrulline from carbamyl phosphate and ornithine. X-linked deficiency of OTC is the most prevalent genetic defect of ureagenesis. Mutations and polymorphisms in the OTC gene identified in deficient patients have indicated the occurrence of many family-specific, unique alleles. Due to the low frequency of recurrent mutations, distinguishing between deleterious mutations and polymorphisms is difficult. Using a human OTC gene containing plasmid driven by a tac promoter, we have devised a simple and efficient method for expressing mutations in the mature human OTC enzyme. To demonstrate this method, PCR engineered site-directed mutagenesismore » was employed to generated cDNA fragments which contained either the R151Q or R277W known mutations found in patients with neonatal and late-onset OTC deficiency, respectively. The normal allele for each mutation was also generated by an identical PCR procedure. Digestion with Bgl II- and Sty I-generated mutant and normal replacement cassettes containing the respective mutant and wild type sequences. Upon transformation of JM109 E.coli cells, OTC enzymatic activity was measured at log and stationary phases of growth using a radiochromatographic method. The R141Q mutation abolished enzymatic activity (<0.02% of normal), whereas the R277W mutation expressed partial activity (2.3% of normal). In addition, a PCR-generated mutation, A280V, resulted in 73% loss of catalytic activity. This OTC expression system is clinically applicable for distinguishing between mutations and polymorphisms, and it can be used to investigate the effects of mutations on various domains of the OTC gene.« less

Crystallographic and mutational analyses of cystathionine β-synthase in the H2 S-synthetic gene cluster in Lactobacillus plantarum.

PubMed

Matoba, Yasuyuki; Yoshida, Tomoki; Izuhara-Kihara, Hisae; Noda, Masafumi; Sugiyama, Masanori

2017-04-01

Cystathionine β-synthase (CBS) catalyzes the formation of l-cystathionine from l-serine and l-homocysteine. The resulting l-cystathionine is decomposed into l-cysteine, ammonia, and α-ketobutylic acid by cystathionine γ-lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H 2 S). In some bacteria, including the plant-derived lactic acid bacterium Lactobacillus plantarum, the CBS- and CGL-encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H 2 S production in bacteria; interestingly, it has been shown that H 2 S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O-acetyl-l-serine sulfhydrylase (OASS) that catalyzes the generation of l-cysteine from O-acetyl-l-serine (l-OAS) and H 2 S. The L. plantarum CBS shows l-OAS- and l-cysteine-dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H 2 S in the presence of l-cysteine and l-homocysteine, together with the formation of l-cystathionine. The high affinity toward l-cysteine as a first substrate and tendency to use l-homocysteine as a second substrate might be associated with its enzymatic ability to generate H 2 S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H 2 S-generating activity. © 2017 The Protein Society.

The sugar code: Why glycans are so important.

PubMed

Gabius, Hans-Joachim

2018-02-01

The cell surface is the platform for presentation of biochemical signals that are required for intercellular communication. Their profile necessarily needs to be responsive to internal and external factors in a highly dynamic manner. The structural features of the signals must meet the criterion of high-density information coding in a minimum of space. Thus, only biomolecules that can generate many different oligomers ('words') from few building blocks ('letters') qualify to meet this challenge. Examining the respective properties of common biocompounds that form natural oligo- and polymers comparatively, starting with nucleotides and amino acids (the first and second alphabets of life), comes up with sugars as clear frontrunner. The enzymatic machinery for the biosynthesis of sugar chains can indeed link monosaccharides, the letters of the third alphabet of life, in a manner to reach an unsurpassed number of oligomers (complex carbohydrates or glycans). Fittingly, the resulting glycome of a cell can be likened to a fingerprint. Conjugates of glycans with proteins and sphingolipids (glycoproteins and glycolipids) are ubiquitous in Nature. This implies a broad (patho)physiologic significance. By looking at the signals, at the writers and the erasers of this information as well as its readers and ensuing consequences, this review intends to introduce a broad readership to the principles of the concept of the sugar code. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of body temperature on chondroitinase ABC's ability to cleave chondroitin sulfate glycosaminoglycans.

PubMed

Tester, Nicole J; Plaas, Anna H; Howland, Dena R

2007-04-01

Chondroitinase ABC (Ch'ase ABC) is a bacterial lyase that degrades chondroitin sulfate (CS), dermatan sulfate, and hyaluronan glycosaminoglycans (GAGs). This enzyme has received significant attention as a potential therapy for promoting central nervous system and peripheral nervous system repair based on its degradation of CS GAGs. Determination of the stability of Ch'ase ABC activity at temperatures equivalent to normal (37 degrees C) and elevated (39 degrees C) body temperatures is important for optimizing its clinical usage. We report here data obtained from examining enzymatic activity at these temperatures across nine lots of commercially available protease-free Ch'ase ABC. CS GAG degrading activity was assayed by using 1) immunohistochemical detection of unsaturated disaccharide stubs generated by digestion of proteoglycans in tissue sections and 2) fluorophore-assisted carbohydrate electrophoresis (FACE) and/or high-performance liquid chromatography (HPLC) to separate and quantify unsaturated disaccharide digestion products. Our results indicate that there is a significant effect of lot and time on enzymatic thermostability. Average enzymatic activity is significantly decreased at 1 and 3 days at 39 degrees C and 37 degrees C, respectively. Furthermore, the average activity seen after 1 day was significantly different between the two temperatures. Addition of bovine serum albumin as a stabilizer significantly preserved enzymatic activity at 1 day, but not 3 days, at 39 degrees C. These results show that the CS GAG degrading activity of Ch'ase ABC is significantly decreased with incubation at body temperature over time and that all lots do not show equal thermostability. These findings are important for the design and interpretation of experimental and potential clinical studies involving Ch'ase ABC. (c) 2007 Wiley-Liss, Inc.

Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3*

PubMed Central

Porté, Sergio; Valencia, Eva; Yakovtseva, Evgenia A.; Borràs, Emma; Shafqat, Naeem; Debreczeny, Judit É.; Pike, Ashley C. W.; Oppermann, Udo; Farrés, Jaume; Fita, Ignacio; Parés, Xavier

2009-01-01

Tumor suppressor p53 regulates the expression of p53-induced genes (PIG) that trigger apoptosis. PIG3 or TP53I3 is the only known member of the medium chain dehydrogenase/reductase superfamily induced by p53 and is used as a proapoptotic marker. Although the participation of PIG3 in the apoptotic pathway is proven, the protein and its mechanism of action were never characterized. We analyzed human PIG3 enzymatic function and found NADPH-dependent reductase activity with ortho-quinones, which is consistent with the classification of PIG3 in the quinone oxidoreductase family. However, the activity is much lower than that of ζ-crystallin, a better known quinone oxidoreductase. In addition, we report the crystallographic structure of PIG3, which allowed the identification of substrate- and cofactor-binding sites, with residues fully conserved from bacteria to human. Tyr-59 in ζ-crystallin (Tyr-51 in PIG3) was suggested to participate in the catalysis of quinone reduction. However, kinetics of Tyr/Phe and Tyr/Ala mutants of both enzymes demonstrated that the active site Tyr is not catalytic but may participate in substrate binding, consistent with a mechanism based on propinquity effects. It has been proposed that PIG3 contribution to apoptosis would be through oxidative stress generation. We found that in vitro activity and in vivo overexpression of PIG3 accumulate reactive oxygen species. Accordingly, an inactive PIG3 mutant (S151V) did not produce reactive oxygen species in cells, indicating that enzymatically active protein is necessary for this function. This supports that PIG3 action is through oxidative stress produced by its enzymatic activity and provides essential knowledge for eventual control of apoptosis. PMID:19349281

Enzymatic hydrolysis of potato pulp.

PubMed

Lesiecki, Mariusz; Białas, Wojciech; Lewandowicz, Grażyna

2012-01-01

Potato pulp constitutes a complicated system of four types of polysaccharides: cellulose, hemicellulose, pectin and starch. Its composition makes it a potential and attractive raw material for the production of the second generation bioethanol. The aim of this research project was to assess the usefulness of commercial enzymatic preparations for the hydrolysis of potato pulp and to evaluate the effectiveness of hydrolysates obtained in this way as raw materials for ethanol fermentation. Sterilised potato pulp was subjected to hydrolysis with commercial enzymatic preparations. The effectiveness of the preparations declared as active towards only one fraction of potato pulp (separate amylase, pectinase and cellulase activity) and mixtures of these preparations was analysed. The monomers content in hydrolysates was determined using HPLC method. The application of amylolytic enzymes for potato pulp hydrolysis resulted in the release of only 18% of raw material with glucose as the dominant (77%) constituent of the formed product. In addition, 16% galactose was also determined in it. The hydrolysis of the cellulose fraction yielded up to 35% raw material and the main constituents of the obtained hydrolysate were glucose (46%) and arabinose (40%). Simultaneous application of amylolytic, cellulolytic and pectinolytic enzymes turned out to be the most effective way of carrying out the process as its efficiency in this case reached 90%. The obtained hydrolysate contained 63% glucose, 25% arabinose and 12% other simple substances. The application of commercial enzymatic preparations made it possible to perform potato pulp hydrolysis with 90% effectiveness. This was achieved by the application of a complex of amylolytic, cellulolytic and pectinolytic enzymes and the hydrolysate obtained in this way contained, primarily, glucose making it a viable substrate for ethanol fermentation.

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

DOE PAGES

Healey, Adam L.; Lee, David J.; Lupoi, Jason S.; ...

2016-11-18

In order for a lignocellulosic bioenergy feedstock to be considered sustainable,it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application,it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin,glucan,and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) amongmore » parental and hybrid populations,whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age,with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase),and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%,respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass,with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass,respectively),with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate,biomass composition,and further optimization of enzymatic saccharification yield,high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.« less

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

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

Healey, Adam L.; Lee, David J.; Lupoi, Jason S.

In order for a lignocellulosic bioenergy feedstock to be considered sustainable,it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application,it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin,glucan,and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) amongmore » parental and hybrid populations,whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age,with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase),and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%,respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass,with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass,respectively),with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate,biomass composition,and further optimization of enzymatic saccharification yield,high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.« less

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa.

PubMed

Healey, Adam L; Lee, David J; Lupoi, Jason S; Papa, Gabriella; Guenther, Joel M; Corno, Luca; Adani, Fabrizio; Singh, Seema; Simmons, Blake A; Henry, Robert J

2016-01-01

In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application, it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin, glucan, and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7-21.3%) among parental and hybrid populations, whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28-38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age, with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%, respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass, respectively), with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate, biomass composition, and further optimization of enzymatic saccharification yield, high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production.

Evaluation of Relationships between Growth Rate, Tree Size, Lignocellulose Composition, and Enzymatic Saccharification in Interspecific Corymbia Hybrids and Parental Taxa

PubMed Central

Healey, Adam L.; Lee, David J.; Lupoi, Jason S.; Papa, Gabriella; Guenther, Joel M.; Corno, Luca; Adani, Fabrizio; Singh, Seema; Simmons, Blake A.; Henry, Robert J.

2016-01-01

In order for a lignocellulosic bioenergy feedstock to be considered sustainable, it must possess a high rate of growth to supply biomass for conversion. Despite the desirability of a fast growth rate for industrial application, it is unclear what effect growth rate has on biomass composition or saccharification. We characterized Klason lignin, glucan, and xylan content with response to growth in Corymbia interspecific F1 hybrid families (HF) and parental species Corymbia torelliana and C. citriodora subspecies variegata and measured the effects on enzymatic hydrolysis from hydrothermally pretreated biomass. Analysis of biomass composition within Corymbia populations found similar amounts of Klason lignin content (19.7–21.3%) among parental and hybrid populations, whereas glucan content was clearly distinguished within C. citriodora subspecies variegata (52%) and HF148 (60%) as compared to other populations (28–38%). Multiple linear regression indicates that biomass composition is significantly impacted by tree size measured at the same age, with Klason lignin content increasing with diameter breast height (DBH) (+0.12% per cm DBH increase), and glucan and xylan typically decreasing per DBH cm increase (-0.7 and -0.3%, respectively). Polysaccharide content within C. citriodora subspecies variegata and HF-148 were not significantly affected by tree size. High-throughput enzymatic saccharification of hydrothermally pretreated biomass found significant differences among Corymbia populations for total glucose production from biomass, with parental Corymbia torelliana and hybrids HF-148 and HF-51 generating the highest amounts of glucose (~180 mg/g biomass, respectively), with HF-51 undergoing the most efficient glucan-to-glucose conversion (74%). Based on growth rate, biomass composition, and further optimization of enzymatic saccharification yield, high production Corymbia hybrid trees are potentially suitable for fast-rotation bioenergy or biomaterial production. PMID:27917179

Fluorinase: a tool for the synthesis of ¹⁸F-labeled sugars and nucleosides for PET.

PubMed

Onega, Mayca; Winkler, Margit; O'Hagan, David

2009-08-01

There is an increasing interest in the preparation of (18)F-labeled radiopharmaceuticals with potential applications in PET for medicinal imaging. Appropriate synthetic methods require a quick and efficient route in which to incorporate the (18)F into a ligand, due to the relatively short half-life of the (18)F isotope. Enzymatic methods are rare in this area; however, the discovery of a fluorinating enzyme from Streptomyces cattleya (EC 2.5.1.63) has opened up the possibility of the enzymatic synthesis and formation of C-(18)F bonds from the [(18)F]fluoride ion. In this article, the development of enzymatic preparations of (18)F-labeled sugars and nucleosides as potential radiotracers using the fluorinase from S. cattleya for PET applications is reviewed. Enzymatic reactions are not traditional in PET synthesis, but this enzyme has some attractive features. The enzyme is available in an overexpressed form from Escherichia coli and it is relatively stable and can be easily purified and manipulated. Most notably, it utilizes [(18)F] fluoride, the form of the isotope normally generated by the cyclotron and usually in very high specific radioactivity. The disadvantage with the enzyme is that it is substrate specific; however, when the fluorinase is used in combination biotransformations with a second or third enzyme, then a range of radiolabeled nucleosides and ribose sugars can be prepared. The fluorinase enzyme has emerged as a curiosity from biosynthesis studies, but it now has some potential as a new catalyst for (18)F incorporation for PET syntheses. The focus is now on delivering a user-friendly catalyst to the PET synthesis community and establishing a clinical role for some of the (18)F-labeled molecules available using this technology.

The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

PubMed

Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

2013-08-01

The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Liberation of fermentable sugars from soybean hull biomass using ionic liquid 1-butyl-3-methylimidazolium acetate and their bioconversion to ethanol.

PubMed

da Cunha-Pereira, Fernanda; Rech, Rosane; Záchia Ayub, Marco Antônio; Pinheiro Dillon, Aldo; Dupont, Jairton

2016-03-01

Optimized hydrolysis of lignocellulosic waste biomass is essential to achieve the liberation of sugars to be used in fermentation process. Ionic liquids (ILs), a new class of solvents, have been tested in the pretreatment of cellulosic materials to improve the subsequent enzymatic hydrolysis of the biomass. Optimized application of ILs on biomass is important to advance the use of this technology. In this research, we investigated the effects of using 1-butyl-3-methylimidazolium acetate ([bmim][Ac]) on the decomposition of soybean hull, an abundant cellulosic industrial waste. Reaction aspects of temperature, incubation time, IL concentration, and solid load were optimized before carrying out the enzymatic hydrolysis of this residue to liberate fermentable glucose. Optimal conditions were found to be 75°C, 165 min incubation time, 57% (mass fraction) of [bmim][Ac], and 12.5% solid loading. Pretreated soybean hull lost its crystallinity, which eased enzymatic hydrolysis, confirmed by Fourier Transform Infrared analysis. The enzymatic hydrolysis of the biomass using an enzyme complex from Penicillium echinulatum liberated 92% of glucose from the cellulose matrix. The hydrolysate was free of any toxic compounds, such as hydroxymethylfurfural and furfural. The obtained hydrolysate was tested for fermentation using Candida shehatae HM 52.2, which was able to convert glucose to ethanol at yields of 0.31. These results suggest the possible use of ILs for the pretreatment of some lignocellulosic waste materials, avoiding the formation of toxic compounds, to be used in second-generation ethanol production and other fermentation processes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:312-320, 2016. © 2015 American Institute of Chemical Engineers.

Growth, chamber building rate and reproduction time of Palaeonummulites venosus under natural conditions.

NASA Astrophysics Data System (ADS)

Kinoshita, Shunichi; Eder, Wolfgang; Wöger, Julia; Hohenegger, Johann; Briguglio, Antonino

2017-04-01

Investigations on Palaeonummulites venosus using the natural laboratory approach for determining chamber building rate, test diameter increase rate, reproduction time and longevity is based on the decomposition of monthly obtained frequency distributions based on chamber number and test diameter into normal-distributed components. The shift of the component parameters 'mean' and 'standard deviation' during the investigation period of 15 months was used to calculate Michaelis-Menten functions applied to estimate the averaged chamber building rate and diameter increase rate under natural conditions. The individual dates of birth were estimated using the inverse averaged chamber building rate and the inverse diameter increase rate fitted by the individual chamber number or the individual test diameter at the sampling date. Distributions of frequencies and densities (i.e. frequency divided by sediment weight) based on chamber building rate and diameter increase rate resulted both in a continuous reproduction through the year with two peaks, the stronger in May /June determined as the beginning of the summer generation (generation1) and the weaker in November determined as the beginning of the winter generation (generation 2). This reproduction scheme explains the existence of small and large specimens in the same sample. Longevity, calculated as the maximum difference in days between the individual's birth date and the sampling date seems to be round about one year, obtained by both estimations based on the chamber building rate and the diameter increase rate.

Detergent-enzymatic decellularization of swine blood vessels: insight on mechanical properties for vascular tissue engineering.

PubMed

Pellegata, Alessandro F; Asnaghi, M Adelaide; Stefani, Ilaria; Maestroni, Anna; Maestroni, Silvia; Dominioni, Tommaso; Zonta, Sandro; Zerbini, Gianpaolo; Mantero, Sara

2013-01-01

Small caliber vessels substitutes still remain an unmet clinical need; few autologous substitutes are available, while synthetic grafts show insufficient patency in the long term. Decellularization is the complete removal of all cellular and nuclear matters from a tissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds for tissue engineering, already used for various tissues with positive outcomes. The aim of this work is to investigate the effect of a detergent-enzymatic decellularization protocol on swine arteries in terms of cell removal, extracellular matrix preservation, and mechanical properties. Furthermore, the effect of storage at -80°C on the mechanical properties of the tissue is evaluated. Swine arteries were harvested, frozen, and decellularized; histological analysis revealed complete cell removal and preserved extracellular matrix. Furthermore, the residual DNA content in decellularized tissues was far low compared to native one. Mechanical testings were performed on native, defrozen, and decellularized tissues; no statistically significant differences were reported for Young's modulus, ultimate stress, compliance, burst pressure, and suture retention strength, while ultimate strain and stress relaxation of decellularized vessels were significantly different from the native ones. Considering the overall results, the process was confirmed to be suitable for the generation of acellular scaffolds for vascular tissue engineering.

Primary midgut, salivary gland, and ovary cultures from Boophilus microplus.

PubMed

Mosqueda, Juan; Cossío-Bayugar, Raquel; Rodríguez, Elba; Falcón, Alfonso; Ramos, Alberto; Figueroa, Julio V; Alvarez, Antonio

2008-12-01

Primary cell cultures from different tick organs are a valuable tool for host parasite research in the study of the protozoan Babesia sp., which infects different organs of the tick. In this work we describe the generation of midgut, salivary gland, and ovary primary cell cultures from dissections of Boophilus microplus. Midguts, salivary glands, and ovaries were dissected from B. microplus ticks on different days after bovine infestation; different enzymatic disaggregating protocols were tested in the presence of proteolytic enzymes, such as trypsin and collagenase type I and II, for tissue disaggregation and primary cell culture generation. The dissected tick organs obtained 18-20 days after bovine infestation showed a major cellular differentiation and were easier to identify by cellular morphology. The enzymatic disaggregation results showed that each tissue required a different proteolytic enzyme for optimal disaggregation; collagenase type I produced the most complete disaggregation for ovaries but not for midgut or salivary glands. Collagenase type II was effective for salivary glands but performed poorly on ovaries and midgets, and typsin was effective for midguts only. The midgut and ovary primary cell cultures were maintained for 4 weeks in optimal conditions after the cells were no longer viable. The salivary gland cell cultures were viable for 8 months.

Removal of bisphenol derivatives through quinone oxidation by polyphenol oxidase and subsequent quinone adsorption on chitosan in the heterogeneous system.

PubMed

Kimura, Yuji; Takahashi, Ayumi; Kashiwada, Ayumi; Yamada, Kazunori

2015-01-01

In this study, the combined use of a biopolymer chitosan and an oxidoreductase polyphenol oxidase (PPO) was systematically investigated for the removal of bisphenol derivatives from aqueous medium. The process parameters, such as the pH value, temperature, and PPO concentration, were estimated to conduct the enzymatic quinone oxidation of bisphenol derivatives by as little enzyme as possible. Bisphenol derivatives effectively underwent PPO-catalysed quinone oxidation without H2O2 unlike other oxidoreductases, such as peroxidase and tyrosinase, and the optimum conditions were determined to be pH 7.0 and 40°C for bisphenol B, bisphenol E, bisphenol O, and bisphenol Z; pH 7.0 and 30°C for bisphenol C and bisphenol F; and pH 8.0 and 40°C for bisphenol T. They were completely removed through adsorption of enzymatically generated quinone derivatives on chitosan beads or chitosan powders. Quinone adsorption on chitosan beads or chitosan powders in the heterogeneous system was found to be a more effective procedure than generation of aggregates in the homogeneous system with chitosan solution. The removal time was shortened by increasing the amount of chitosan beads or decreasing the size of the chitosan powders.

Physiological and biochemical changes during drought and recovery periods at tillering and jointing stages in wheat (Triticum aestivum L.).

PubMed

Abid, Muhammad; Ali, Shafaqat; Qi, Lei Kang; Zahoor, Rizwan; Tian, Zhongwei; Jiang, Dong; Snider, John L; Dai, Tingbo

2018-03-15

Defining the metabolic strategies used by wheat to tolerate and recover from drought events will be important for ensuring yield stability in the future, but studies addressing this critical research topic are limited. To this end, the current study quantified the physiological, biochemical, and agronomic responses of a drought tolerant and drought sensitive cultivar to periods of water deficit and recovery. Drought stress caused a reversible decline in leaf water relations, membrane stability, and photosynthetic activity, leading to increased reactive oxygen species (ROS) generation, lipid peroxidation and membrane injury. Plants exhibited osmotic adjustment through the accumulation of soluble sugars, proline, and free amino acids and increased enzymatic and non-enzymatic antioxidant activities. After re-watering, leaf water potential, membrane stability, photosynthetic processes, ROS generation, anti-oxidative activities, lipid peroxidation, and osmotic potential completely recovered for moderately stressed plants and did not fully recover in severely stressed plants. Higher photosynthetic rates during drought and rapid recovery after re-watering produced less-pronounced yield declines in the tolerant cultivar than the sensitive cultivar. These results suggested that the plant's ability to maintain functions during drought and to rapidly recover after re-watering during vegetative periods are important for determining final productivity in wheat.

Disposal of Kitchen Waste from High Rise Apartment

NASA Astrophysics Data System (ADS)

Ori, Kirki; Bharti, Ajay; Kumar, Sunil

2017-09-01

The high rise building has numbers of floor and rooms having variety of users or tenants for residential purposes. The huge quantities of heterogenous mixtures of domestic food waste are generated from every floor of the high rise residential buildings. Disposal of wet and biodegradable domestic kitchen waste from high rise buildings are more expensive in regards of collection and vertical transportation. This work is intended to address the technique to dispose of the wet organic food waste from the high rise buildings or multistory building at generation point with the advantage of gravity and vermicomposting technique. This innovative effort for collection and disposal of wet organic solid waste from high rise apartment is more economical and hygienic in comparison with present system of disposal.

ETR ELECTRICAL BUILDING, TRA648. EMERGENCY STANDBY GENERATOR AND DIESEL UNIT. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

ETR ELECTRICAL BUILDING, TRA-648. EMERGENCY STANDBY GENERATOR AND DIESEL UNIT. METAL ROOF AND PUMICE BLOCK WALLS. CAMERA FACING SOUTHWEST. INL NEGATIVE NO. 56-3708. R.G. Larsen, Photographer, 11/13/1956 - Idaho National Engineering Laboratory, Test Reactor Area, Materials & Engineering Test Reactors, Scoville, Butte County, ID

78 FR 65639 - Questar Pipeline Company; Notice of Request Under Blanket Authorization

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-01

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. CP14-6-000] Questar Pipeline... appurtenant facilities located in Moffat County, Colorado. Specifically, Questar proposes to abandon one Solar Saturn 1200 compressor, a compressor building, two generators and a generator building, a liquids storage...

Exploitation of Digital Surface Models Generated from WORLDVIEW-2 Data for SAR Simulation Techniques

NASA Astrophysics Data System (ADS)

Ilehag, R.; Auer, S.; d'Angelo, P.

2017-05-01

GeoRaySAR, an automated SAR simulator developed at DLR, identifies buildings in high resolution SAR data by utilizing geometric knowledge extracted from digital surface models (DSMs). Hitherto, the simulator has utilized DSMs generated from LiDAR data from airborne sensors with pre-filtered vegetation. Discarding the need for pre-optimized model input, DSMs generated from high resolution optical data (acquired with WorldView-2) are used for the extraction of building-related SAR image parts in this work. An automatic preprocessing of the DSMs has been developed for separating buildings from elevated vegetation (trees, bushes) and reducing the noise level. Based on that, automated simulations are triggered considering the properties of real SAR images. Locations in three cities, Munich, London and Istanbul, were chosen as study areas to determine advantages and limitations related to WorldView-2 DSMs as input for GeoRaySAR. Beyond, the impact of the quality of the DSM in terms of building extraction is evaluated as well as evaluation of building DSM, a DSM only containing buildings. The results indicate that building extents can be detected with DSMs from optical satellite data with various success, dependent on the quality of the DSM as well as on the SAR imaging perspective.

Building Momentum: National Trends and Prospects for High-Performance Green Buildings.

ERIC Educational Resources Information Center

2003

This report is an outgrowth of the Green Building Roundtable of the U.S. Senate Committee on Environment and Public Works held in conjunction with the U.S. Green Building Council on April 24, 2002. The roundtable brought together diverse interests to educate members of Congress on green building trends and generated discussion about the economic…

ATLAS software configuration and build tool optimisation

NASA Astrophysics Data System (ADS)

Rybkin, Grigory; Atlas Collaboration

2014-06-01

ATLAS software code base is over 6 million lines organised in about 2000 packages. It makes use of some 100 external software packages, is developed by more than 400 developers and used by more than 2500 physicists from over 200 universities and laboratories in 6 continents. To meet the challenge of configuration and building of this software, the Configuration Management Tool (CMT) is used. CMT expects each package to describe its build targets, build and environment setup parameters, dependencies on other packages in a text file called requirements, and each project (group of packages) to describe its policies and dependencies on other projects in a text project file. Based on the effective set of configuration parameters read from the requirements files of dependent packages and project files, CMT commands build the packages, generate the environment for their use, or query the packages. The main focus was on build time performance that was optimised within several approaches: reduction of the number of reads of requirements files that are now read once per package by a CMT build command that generates cached requirements files for subsequent CMT build commands; introduction of more fine-grained build parallelism at package task level, i.e., dependent applications and libraries are compiled in parallel; code optimisation of CMT commands used for build; introduction of package level build parallelism, i. e., parallelise the build of independent packages. By default, CMT launches NUMBER-OF-PROCESSORS build commands in parallel. The other focus was on CMT commands optimisation in general that made them approximately 2 times faster. CMT can generate a cached requirements file for the environment setup command, which is especially useful for deployment on distributed file systems like AFS or CERN VMFS. The use of parallelism, caching and code optimisation significantly-by several times-reduced software build time, environment setup time, increased the efficiency of multi-core computing resources utilisation, and considerably improved software developer and user experience.

Calibrating Building Energy Models Using Supercomputer Trained Machine Learning Agents

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

Sanyal, Jibonananda; New, Joshua Ryan; Edwards, Richard

2014-01-01

Building Energy Modeling (BEM) is an approach to model the energy usage in buildings for design and retrofit purposes. EnergyPlus is the flagship Department of Energy software that performs BEM for different types of buildings. The input to EnergyPlus can often extend in the order of a few thousand parameters which have to be calibrated manually by an expert for realistic energy modeling. This makes it challenging and expensive thereby making building energy modeling unfeasible for smaller projects. In this paper, we describe the Autotune research which employs machine learning algorithms to generate agents for the different kinds of standardmore » reference buildings in the U.S. building stock. The parametric space and the variety of building locations and types make this a challenging computational problem necessitating the use of supercomputers. Millions of EnergyPlus simulations are run on supercomputers which are subsequently used to train machine learning algorithms to generate agents. These agents, once created, can then run in a fraction of the time thereby allowing cost-effective calibration of building models.« less

Automatic generation of smart earthquake-resistant building system: Hybrid system of base-isolation and building-connection.

PubMed

Kasagi, M; Fujita, K; Tsuji, M; Takewaki, I

2016-02-01

A base-isolated building may sometimes exhibit an undesirable large response to a long-duration, long-period earthquake ground motion and a connected building system without base-isolation may show a large response to a near-fault (rather high-frequency) earthquake ground motion. To overcome both deficiencies, a new hybrid control system of base-isolation and building-connection is proposed and investigated. In this new hybrid building system, a base-isolated building is connected to a stiffer free wall with oil dampers. It has been demonstrated in a preliminary research that the proposed hybrid system is effective both for near-fault (rather high-frequency) and long-duration, long-period earthquake ground motions and has sufficient redundancy and robustness for a broad range of earthquake ground motions.An automatic generation algorithm of this kind of smart structures of base-isolation and building-connection hybrid systems is presented in this paper. It is shown that, while the proposed algorithm does not work well in a building without the connecting-damper system, it works well in the proposed smart hybrid system with the connecting damper system.

164. Photocopied July 1978. VIEW OF STEAMTURBINE BUILDING AT STAMP ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

164. Photocopied July 1978. VIEW OF STEAM-TURBINE BUILDING AT STAMP MILL. BUILDING CONSTRUCTED IN 1921 TO USE EXHAUST STEAM TO GENERATE ELECTRICITY. C. 1925. - Quincy Mining Company, Hancock, Houghton County, MI

19. Launch Area, general view of Missile Assembly Building and ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

19. Launch Area, general view of Missile Assembly Building and Generator Building VIEW SOUTHWEST - NIKE Missile Battery PR-79, Launch Area, East Windsor Road south of State Route 101, Foster, Providence County, RI

Solid-State Treatment of Castor Cake Employing the Enzymatic Cocktail Produced from Pleurotus djamor Fungi.

PubMed

Sánchez-Cantú, Manuel; Ortiz-Moreno, Liliana; Ramos-Cassellis, María E; Marín-Castro, Marco; De la Cerna-Hernández, C

2018-06-01

In this work, the enzymatic cocktail produced by Pleurotus djamor fungi extracted at pH of 4.8 and 5.3 was employed for castor cake solid-state treatment. Proximal, X-ray powder diffraction and scanning electron microscopy analysis of the pristine castor cake were carried out. First, Pleurotus djamor stain was inoculated in castor cake for the enzymatic production and the enzymatic activity was determined. The maximum enzymatic activity was identified at days 14 (65.9 UI/gss) and 11 (140.3 UI/gss) for the enzymatic cocktail obtained at pH 5.3 and 4.8, respectively. Then, the enzymatic cocktail obtained at the highest enzymatic activity days was employed directly over castor cake. Lignin was degraded throughout incubation time achieving a 47 and 45% decrease for the cocktail produced at pH 4.8 and 5.3, correspondingly. These results were corroborated by the SEM and XRD analysis where a higher porosity and xylan degradation were perceived throughout the enzymatic treatment.

Magnetic resonance imaging findings of central nervous system in lysosomal storage diseases: A pictorial review.

PubMed

Fagan, Nathan; Alexander, Allen; Irani, Neville; Saade, Charbel; Naffaa, Lena

2017-06-01

Lysosomal storage diseases (LSD) are a complex group of genetic disorders that are a result of inborn errors of metabolism. These errors result in a variety of metabolic dysfunction and build-up certain molecules within the tissues of the central nervous system (CNS). Although, they have discrete enzymatic deficiencies, symptomology and CNS imaging findings can overlap with each other, which can become challenging to radiologists. The purpose of this paper is to review the most common CNS imaging findings in LSD in order to familiarize the radiologist with their imaging findings and help narrow down the differential diagnosis. © 2016 The Royal Australian and New Zealand College of Radiologists.

Self-assembly of free-standing RNA membranes

NASA Astrophysics Data System (ADS)

Han, Daehoon; Park, Yongkuk; Kim, Hyejin; Lee, Jong Bum

2014-07-01

RNA has emerged as a promising material for nanostructure and microstructure engineering. Although rare, some macroscopic RNA structures have also been constructed using lipid or polymer materials. Here, we report the first example of an enzymatically generated RNA membrane. This robust and free-standing RNA membrane has a macroscopic structure and is generated without any polymer support or complexation. Our RNA membrane is fabricated following two sequential processes, complementary rolling circle transcription and evaporation-induced self-assembly, and its structural and functional properties are rationally controlled by adjusting RNA base pairing. In this study, three types of RNA membranes are fabricated and are used to demonstrate potential applications.

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

PubMed Central

Gutiérrez‐Sanz, Óscar; Natale, Paolo; Márquez, Ileana; Marques, Marta C.; Zacarias, Sonia; Pita, Marcos; Pereira, Inês A. C.

2016-01-01

Abstract ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1F0‐ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode‐assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products. PMID:26991333

Response measurements for two building structures excited by noise from a large horizontal axis wind turbine generator

NASA Technical Reports Server (NTRS)

Hubbard, H. H.; Shepherd, K. P.

1984-01-01

Window and wall acceleration measurements and interior noise measurements ere made for two different building structures during excitation by noise from the WTS-4 horizontal axis wind turbine generator operating in a normal power generation mode. With turbine noise input pulses resulted in acceleration pulses for the wall and window elements of the two tests buildings. Response spectra suggest that natural vibration modes of the structures are excited. Responses of a house trailer were substantially greater than those for a building of sturdier construction. Peak acceleration values correlate well with similar data for houses excited by flyover noise from commercial and military airplanes and helicopters, and sonic booms from supersonic aircraft. Interior noise spectra have peaks at frequencies corresponding to structural vibration modes and room standing waves; and the levels for particular frequencies and locations can be higher than the outside levels.

Enzymatic characteristics of I213T mutant presenilin-1/gamma-secretase in cell models and knock-in mouse brains: familial Alzheimer disease-linked mutation impairs gamma-site cleavage of amyloid precursor protein C-terminal fragment beta.

PubMed

Shimojo, Masafumi; Sahara, Naruhiko; Mizoroki, Tatsuya; Funamoto, Satoru; Morishima-Kawashima, Maho; Kudo, Takashi; Takeda, Masatoshi; Ihara, Yasuo; Ichinose, Hiroshi; Takashima, Akihiko

2008-06-13

Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset.

Building Energy Asset Score for Building Owners

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

Building Technologies Office

2015-01-01

The Building Energy Asset Score is a national standardized tool for evaluating the physical and structural energy efficiency of commercial and multifamily residential buildings. The Asset Score generates a simple energy efficiency rating that enables comparison among buildings, and identifies opportunities for users to invest in energy efficiency upgrades. It is web-based and free to use. This fact sheet discusses the value of the score for building owners.

Elasticity, biodegradability and cell adhesive properties of chitosan/hyaluronan multilayer films

NASA Astrophysics Data System (ADS)

Schneider, Aurore; Richert, Ludovic; Francius, Gregory; Voegel, Jean-Claude; Picart, Catherine

2007-03-01

In the bioengineering field, a recent and promising approach to modifying biomaterial surfaces is the layer-by-layer (LbL) technique used to build thin polyelectrolyte multilayer films. In this work, we focused on polyelectrolyte multilayer films made of two polysaccharides, chitosan (CHI) and hyaluronan (HA), and on the control of their physico-chemical and cell adhesive properties by chemical cross-linking. CHI/HA films were cross-linked using a water soluble carbodiimide and observed by confocal laser scanning microscopy (CLSM) with a fluorescently labeled CHI. Film thicknesses were similar for native and cross-linked films. The film nanometer roughness was measured by atomic force microscopy and was found to be higher for cross-linked films. Cross-linking the films also leads to a drastic change in film stiffness. The elastic modulus of the films (Young's modulus) as measured by AFM nano-indentation was about tenfold increased for cross-linked films as compared to native ones. From a biological point of view, cross-liked films are more resistant to enzymatic degradation by hyaluronidase. Furthermore, the increase in film stiffness has a favorable effect on the adhesion and spreading of chondrosarcoma cells. Thus, the CHI/HA cross-linked films could be used for various applications due to their adhesive properties and to their mechanical properties (including stability in enzymatic media).

Fabrication of nanoporous thin-film working electrodes and their biosensing applications.

PubMed

Li, Tingjie; Jia, Falong; Fan, Yaxi; Ding, Zhifeng; Yang, Jun

2013-04-15

Electrochemical detection for point-of-care diagnostics is of great interest due to its high sensitivity, fast analysis time and ability to operate on a small scale. Herein, we report the fabrication of a nanoporous thin-film electrode and its application in the configuration of a simple and robust enzymatic biosensor. The nanoporous thin-film was formed in a planar gold electrode through an alloying/dealloying process. The nanoporous electrode has an electroactive surface area up to 40 times higher than that of a flat gold electrode of the same size. The nanoporous electrode was used as a substrate to build an enzymatic electrochemical biosensor for the detection of glucose in standard samples and control serum samples. The example glucose biosensor has a linear response up to 30 mM, with a high sensitivity of 0.50 μA mM⁻¹ mm⁻², and excellent anti-interference ability against lactate, uric acid and ascorbic acid. Abundant catalyst and enzyme were stably entrapped in the nanoporous structure, leading to high stability and reproducibility of the biosensor. Development of such nanoporous structure enables the miniaturization of high-performance electrochemical biosensors for point-of-care diagnostics or environmental field testing. Copyright © 2012 Elsevier B.V. All rights reserved.

Enzymatic preparation of optically pure (+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (-)-γ-lactamase from Bradyrhizobium japonicum USDA 6.

PubMed

Zhu, Shaozhou; Ren, Lu; Yu, Songzhu; Gong, Cuiyu; Song, Dawei; Zheng, Guojun

2014-10-15

Whole cells of Bradyrhizobium japonicum USDA 6 showed both (+)-γ-lactamase activity and (-)-γ-lactamase activity. Insight into the genome of B. japonicum USDA 6 revealed two potential γ-lactamases: a type I (+)-γ-lactamase and a (-)-γ-lactamase, making it the first strain to contain two totally different enantioselective lactamases. Both recombinant enzymes could easily be used to prepare either optically pure (+)-γ-lactam ((+)-2-azabicyclo[2.2.1]hept-5-en-3-one) or optically pure (-)-γ-lactam ((-)-2-azabicyclo[2.2.1]hept-5-en-3-one), which are versatile synthetic building blocks for the synthesis of various carbocyclic nucleosides and carbocyclic sugar analogues. Bioinformatic analysis showed that the type I (+)-γ-lactamase belongs to the amidase signature family, with 504 amino acids; the (-)-γ-lactamase, which consists of 274 amino acids, belongs to the hydrolase family. Here, we report that B. japonicum USDA contains a (-)-γ-lactamase in addition to a (+)-γ-lactamase, and it is the (-)-γ-lactamase from this strain that is examined in detail in this Letter. Enzymatic synthesis of optically pure (+)-γ-lactam with nearly 50% isolated yield and >99% ee was achieved. Copyright © 2014 Elsevier Ltd. All rights reserved.

Global optimization framework for solar building design

NASA Astrophysics Data System (ADS)

Silva, N.; Alves, N.; Pascoal-Faria, P.

2017-07-01

The generative modeling paradigm is a shift from static models to flexible models. It describes a modeling process using functions, methods and operators. The result is an algorithmic description of the construction process. Each evaluation of such an algorithm creates a model instance, which depends on its input parameters (width, height, volume, roof angle, orientation, location). These values are normally chosen according to aesthetic aspects and style. In this study, the model's parameters are automatically generated according to an objective function. A generative model can be optimized according to its parameters, in this way, the best solution for a constrained problem is determined. Besides the establishment of an overall framework design, this work consists on the identification of different building shapes and their main parameters, the creation of an algorithmic description for these main shapes and the formulation of the objective function, respecting a building's energy consumption (solar energy, heating and insulation). Additionally, the conception of an optimization pipeline, combining an energy calculation tool with a geometric scripting engine is presented. The methods developed leads to an automated and optimized 3D shape generation for the projected building (based on the desired conditions and according to specific constrains). The approach proposed will help in the construction of real buildings that account for less energy consumption and for a more sustainable world.

Promising Practices: Building the Next Generation of School Leaders

ERIC Educational Resources Information Center

Bryant, Jennifer Edic; Escalante, Karen; Selva, Ashley

2017-01-01

This study applies transformational leadership theory practices to examine the purposeful ways in which principals work to build the next generation of teacher leaders in response to the shortage of K-12 principals. Given the impact principals have on student development and the shortage of those applying for the principalship, the purpose of this…

36. SITE BUILDING 004 ELECTRIC POWER STATION CLOSE ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

36. SITE BUILDING 004 - ELECTRIC POWER STATION - CLOSE UP VIEW OF 1200 HORSEPOWER STANDBY POWER DIESEL ENGINE/GENERATOR SETS. - Cape Cod Air Station, Technical Facility-Scanner Building & Power Plant, Massachusetts Military Reservation, Sandwich, Barnstable County, MA

37. SITE BUILDING 004 ELECTRIC POWER STATION ELEVATED ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

37. SITE BUILDING 004 - ELECTRIC POWER STATION - ELEVATED VIEW OF FIVE (5) 1200 HORSEPOWER STANDBY - POWER DIESEL ENGINE/GENERATOR SETS. - Cape Cod Air Station, Technical Facility-Scanner Building & Power Plant, Massachusetts Military Reservation, Sandwich, Barnstable County, MA

Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Direct local building inundation depth determination in 3-D point clouds generated from user-generated flood images

NASA Astrophysics Data System (ADS)

Griesbaum, Luisa; Marx, Sabrina; Höfle, Bernhard

2017-07-01

In recent years, the number of people affected by flooding caused by extreme weather events has increased considerably. In order to provide support in disaster recovery or to develop mitigation plans, accurate flood information is necessary. Particularly pluvial urban floods, characterized by high temporal and spatial variations, are not well documented. This study proposes a new, low-cost approach to determining local flood elevation and inundation depth of buildings based on user-generated flood images. It first applies close-range digital photogrammetry to generate a geo-referenced 3-D point cloud. Second, based on estimated camera orientation parameters, the flood level captured in a single flood image is mapped to the previously derived point cloud. The local flood elevation and the building inundation depth can then be derived automatically from the point cloud. The proposed method is carried out once for each of 66 different flood images showing the same building façade. An overall accuracy of 0.05 m with an uncertainty of ±0.13 m for the derived flood elevation within the area of interest as well as an accuracy of 0.13 m ± 0.10 m for the determined building inundation depth is achieved. Our results demonstrate that the proposed method can provide reliable flood information on a local scale using user-generated flood images as input. The approach can thus allow inundation depth maps to be derived even in complex urban environments with relatively high accuracies.

A high-throughput assay for enzymatic polyester hydrolysis activity by fluorimetric detection.

PubMed

Wei, Ren; Oeser, Thorsten; Billig, Susan; Zimmermann, Wolfgang

2012-12-01

A fluorimetric assay for the fast determination of the activity of polyester-hydrolyzing enzymes in a large number of samples has been developed. Terephthalic acid (TPA) is a main product of the enzymatic hydrolysis of polyethylene terephthalate (PET), a synthetic polyester. Terephthalate has been quantified following its conversion to the fluorescent 2-hydroxyterephthalate by an iron autoxidation-mediated generation of free hydroxyl radicals. The assay proved to be robust at different buffer concentrations, reaction times, pH values, and in the presence of proteins. A validation of the assay was performed by analyzing TPA formation from PET films and nanoparticles catalyzed by a polyester hydrolase from Thermobifida fusca KW3 in a 96-well microplate format. The results showed a close correlation (R(2) = 0.99) with those obtained by a considerably more tedious and time-consuming HPLC method, suggesting the aptness of the fluorimetric assay for a high-throughput screening for polyester hydrolases. The method described in this paper will facilitate the detection and development of biocatalysts for the modification and degradation of synthetic polymers. The fluorimetric assay can be used to quantify the amount of TPA obtained as the final degradation product of the enzymatic hydrolysis of PET. In a microplate format, this assay can be applied for the high-throughput screening of polyester hydrolases. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elements of the cellular metabolic structure

PubMed Central

De la Fuente, Ildefonso M.

2015-01-01

A large number of studies have demonstrated the existence of metabolic covalent modifications in different molecular structures, which are able to store biochemical information that is not encoded by DNA. Some of these covalent mark patterns can be transmitted across generations (epigenetic changes). Recently, the emergence of Hopfield-like attractor dynamics has been observed in self-organized enzymatic networks, which have the capacity to store functional catalytic patterns that can be correctly recovered by specific input stimuli. Hopfield-like metabolic dynamics are stable and can be maintained as a long-term biochemical memory. In addition, specific molecular information can be transferred from the functional dynamics of the metabolic networks to the enzymatic activity involved in covalent post-translational modulation, so that determined functional memory can be embedded in multiple stable molecular marks. The metabolic dynamics governed by Hopfield-type attractors (functional processes), as well as the enzymatic covalent modifications of specific molecules (structural dynamic processes) seem to represent the two stages of the dynamical memory of cellular metabolism (metabolic memory). Epigenetic processes appear to be the structural manifestation of this cellular metabolic memory. Here, a new framework for molecular information storage in the cell is presented, which is characterized by two functionally and molecularly interrelated systems: a dynamic, flexible and adaptive system (metabolic memory) and an essentially conservative system (genetic memory). The molecular information of both systems seems to coordinate the physiological development of the whole cell. PMID:25988183

Compounds inhibiting the bioconversion of hydrothermally pretreated lignocellulose.

PubMed

Ko, Ja Kyong; Um, Youngsoon; Park, Yong-Cheol; Seo, Jin-Ho; Kim, Kyoung Heon

2015-05-01

Hydrothermal pretreatment using liquid hot water, steam explosion, or dilute acids enhances the enzymatic digestibility of cellulose by altering the chemical and/or physical structures of lignocellulosic biomass. However, compounds that inhibit both enzymes and microbial activity, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are also generated during pretreatment. Insoluble lignin, which predominantly remains within the pretreated solids, also acts as a significant inhibitor of cellulases during hydrolysis of cellulose. Exposed lignin, which is modified to be more recalcitrant to enzymes during pretreatment, adsorbs cellulase nonproductively and reduces the availability of active cellulase for hydrolysis of cellulose. Similarly, lignin-derived phenolics inhibit or deactivate cellulase and β-glucosidase via irreversible binding or precipitation. Meanwhile, the performance of fermenting microorganisms is negatively affected by phenolics, sugar degradation products, and weak acids. This review describes the current knowledge regarding the contributions of inhibitors present in whole pretreatment slurries to the enzymatic hydrolysis of cellulose and fermentation. Furthermore, we discuss various biological strategies to mitigate the effects of these inhibitors on enzymatic and microbial activity to improve the lignocellulose-to-biofuel process robustness. While the inhibitory effect of lignin on enzymes can be relieved through the use of lignin blockers and by genetically engineering the structure of lignin or of cellulase itself, soluble inhibitors, including phenolics, furan aldehydes, and weak acids, can be detoxified by microorganisms or laccase.

The Effect of Ionic Liquid Pretreatment on the Bioconversion of Tomato Processing Waste to Fermentable Sugars and Biogas.

PubMed

Allison, Brittany J; Cádiz, Juan Canales; Karuna, Nardrapee; Jeoh, Tina; Simmons, Christopher W

2016-08-01

Tomato pomace is an abundant lignocellulosic waste stream from industrial tomato processing and therefore a potential feedstock for production of renewable biofuels. However, little research has been conducted to determine if pretreatment can enhance release of fermentable sugars from tomato pomace. Ionic liquids (ILs) are an emerging pretreatment technology for lignocellulosic biomass to increase enzymatic digestibility and biofuel yield while utilizing recyclable chemicals with low toxicity. In this study, pretreatment of tomato pomace with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was investigated. Changes in pomace enzymatic digestibility were affected by pretreatment time and temperature. Certain pretreatment conditions significantly improved reducing sugar yield and hydrolysis time compared to untreated pomace. Compositional analyses suggested that pretreatment primarily removed water-soluble compounds and enriched for lignocellulose in pomace, with only subtle changes to the composition of the lignocellulose. While tomato pomace was effectively pretreated with [C2mim][OAc] to improve enzymatic digestibility, as of yet, unknown factors in the pomace caused ionic liquid pretreatment to negatively affect anaerobic digestion of pretreated material. This result, which is unique compared to similar studies on IL pretreatment of grasses and woody biomass, highlights the need for additional research to determine how the unique chemical composition of tomato pomace and other lignocellulosic fruit residues may interact with ionic liquids to generate inhibitors for downstream fermentation to biofuels.

Bioremediation of cooking oil waste using lipases from wastes

PubMed Central

do Prado, Débora Zanoni; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases. PMID:29073166

Molecular control of brain size: Regulators of neural stem cell life, death and beyond

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

Joseph, Bertrand; Hermanson, Ola, E-mail: ola.hermanson@ki.se

2010-05-01

The proper development of the brain and other organs depends on multiple parameters, including strictly controlled expansion of specific progenitor pools. The regulation of such expansion events includes enzymatic activities that govern the correct number of specific cells to be generated via an orchestrated control of cell proliferation, cell cycle exit, differentiation, cell death etc. Certain proteins in turn exert direct control of these enzymatic activities and thus progenitor pool expansion and organ size. The members of the Cip/Kip family (p21Cip1/p27Kip1/p57Kip2) are well-known regulators of cell cycle exit that interact with and inhibit the activity of cyclin-CDK complexes, whereas membersmore » of the p53/p63/p73 family are traditionally associated with regulation of cell death. It has however become clear that the roles for these proteins are not as clear-cut as initially thought. In this review, we discuss the roles for proteins of the Cip/Kip and p53/p63/p73 families in the regulation of cell cycle control, differentiation, and death of neural stem cells. We suggest that these proteins act as molecular interfaces, or 'pilots', to assure the correct assembly of protein complexes with enzymatic activities at the right place at the right time, thereby regulating essential decisions in multiple cellular events.« less

Flexible electronics-compatible non-enzymatic glucose sensing via transparent CuO nanowire networks on PET films

NASA Astrophysics Data System (ADS)

Bell, Caroline; Nammari, Abdullah; Uttamchandani, Pranay; Rai, Amit; Shah, Pujan; Moore, Arden L.

2017-06-01

Diabetic individuals need simple, accurate, and cost effective means by which to independently assess their glucose levels in a non-invasive way. In this work, a sensor based on randomly oriented CuO nanowire networks supported by a polyethylene terephthalate thin film is evaluated as a flexible, transparent, non-enzymatic glucose sensing system analogous to those envisioned for future wearable diagnostic devices. The amperometric sensing characteristics of this type of device architecture are evaluated both before and after bending, with the system’s glucose response, sensitivity, lower limit of detection, and effect of applied bias being experimentally determined. The obtained data shows that the sensor is capable of measuring changes in glucose levels within a physiologically relevant range (0-12 mM glucose) and at lower limits of detection (0.05 mM glucose at +0.6 V bias) consistent with patient tears and saliva. Unlike existing studies utilizing a conductive backing layer or macroscopic electrode setup, this sensor demonstrates a percolation network-like trend of current versus glucose concentration. In this implementation, controlling the architectural details of the CuO nanowire network could conceivably allow the sensor’s sensitivity and optimal sensing range to be tuned. Overall, this work shows that integrating CuO nanowires into a sensor architecture compatible with transparent, flexible electronics is a promising avenue to realizing next generation wearable non-enzymatic glucose diagnostic devices.

Bioremediation of cooking oil waste using lipases from wastes.

PubMed

Okino-Delgado, Clarissa Hamaio; Prado, Débora Zanoni do; Facanali, Roselaine; Marques, Márcia Mayo Ortiz; Nascimento, Augusto Santana; Fernandes, Célio Junior da Costa; Zambuzzi, William Fernando; Fleuri, Luciana Francisco

2017-01-01

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

Fungal secretomes enhance sugar beet pulp hydrolysis.

PubMed

Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

2014-04-01

The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free fluorescent enzymatic assay of citrate synthase by CoA-Au(I) co-ordination polymer and its application in a multi-enzyme logic gate cascade.

PubMed

Li, Yong; Wang, Huixia; Dai, Futao; Li, Pei; Jin, Xin; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

2016-12-15

Citrate synthase (CS) is one of the key metabolic enzymes in the Krebs tricarboxylic acid (TCA) cycle. It regulates energy generation in mitochondrial respiration by catalysing the reaction between oxaloacetic acid (OAA) and acetyl coenzyme A (Ac-CoA) to generate citrate and coenzyme A (CoA). CS has been shown to be a biomarker of neurological diseases and various kinds of cancers. Here, a label-free fluorescent assay has been developed for homogeneously detecting CS and its inhibitor based on the in situ generation of CoA-Au(I) co-ordination polymer (CP) and the fluorescence signal-on by SYBR Green II-stained CoA-Au(I) CP. Because of the unique property of the CoA-Au(I) CP, this CS activity assay method could achieve excellent selectivity and sensitivity, with a linear range from 0.0033 U/μL to 0.264 U/μL and a limit of detection to be 0.00165 U/μL. Meanwhile, this assay method has advantages of being facile and cost effective with quick detection. Moreover, based on this method, a biomimetic logic system was established by rationally exploiting the cascade enzymatic interactions in TCA cycle for chemical information processing. In the TCA cycle-derived logic system, an AND-AND-AND-cascaded gate was rigorously operated step by step in one pot, and is outputted by a label-free fluorescent signal with visualized readout. Copyright © 2016 Elsevier B.V. All rights reserved.

Establishing a National 3d Geo-Data Model for Building Data Compliant to Citygml: Case of Turkey

NASA Astrophysics Data System (ADS)

Ates Aydar, S.; Stoter, J.; Ledoux, H.; Demir Ozbek, E.; Yomralioglu, T.

2016-06-01

This paper presents the generation of the 3D national building geo-data model of Turkey, which is compatible with the international OGC CityGML Encoding Standard. We prepare an ADE named CityGML-TRKBIS.BI that is produced by extending existing thematic modules of CityGML according to TRKBIS needs. All thematic data groups in TRKBIS geo-data model have been remodelled in order to generate the national large scale 3D geo-data model for Turkey. Specific attention has been paid to data groups that have different class structure according to related CityGML data themes such as building data model. Current 2D geo-information model for building data theme of Turkey (TRKBIS.BI) was established based on INSPIRE specifications for building (Core 2D and Extended 2D profiles), ISO/TC 211 standards and OGC web services. New version of TRKBIS.BI which is established according to semantic and geometric rules of CityGML will represent 2D-2.5D and 3D objects. After a short overview on generic approach, this paper describes extending CityGML building data theme according to TRKBIS.BI through several steps. First, building models of both standards were compared according to their data structure, classes and attributes. Second, CityGML building model was extended with respect to TRKBIS needs and CityGML-TRKBIS Building ADE was established in UML. This study provides new insights into 3D applications in Turkey. The generated 3D geo-data model for building thematic class will be used as a common exchange format that meets 2D, 2.5D and 3D implementation needs at national level.

7. PHOTOCOPY, PLANS, ELEVATIONS, AND SECTION DRAWING FOR MISSILE TEST ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

7. PHOTOCOPY, PLANS, ELEVATIONS, AND SECTION DRAWING FOR MISSILE TEST AND ASSEMBLY BUILDING. - NIKE Missile Base SL-40, Missile Test & Assembly Building, South end of launch area, northeast of Generator Building No. 3, Hecker, Monroe County, IL

35. SITE BUILDING 004 ELECTRIC POWER STATION CONTROL ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

35. SITE BUILDING 004 - ELECTRIC POWER STATION - CONTROL ROOM OF ELECTRIC POWER STATION WITH DIESEL ENGINE POWERED ELECTRIC GENERATION EQUIPMENT IN BACKGROUND. - Cape Cod Air Station, Technical Facility-Scanner Building & Power Plant, Massachusetts Military Reservation, Sandwich, Barnstable County, MA

38. SITE BUILDING 004 ELECTRIC POWER STATION AT INTERIOR ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

38. SITE BUILDING 004 - ELECTRIC POWER STATION AT INTERIOR - OBLIQUE VIEW AT FLOOR LEVEL SHOWING DIESEL ENGINE/GENERATOR SET NUMBER 5. - Cape Cod Air Station, Technical Facility-Scanner Building & Power Plant, Massachusetts Military Reservation, Sandwich, Barnstable County, MA

A simulation-based efficiency comparison of AC and DC power distribution networks in commercial buildings

DOE PAGES

Gerber, Daniel L.; Vossos, Vagelis; Feng, Wei; ...

2017-06-12

Direct current (DC) power distribution has recently gained traction in buildings research due to the proliferation of on-site electricity generation and battery storage, and an increasing prevalence of internal DC loads. The research discussed in this paper uses Modelica-based simulation to compare the efficiency of DC building power distribution with an equivalent alternating current (AC) distribution. The buildings are all modeled with solar generation, battery storage, and loads that are representative of the most efficient building technology. A variety of paramet ric simulations determine how and when DC distribution proves advantageous. These simulations also validate previous studies that use simplermore » approaches and arithmetic efficiency models. This work shows that using DC distribution can be considerably more efficient: a medium sized office building using DC distribution has an expected baseline of 12% savings, but may also save up to 18%. In these results, the baseline simulation parameters are for a zero net energy (ZNE) building that can island as a microgrid. DC is most advantageous in buildings with large solar capacity, large battery capacity, and high voltage distribution.« less

A simulation-based efficiency comparison of AC and DC power distribution networks in commercial buildings

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

Gerber, Daniel L.; Vossos, Vagelis; Feng, Wei

Direct current (DC) power distribution has recently gained traction in buildings research due to the proliferation of on-site electricity generation and battery storage, and an increasing prevalence of internal DC loads. The research discussed in this paper uses Modelica-based simulation to compare the efficiency of DC building power distribution with an equivalent alternating current (AC) distribution. The buildings are all modeled with solar generation, battery storage, and loads that are representative of the most efficient building technology. A variety of paramet ric simulations determine how and when DC distribution proves advantageous. These simulations also validate previous studies that use simplermore » approaches and arithmetic efficiency models. This work shows that using DC distribution can be considerably more efficient: a medium sized office building using DC distribution has an expected baseline of 12% savings, but may also save up to 18%. In these results, the baseline simulation parameters are for a zero net energy (ZNE) building that can island as a microgrid. DC is most advantageous in buildings with large solar capacity, large battery capacity, and high voltage distribution.« less

Optimized design and control of an off grid solar PV/hydrogen fuel cell power system for green buildings

NASA Astrophysics Data System (ADS)

Ghenai, C.; Bettayeb, M.

2017-11-01

Modelling, simulation, optimization and control strategies are used in this study to design a stand-alone solar PV/Fuel Cell/Battery/Generator hybrid power system to serve the electrical load of a commercial building. The main objective is to design an off grid energy system to meet the desired electric load of the commercial building with high renewable fraction, low emissions and low cost of energy. The goal is to manage the energy consumption of the building, reduce the associate cost and to switch from grid-tied fossil fuel power system to an off grid renewable and cleaner power system. Energy audit was performed in this study to determine the energy consumption of the building. Hourly simulations, modelling and optimization were performed to determine the performance and cost of the hybrid power configurations using different control strategies. The results show that the hybrid off grid solar PV/Fuel Cell/Generator/Battery/Inverter power system offers the best performance for the tested system architectures. From the total energy generated from the off grid hybrid power system, 73% is produced from the solar PV, 24% from the fuel cell and 3% from the backup Diesel generator. The produced power is used to meet all the AC load of the building without power shortage (<0.1%). The hybrid power system produces 18.2% excess power that can be used to serve the thermal load of the building. The proposed hybrid power system is sustainable, economically viable and environmentally friendly: High renewable fraction (66.1%), low levelized cost of energy (92 /MWh), and low carbon dioxide emissions (24 kg CO2/MWh) are achieved.

77 FR 28626 - Biweekly Notice, Applications and Amendments to Facility Operating Licenses and Combined Licenses...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-15

... Water System, the Auxiliary Building Filtered Ventilation Exhaust System, or the Diesel Generators... nuclear service water system (NSWS) for a time period of 14 days. Basis for proposed no significant... the diesel generator buildings in the event of a leak or a break in the system piping. The probability...

The Knowledge Building Paradigm: A Model of Learning for Net Generation Students

ERIC Educational Resources Information Center

Philip, Donald

2005-01-01

In this article Donald Philip describes Knowledge Building, a pedagogy based on the way research organizations function. The global economy, Philip argues, is driving a shift from older, industrial models to the model of the business as a learning organization. The cognitive patterns of today's Net Generation students, formed by lifetime exposure…

Using a Capability Maturity Model to Build on the Generational Approach to Student Engagement Practices

ERIC Educational Resources Information Center

Nelson, K.; Clarke, J.; Stoodley, I.; Creagh, T.

2015-01-01

The generational approach to conceptualising first-year student learning behaviour has made a useful contribution to understanding student engagement. It has an explicit focus on student behaviour and we suggest that a Capability Maturity Model interpretation may provide a complementary extension of that understanding as it builds on the…

An empirical study on energy efficiency improvement through photovoltaic systems and a LED lighting control system

NASA Astrophysics Data System (ADS)

Choi, Young Kwan; Lee, Jae Hyeong

2015-09-01

In this research, a facility was constructed and its performance was analyzed to improve the energy efficiency of a vertical-type water treatment building. After the design and construction of a fixed tilt Photovoltaic in Building (PVIB) on the rooftop using a crystalline silicon solar cell module and photovoltaic generator integrated with the building by using a Building Integrated Photovoltaic System (BIPV), a thin-film module on the rooftop and outer wall of water treatment building, and the generation efficiency was analyzed. Also, a DC distribution was established for use of a brushless DC (BLDC) pump motor, and the existing lighting-facility-based manual on-off method was turned into a system for energy conservation by controlling light emitting diode (LED) through a wireless motion sensor and dimming control. In addition, a Building Energy Management System (BEMS) for a real-time analysis of the energy efficiency for a vertical0type water treatment building was prepared and tested. The vertical-type water treatment building developed in this study is currently operating the BEMS. The vertical-type water treatment building reported in this paper is expected to reduce energy consumption by about 30% compared to existing water treatment systems.

1. SOUTH AND EAST SIDES OF BUILDING 522. VIEW TO ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. SOUTH AND EAST SIDES OF BUILDING 522. VIEW TO NORTHWEST. - Rocky Mountain Arsenal, White Phosphorous Filling-Acetylene Generation Building-Warehouse, 840 feet South of December Seventh Avenue; 1030 feet East of D Street, Commerce City, Adams County, CO

2. NORTH AND WEST SIDES OF BUILDING 522. VEW TO ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

2. NORTH AND WEST SIDES OF BUILDING 522. VEW TO SOUTHEAST. - Rocky Mountain Arsenal, White Phosphorous Filling-Acetylene Generation Building-Warehouse, 840 feet South of December Seventh Avenue; 1030 feet East of D Street, Commerce City, Adams County, CO

15. SITE BUILDING 004 ELECTRIC POWER STATION VIEW ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

15. SITE BUILDING 004 - ELECTRIC POWER STATION - VIEW IS LOOKING SOUTH 55° EAST AT FIVE DIESEL ENGINE/ GENERATOR SILENCER SYSTEM EXHAUST STACKS. - Cape Cod Air Station, Technical Facility-Scanner Building & Power Plant, Massachusetts Military Reservation, Sandwich, Barnstable County, MA

Cost Analysis of Cot-Side Screening Methods for Neonatal Hypoglycaemia.

PubMed

Glasgow, Matthew J; Harding, Jane E; Edlin, Richard

2018-06-12

Babies at risk of neonatal hypoglycaemia are often screened using cot-side glucometers, but non-enzymatic glucometers are inaccurate, potentially resulting in over-treatment and under-treatment, and low values require laboratory confirmation. More accurate enzymatic glucometers are available but at apparently higher costs. Our objective was to compare the cost of screening for neonatal hypoglycaemia using point-of-care enzymatic and non-enzymatic glucometers. We used a decision tree to model costs, including consumables and staff time. Sensitivity analyses assessed the impact of staff time, staff costs, probability that low results are confirmed via laboratory testing, false-positive and false-negative rates of non-enzymatic glucometers, and the blood glucose concentration threshold. In the primary analysis, screening using an enzymatic glucometer cost NZD 86.94 (USD 63.47) while using a non-enzymatic glucometer cost NZD 97.08 (USD 70.87) per baby. Sensitivity analyses showed that using an enzymatic glucometer is cost saving with wide variations in staff time and costs, irrespective of the false-positive level of non-enzymatic glucometers, and where ≥78% of low values are laboratory confirmed. Where non-enzymatic glucometers may be less costly (e.g., false-negative rate exceeds 15%), instances of hypoglycaemia will be missed. Reducing the blood glucose concentration threshold to 1.94 mmol/L reduced the incidence of hypoglycaemia from 52 to 13%, and the cost of screening using a non-enzymatic glucometer to NZD 47.71 (USD 34.83). In view of their lower cost in most circumstances and greater accuracy, enzymatic glucometers should be routinely utilised for point-of-care screening for neonatal hypoglycaemia. © 2018 S. Karger AG, Basel.

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

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

Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E.

Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. In the bacterial FASII pathway we found it a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here, we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promisingmore » low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, the improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.« less

Structural and biological evaluation of a novel series of benzimidazole inhibitors of Francisella tularensis enoyl-ACP reductase (FabI)

DOE PAGES

Mehboob, Shahila; Song, Jinhua; Hevener, Kirk E.; ...

2015-01-29

Francisella tularensis, the causative agent of tularemia, presents a significant biological threat and is a Category A priority pathogen due to its potential for weaponization. In the bacterial FASII pathway we found it a viable target for the development of novel antibacterial agents treating Gram-negative infections. Here, we report the advancement of a promising series of benzimidazole FabI (enoyl-ACP reductase) inhibitors to a second-generation using a systematic, structure-guided lead optimization strategy, and the determination of several co-crystal structures that confirm the binding mode of designed inhibitors. Furthermore, these compounds display an improved low nanomolar enzymatic activity as well as promisingmore » low microgram/mL antibacterial activity against both F. tularensis and Staphylococcus aureus and its methicillin-resistant strain (MRSA). Finally, the improvements in activity accompanying structural modifications lead to a better understanding of the relationship between the chemical structure and biological activity that encompasses both enzymatic and whole-cell activity.« less

Analysis of Resistant Starches in Rat Cecal Contents Using Fourier Transform Infrared Photoacoustic Spectroscopy

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

Anderson, Timothy J.; Ai, Yongfeng; Jones, Roger W.

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) qualitatively and quantitatively measured resistant starch (RS) in rat cecal contents. Fisher 344 rats were fed diets of 55% (w/w, dry basis) starch for 8 weeks. Cecal contents were collected from sacrificed rats. A corn starch control was compared against three RS diets. The RS diets were high-amylose corn starch (HA7), HA7 chemically modified with octenyl succinic anhydride, and stearic-acid-complexed HA7 starch. To calibrate the FTIR-PAS analysis, samples from each diet were analyzed using an enzymatic assay. A partial least-squares cross-validation plot generated from the enzymatic assay and FTIR-PAS spectral results for starch fitmore » the ideal curve with a R2 of 0.997. A principal component analysis plot of components 1 and 2 showed that spectra from diets clustered significantly from each other. This study clearly showed that FTIR-PAS can accurately quantify starch content and identify the form of starch in complex matrices.« less

An histidine covalent receptor/butenolide complex mediates strigolactone perception

PubMed Central

Badet-Denisot, Marie-Ange; Pillot, Jean-Paul; Cornu, David; Le Caer, Jean-Pierre; Burger, Marco; Pelissier, Frank; Retailleau, Pascal; Turnbull, Colin; Bonhomme, Sandrine; Chory, Joanne; Rameau, Catherine; Boyer, François-Didier

2016-01-01

Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D-ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases and is known to hydrolyze the bond between the ABC lactone and the D-ring. Here we characterize the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using novel profluorescent probes with strigolactone-like bioactivity, we show that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We further demonstrate the formation of a covalent RMS3/D-ring complex, essential for bioactivity, in which the D-ring is attached to Histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception where the receptor performs an irreversible enzymatic reaction to generate its own ligand. PMID:27479744

Novel insights into redox system and the mechanism of redox regulation.

PubMed

Wang, Xin; Hai, Chunxu

2016-07-01

In view of the critical role of redox system in numerous physiological and pathophysiological processes, it is important to clearly understand the family members and regulatory mechanism of redox system. In this work, we will systematically review the current data detailing the reactive oxygen species (ROS), enzymatic and non-enzymatic antioxidants and redox sensitive transcription factors and we give a brief description of redox-mediated epigenetic and post-translational regulation. We propose that the redox system functions as a "Redox Chain", consisting of "ROS-generating Enzyme Chain", "Combined Antioxidant Chain" and "Transcription Factor Chain". We suggest that an individualized assessment of the redox status in the body should be conducted for the redox intervention of a patient. The strategy of intervention is to maintain redox homeostasis via either facilitation of ROS signaling or enhancement of antioxidant defense. These findings provide valuable new insights into redox system and open up new paths for the control of redox-related disorders.

An Electrochemical Impedance Spectroscopy-Based Technique to Identify and Quantify Fermentable Sugars in Pineapple Waste Valorization for Bioethanol Production

PubMed Central

Conesa, Claudia; García-Breijo, Eduardo; Loeff, Edwin; Seguí, Lucía; Fito, Pedro; Laguarda-Miró, Nicolás

2015-01-01

Electrochemical Impedance Spectroscopy (EIS) has been used to develop a methodology able to identify and quantify fermentable sugars present in the enzymatic hydrolysis phase of second-generation bioethanol production from pineapple waste. Thus, a low-cost non-destructive system consisting of a stainless double needle electrode associated to an electronic equipment that allows the implementation of EIS was developed. In order to validate the system, different concentrations of glucose, fructose and sucrose were added to the pineapple waste and analyzed both individually and in combination. Next, statistical data treatment enabled the design of specific Artificial Neural Networks-based mathematical models for each one of the studied sugars and their respective combinations. The obtained prediction models are robust and reliable and they are considered statistically valid (CCR% > 93.443%). These results allow us to introduce this EIS-based technique as an easy, fast, non-destructive, and in-situ alternative to the traditional laboratory methods for enzymatic hydrolysis monitoring. PMID:26378537

Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols.

PubMed

Zhang, Zhengke; Huber, Donald J; Qu, Hongxia; Yun, Ze; Wang, Hui; Huang, Zihui; Huang, Hua; Jiang, Yueming

2015-03-15

'Guiwei' litchi fruit were treated with 5 ga.i. L(-1) apple polyphenols (APP) and then stored at 25°C to investigate the effects on pericarp browning. APP treatment effectively reduced pericarp browning and retarded the loss of red colour. APP-treated fruit exhibited higher levels of anthocyanins and cyanidin-3-rutinoside, which correlated with suppressed anthocyanase activity. APP treatment also maintained membrane integrity and reduced oxidative damage, as indicated by a lower relative leakage rate, malondialdehyde content, and reactive oxygen species (ROS) generation. The data suggest that decompartmentalisation of peroxidase and polyphenoloxidase and respective browning substrates was reduced. In addition, APP treatment enhanced the activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), as well as non-enzymatic antioxidant capacity (DPPH radical-scavenging activity and reducing power), which might be beneficial in scavenging ROS. We propose that APP treatment is a promising safe strategy for controlling postharvest browning of litchi fruit. Copyright © 2014 Elsevier Ltd. All rights reserved.

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides.

PubMed

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

Lipid mediators and their metabolism in the nucleous: implications for Alzheimer's disease.

PubMed

Farooqui, Akhlaq A

2012-01-01

Lipid mediators are important endogenous regulators derived from enzymatic degradation of glycerophospholipids, sphingolipids, and cholesterol by phospholipases, sphingomyelinases, and cytochrome P450 hydroxylases, respectively. In neural cells, lipid mediators are associated with proliferation, differentiation, oxidative stress, inflammation, and apoptosis. A major group of lipid mediators, which originates from the enzymatic oxidation of arachidonic acid, is called eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes, and lipoxins). The corresponding lipid mediators of docosahexaenoic acid metabolism are named as docosanoids. They include resolvins, protectins (neuroprotectins), and maresins. Docosanoids produce antioxidant, anti-inflammatory, and antiapoptotic effects in brain tissue. Other glycerophospholipid-derived lipid mediators are platelet activating factor, lysophosphatidic acid, and endocannabinoids. Degradation of sphingolipids also results in the generation of sphingolipid-derived lipid mediators, such as ceramide, ceramide 1-phosphate, sphingosine, and sphingosine 1-phosphate. These mediators are involved in differentiation, growth, cell migration, and apoptosis. Similarly, cholesterol-derived lipid mediators, hydroxycholesterol, produce apoptosis. Abnormal metabolism of lipid mediators may be closely associated with pathogenesis of Alzheimer's disease.

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed.

PubMed

Clough, Richard C; Pappu, Kameshwari; Thompson, Kevin; Beifuss, Katherine; Lane, Jeff; Delaney, Donna E; Harkey, Robin; Drees, Carol; Howard, John A; Hood, Elizabeth E

2006-01-01

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.

Direct nitration and azidation of aliphatic carbons by an iron-dependent halogenase

PubMed Central

Chang, Wei-chen; Layne, Andrew P; Miles, Linde A; Krebs, Carsten

2014-01-01

Iron-dependent halogenases employ cis-halo-Fe(IV)-oxo (haloferryl) complexes to functionalize unactivated aliphatic carbon centers, a capability elusive to synthetic chemists. Halogenation requires (1) coordination of a halide anion (Cl− or Br−) to the enzyme's Fe(II) cofactor; (2) coupled activation of O2 and decarboxylation of α-ketoglutarate to generate the haloferryl intermediate; (3) abstraction of hydrogen (H•) from the substrate by the ferryl oxo group; and (4) transfer of the cis halogen as Cl• or Br• to the substrate radical. This enzymatic solution to an unsolved chemical challenge is potentially generalizable to installation of other functional groups, provided that the corresponding anions can support the four requisite steps. We show here that the wild-type halogenase SyrB2 can indeed direct aliphatic nitration and azidation reactions by the same chemical logic. The discovery and enhancement by mutagenesis of these previously unknown reaction types suggests unrecognized or untapped versatility in ferryl-mediated enzymatic C–H-bond activation. PMID:24463698

Enhancing enzymatic hydrolysis of coconut husk through Pseudomonas aeruginosa AP 029/GLVIIA rhamnolipid preparation.

PubMed

de Araújo, Cynthia Kérzia Costa; de Oliveira Campos, Alan; de Araújo Padilha, Carlos Eduardo; de Sousa Júnior, Francisco Canindé; do Nascimento, Ruthinéia Jéssica Alves; de Macedo, Gorete Ribeiro; Dos Santos, Everaldo Silvino

2017-08-01

This work investigated the influence of chemical (Triton X-100) and biological surfactant preparation (rhamnolipids) in coconut husk hydrolysis that was subjected to pretreatment with acid-alkali or alkaline hydrogen peroxide. The natural and pretreated biomass was characterized using the National Renewable Energy Laboratory protocol analysis as well as X-ray diffraction and scanning electron microscopy. The results demonstrated that in terms of the total reducing sugars, there was no significant difference between the hydrolysis using Triton X-100 and rhamnolipids, regardless of the pretreatment. A cellulosic conversion value as high as 33.0% was obtained in experiments with rhamnolipids. The coconut husk was observed to be a potential biomass that could produce second generation ethanol, and the rhamnolipid preparation can be used to support for the enzymatic hydrolysis, enhancing the advantage of cellulose conversion into glucose over chemical surfactants because it is an environmentally friendly approach. Copyright © 2017 Elsevier Ltd. All rights reserved.

How water molecules affect the catalytic activity of hydrolases--a XANES study of the local structures of peptide deformylase.

PubMed

Cui, Peixin; Wang, Yu; Chu, Wangsheng; Guo, Xiaoyun; Yang, Feifei; Yu, Meijuan; Zhao, Haifeng; Dong, Yuhui; Xie, Yaning; Gong, Weimin; Wu, Ziyu

2014-12-12

Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis and water molecules play a key role in these hydrolases. Using X-ray absorption near edge spectroscopy (XANES) and ab initio calculations we accurately probe the local atomic environment of the metal ion binding in the active site of PDF at different pH values and with different metal ions. This new approach is an effective way to monitor existing correlations among functions and structural changes. We show for the first time that the enzymatic activity depends on pH values and metal ions via the bond length of the nearest coordinating water (Wat1) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits an enhanced enzymatic activity only when the distance of the Wat1 molecule with the metal ion falls in the limited range from 2.15 to 2.55 Å.

Production of Chitin from Penaeus vannamei By-Products to Pilot Plant Scale Using a Combination of Enzymatic and Chemical Processes and Subsequent Optimization of the Chemical Production of Chitosan by Response Surface Methodology.

PubMed

Vázquez, José A; Ramos, Patrícia; Mirón, Jesús; Valcarcel, Jesus; Sotelo, Carmen G; Pérez-Martín, Ricardo I

2017-06-16

The waste generated from shrimp processing contains valuable materials such as protein, carotenoids, and chitin. The present study describes a process at pilot plant scale to recover chitin from the cephalothorax of Penaeus vannamei using mild conditions. The application of a sequential enzymatic-acid-alkaline treatment yields 30% chitin of comparable purity to commercial sources. Effluents from the process are rich in protein and astaxanthin, and represent inputs for further by-product recovery. As a last step, chitin is deacetylated to produce chitosan; the optimal conditions are established by applying a response surface methodology (RSM). Under these conditions, deacetylation reaches 92% as determined by Proton Nuclear Magnetic Resonance (¹H-NMR), and the molecular weight (Mw) of chitosan is estimated at 82 KDa by gel permeation chromatography (GPC). Chitin and chitosan microstructures are characterized by Scanning Electron Microscopy (SEM).

Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides

NASA Astrophysics Data System (ADS)

Asai, Teigo; Tsukada, Kento; Ise, Satomi; Shirata, Naoki; Hashimoto, Makoto; Fujii, Isao; Gomi, Katsuya; Nakagawara, Kosuke; Kodama, Eiichi N.; Oshima, Yoshiteru

2015-09-01

The structural complexity and diversity of natural products make them attractive sources for potential drug discovery, with their characteristics being derived from the multi-step combination of enzymatic and non-enzymatic conversions of intermediates in each biosynthetic pathway. Intermediates that exhibit multipotent behaviour have great potential for use as starting points in diversity-oriented synthesis. Inspired by the biosynthetic pathways that form complex metabolites from simple intermediates, we developed a semi-synthetic process that combines heterologous biosynthesis and artificial diversification. The heterologous biosynthesis of fungal polyketide intermediates led to the isolation of novel oligomers and provided evidence for ortho-quinonemethide equivalency in their isochromene form. The intrinsic reactivity of the isochromene polyketide enabled us to access various new chemical entities by modifying and remodelling the polyketide core and through coupling with indole molecules. We thus succeeded in generating exceptionally diverse pseudo-natural polyketides through this process and demonstrated an advanced method of using biosynthetic intermediates.

Enzymatic saccharification and bioethanol production from Cynara cardunculus pretreated by steam explosion.

PubMed

Fernandes, Maria C; Ferro, Miguel D; Paulino, Ana F C; Mendes, Joana A S; Gravitis, Janis; Evtuguin, Dmitry V; Xavier, Ana M R B

2015-06-01

The correct choice of the specific lignocellulosic biomass pretreatment allows obtaining high biomass conversions for biorefinery implementations and cellulosic bioethanol production from renewable resources. Cynara cardunculus (cardoon) pretreated by steam explosion (SE) was involved in second-generation bioethanol production using separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) processes. Steam explosion pretreatment led to partial solubilisation of hemicelluloses and increased the accessibility of residual polysaccharides towards enzymatic hydrolysis revealing 64% of sugars yield against 11% from untreated plant material. Alkaline extraction after SE pretreatment of cardoon (CSEOH) promoted partial removal of degraded lignin, tannins, extractives and hemicelluloses thus allowing to double glucose concentration upon saccharification step. Bioethanol fermentation in SSF mode was faster than SHF process providing the best results: ethanol concentration 18.7 g L(-1), fermentation efficiency of 66.6% and a yield of 26.6g ethanol/100 g CSEOH or 10.1 g ethanol/100 g untreated cardoon. Copyright © 2015 Elsevier Ltd. All rights reserved.

Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity.

PubMed

Humenik, Martin; Mohrand, Madeleine; Scheibel, Thomas

2018-04-18

The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allows the generation of fibrous biomaterials with catalytic or light emitting properties.

Combined acid/alkaline-peroxide pretreatment of olive tree biomass for bioethanol production.

PubMed

Martínez-Patiño, José Carlos; Ruiz, Encarnación; Romero, Inmaculada; Cara, Cristóbal; López-Linares, Juan Carlos; Castro, Eulogio

2017-09-01

Olive tree biomass (OTB) can be used for producing second generation bioethanol. In this work, extracted OTB was subjected to fractionation using a sequential acid/alkaline oxidative pretreatment. In the first acid stage, the effects of sulfuric acid concentration and reaction times at 130°C were investigated. Up to 71% solubilization of hemicellulosic sugars was achieved under optimized conditions (2.4% H 2 SO 4 , 84min). In the second stage, the influence of hydrogen peroxide concentration and process time were evaluated at 80°C. Approximately 80% delignification was achieved under the best operational conditions (7% H 2 O 2 , 90min) within the experimental range studied. This pretreatment produced a substrate with 72% cellulose that was highly accessible to enzymatic attack, yielding 82g glucose/100g glucose in delignified OTB. Ethanol production from both hemicellulosic sugars solubilized in the acid pretreatment and glucose from enzymatic hydrolysis of delignified OTB yielded 15g ethanol/100g OTB. Copyright © 2017 Elsevier Ltd. All rights reserved.

Laser microsurgery of higher plant cell walls permits patch-clamp access

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Taylor, A. R.; Brownlee, C.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1996-01-01

Plasma membranes of guard cells in epidermal peels of Vicia faba and Commelina communis can be made accessible to a patch-clamp pipet by removing a small portion (1-3 micrometers in diameter) of the guard cell wall using a microbeam of ultraviolet light generated by a nitrogen laser. Using this laser microsurgical technique, we have measured channel activity across plasma membranes of V. faba guard cells in both cell-attached and isolated patch configurations. Measurements made in the inside-out patch configuration revealed two distinct K(+)-selective channels. Major advantages of the laser microsurgical technique include the avoidance of enzymatic protoplast isolation, the ability to study cell types that have been difficult to isolate as protoplasts or for which enzymatic isolation protocols result in protoplasts not amenable to patch-clamp studies, the maintenance of positional information in single-channel measurements, reduced disruption of cell-wall-mediated signaling pathways, and the ability to investigate intercellular signaling through studies of cells remaining situated within tissue.

Hyperthyroidism in the developing rat testis is associated with oxidative stress and hyperphosphorylated vimentin accumulation.

PubMed

Zamoner, Ariane; Barreto, Kátia Padilha; Filho, Danilo Wilhelm; Sell, Fabíola; Woehl, Viviane Mara; Guma, Fátima Costa Rodrigues; Silva, Fátima Regina Mena Barreto; Pessoa-Pureur, Regina

2007-03-15

Hyperthyroidism was induced in rats and somatic indices and metabolic parameters were analyzed in testis. In addition, the morphological analysis evidenced testes maturation and intense protein synthesis and processing, supporting the enhancement in vimentin synthesis in hyperthyroid testis. Furthermore, vimentin phosphorylation was increased, indicating an accumulation of phosphorylated vimentin associated to the cytoskeleton, which could be a consequence of the extracellular-regulated kinase (ERK) activation regulating the cytoskeleton. Biomarkers of oxidative stress demonstrated an increased basal metabolic rate measured by tissue oxygen consumption, as well as, increased TBARS levels. In addition, the enzymatic and non-enzymatic antioxidant defences appeared to respond according to the augmented oxygen consumption. We observed decreased total glutathione levels, with enhancement of reduced glutathione, whereas most of the antioxidant enzyme activities were induced. Otherwise, superoxide dismutase activity was inhibited. These results support the idea that an increase in mitochondrial ROS generation, underlying cellular oxidative damage, is a side effect of hyperthyroid-induced biochemical changes by which rat testis increase their metabolic capacity.

Solar assisted alkali pretreatment of garden biomass: Effects on lignocellulose degradation, enzymatic hydrolysis, crystallinity and ultra-structural changes in lignocellulose.

PubMed

Gabhane, Jagdish; William, S P M Prince; Vaidya, Atul N; Das, Sera; Wate, Satish R

2015-06-01

A comprehensive study was carried out to assess the effectiveness of solar assisted alkali pretreatment (SAAP) on garden biomass (GB). The pretreatment efficiency was assessed based on lignocellulose degradation, conversion of cellulose into reducing sugars, changes in the ultra-structure and functional groups of lignocellulose and impact on the crystallinity of cellulose, etc. SAAP was found to be efficient for the removal of lignin and hemicellulose that facilitated enzymatic hydrolysis of cellulose. FTIR and XRD studies provided details on the effectiveness of SAAP on lignocellulosic moiety and crystallinity of cellulose. Scanning electron microscopic analysis showed ultra-structural disturbances in the microfibrils of GB as a result of pretreatment. The mass balance closer of 97.87% after pretreatment confirmed the reliability of SAAP pretreatment. Based on the results, it is concluded that SAAP is not only an efficient means of pretreatment but also economical as it involved no energy expenditure for heat generation during pretreatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Gluten-Free Diet: Testing Alternative Cereals Tolerated by Celiac Patients

PubMed Central

Comino, Isabel; de Lourdes Moreno, María; Real, Ana; Rodríguez-Herrera, Alfonso; Barro, Francisco; Sousa, Carolina

2013-01-01

A strict gluten-free diet (GFD) is the only currently available therapeutic treatment for patients with celiac disease, an autoimmune disorder of the small intestine associated with a permanent intolerance to gluten proteins. The complete elimination of gluten proteins contained in cereals from the diet is the key to celiac disease management. However, this generates numerous social and economic repercussions due to the ubiquity of gluten in foods. The research presented in this review focuses on the current status of alternative cereals and pseudocereals and their derivatives obtained by natural selection, breeding programs and transgenic or enzymatic technology, potential tolerated by celiac people. Finally, we describe several strategies for detoxification of dietary gluten. These included enzymatic cleavage of gliadin fragment by Prolyl endopeptidases (PEPs) from different organisms, degradation of toxic peptides by germinating cereal enzymes and transamidation of cereal flours. This information can be used to search for and develop cereals with the baking and nutritional qualities of toxic cereals, but which do not exacerbate this condition. PMID:24152755

Impact of HVAC filter on indoor air quality in terms of ozone removal and carbonyls generation

NASA Astrophysics Data System (ADS)

Lin, Chi-Chi; Chen, Hsuan-Yu

2014-06-01

This study aims at detecting ozone removal rates and corresponding carbonyls generated by ozone reaction with HVAC filters from various building, i.e., shopping mall, school, and office building. Studies were conducted in a small-scale environmental chamber. By examining dust properties including organic carbon proportion and specific surface area of dusts adsorbed on filters along with ozone removal rates and carbonyls generation rate, the relationship among dust properties, ozone removal rates, and carbonyls generation was identified. The results indicate a well-defined positive correlation between ozone removal efficiency and carbonyls generation on filters, as well as a positive correlation among the mass of organic carbon on filters, ozone removal efficiency and carbonyls generations.

Semi-Automatic Modelling of Building FAÇADES with Shape Grammars Using Historic Building Information Modelling

NASA Astrophysics Data System (ADS)

Dore, C.; Murphy, M.

2013-02-01

This paper outlines a new approach for generating digital heritage models from laser scan or photogrammetric data using Historic Building Information Modelling (HBIM). HBIM is a plug-in for Building Information Modelling (BIM) software that uses parametric library objects and procedural modelling techniques to automate the modelling stage. The HBIM process involves a reverse engineering solution whereby parametric interactive objects representing architectural elements are mapped onto laser scan or photogrammetric survey data. A library of parametric architectural objects has been designed from historic manuscripts and architectural pattern books. These parametric objects were built using an embedded programming language within the ArchiCAD BIM software called Geometric Description Language (GDL). Procedural modelling techniques have been implemented with the same language to create a parametric building façade which automatically combines library objects based on architectural rules and proportions. Different configurations of the façade are controlled by user parameter adjustment. The automatically positioned elements of the façade can be subsequently refined using graphical editing while overlaying the model with orthographic imagery. Along with this semi-automatic method for generating façade models, manual plotting of library objects can also be used to generate a BIM model from survey data. After the 3D model has been completed conservation documents such as plans, sections, elevations and 3D views can be automatically generated for conservation projects.

3. DETAIL OF SOUTH AND WEST SIDES OF BUILDING 522. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. DETAIL OF SOUTH AND WEST SIDES OF BUILDING 522. VIEW TO NORTHEAST. - Rocky Mountain Arsenal, White Phosphorous Filling-Acetylene Generation Building-Warehouse, 840 feet South of December Seventh Avenue; 1030 feet East of D Street, Commerce City, Adams County, CO

Emerging Challenges and Opportunities in Building Information Modeling for the US Army Installation Management Command

DTIC Science & Technology

2012-07-01

Information Modeling ( BIM ) is the process of generating and managing building data during a facility’s entire life cycle. New BIM standards for...cycle Building Information Modeling ( BIM ) as a new standard for building information data repositories can serve as the foun- dation for automation and... Building Information Modeling ( BIM ) is defined as “a digital representa- tion of physical and functional

Multi-Level Building Reconstruction for Automatic Enhancement of High Resolution Dsms

NASA Astrophysics Data System (ADS)

Arefi, H.; Reinartz, P.

2012-07-01

In this article a multi-level approach is proposed for reconstruction-based improvement of high resolution Digital Surface Models (DSMs). The concept of Levels of Detail (LOD) defined by CityGML standard has been considered as basis for abstraction levels of building roof structures. Here, the LOD1 and LOD2 which are related to prismatic and parametric roof shapes are reconstructed. Besides proposing a new approach for automatic LOD1 and LOD2 generation from high resolution DSMs, the algorithm contains two generalization levels namely horizontal and vertical. Both generalization levels are applied to prismatic model of buildings. The horizontal generalization allows controlling the approximation level of building footprints which is similar to cartographic generalization concept of the urban maps. In vertical generalization, the prismatic model is formed using an individual building height and continuous to included all flat structures locating in different height levels. The concept of LOD1 generation is based on approximation of the building footprints into rectangular or non-rectangular polygons. For a rectangular building containing one main orientation a method based on Minimum Bounding Rectangle (MBR) in employed. In contrast, a Combined Minimum Bounding Rectangle (CMBR) approach is proposed for regularization of non-rectilinear polygons, i.e. buildings without perpendicular edge directions. Both MBRand CMBR-based approaches are iteratively employed on building segments to reduce the original building footprints to a minimum number of nodes with maximum similarity to original shapes. A model driven approach based on the analysis of the 3D points of DSMs in a 2D projection plane is proposed for LOD2 generation. Accordingly, a building block is divided into smaller parts according to the direction and number of existing ridge lines. The 3D model is derived for each building part and finally, a complete parametric model is formed by merging all the 3D models of the individual parts and adjusting the nodes after the merging step. In order to provide an enhanced DSM, a surface model is provided for each building by interpolation of the internal points of the generated models. All interpolated models are situated on a Digital Terrain Model (DTM) of corresponding area to shape the enhanced DSM. Proposed DSM enhancement approach has been tested on a dataset from Munich central area. The original DSM is created using robust stereo matching of Worldview-2 stereo images. A quantitative assessment of the new DSM by comparing the heights of the ridges and eaves shows a standard deviation of better than 50cm.

Systematic Evaluation of the Efficacy of Chlorine Dioxide in Decontamination of Building Interior Surfaces Contaminated with Anthrax Spores▿

PubMed Central

Rastogi, Vipin K.; Ryan, Shawn P.; Wallace, Lalena; Smith, Lisa S.; Shah, Saumil S.; Martin, G. Blair

2010-01-01

Efficacy of chlorine dioxide (CD) gas generated by two distinct generation systems, Sabre (wet system with gas generated in water) and ClorDiSys (dry system with gas generated in air), was evaluated for inactivation of Bacillus anthracis spores on six building interior surfaces. The six building materials included carpet, acoustic ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. There was no statistically significant difference in the data due to the CD generation technology at a 95% confidence level. Note that a common method of CD gas measurement was used for both wet and dry CD generation types. Doses generated by combinations of different concentrations of CD gas (500, 1,000, 1,500, or 3,000 parts per million of volume [ppmv]) and exposure times (ranging between 0.5 and 12 h) were used to evaluate the relative role of fumigant exposure period and total dose in the decontamination of building surfaces. The results showed that the time required to achieve at least a 6-log reduction in viable spores is clearly a function of the material type on which the spores are inoculated. The wood and cinder block coupons required a longer exposure time to achieve a 6-log reduction. The only material showing a clear statistical difference in rate of decay of viable spores as a function of concentration was cinder block. For all other materials, the profile of spore kill (i.e., change in number of viable spores with exposure time) was not dependent upon fumigant concentration (500 to 3,000 ppmv). The CD dose required for complete spore kill on biological indicators (typically, 1E6 spores of Bacillus atrophaeus on stainless steel) was significantly less than that required for decontamination of most of the building materials tested. PMID:20305025

Systematic evaluation of the efficacy of chlorine dioxide in decontamination of building interior surfaces contaminated with anthrax spores.

PubMed

Rastogi, Vipin K; Ryan, Shawn P; Wallace, Lalena; Smith, Lisa S; Shah, Saumil S; Martin, G Blair

2010-05-01

Efficacy of chlorine dioxide (CD) gas generated by two distinct generation systems, Sabre (wet system with gas generated in water) and ClorDiSys (dry system with gas generated in air), was evaluated for inactivation of Bacillus anthracis spores on six building interior surfaces. The six building materials included carpet, acoustic ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. There was no statistically significant difference in the data due to the CD generation technology at a 95% confidence level. Note that a common method of CD gas measurement was used for both wet and dry CD generation types. Doses generated by combinations of different concentrations of CD gas (500, 1,000, 1,500, or 3,000 parts per million of volume [ppmv]) and exposure times (ranging between 0.5 and 12 h) were used to evaluate the relative role of fumigant exposure period and total dose in the decontamination of building surfaces. The results showed that the time required to achieve at least a 6-log reduction in viable spores is clearly a function of the material type on which the spores are inoculated. The wood and cinder block coupons required a longer exposure time to achieve a 6-log reduction. The only material showing a clear statistical difference in rate of decay of viable spores as a function of concentration was cinder block. For all other materials, the profile of spore kill (i.e., change in number of viable spores with exposure time) was not dependent upon fumigant concentration (500 to 3,000 ppmv). The CD dose required for complete spore kill on biological indicators (typically, 1E6 spores of Bacillus atrophaeus on stainless steel) was significantly less than that required for decontamination of most of the building materials tested.

Stimulation and inhibition of enzymatic hydrolysis by organosolv lignins as determined by zeta potential and hydrophobicity

Treesearch

Yang Huang; Shaolong Sun; Chen Huang; Qiang Yong; Thomas Elder; Maobing Tu

2017-01-01

Background: Lignin typically inhibits enzymatic hydrolysis of cellulosic biomass, but certain organosolv lignins or lignosulfonates enhance enzymatic hydrolysis. The hydrophobic and electrostatic interactions between lignin and cellulases play critical roles in the enzymatic hydrolysis process. However, how to incorporate these two...

Investigating the Functional Role of Prostate-Specific Membrane Antigen and its Enzymatic Activity in Prostate Cancer Metastasis

DTIC Science & Technology

2007-02-01

saponin (Calbiochem, San Diego, CA) in PBS. Results, Significance, Obstacles and Alternative Approaches: We have generated several different fluorescent...1 integrin antibody P4C10 (Life technologies ). We will conjugate the fluorescent probes to these functional blocking antibodies for live cell...characterization of the prostate-specific membrane antigen (PSMA) in tissue extracts and body fluids. Int. J. Cancer. 62:552-558. 1995. 9. Wright GL Jr

Investigating the Functional Role of Prostate-Specific Membrane Antigen and its Enzymatic Activity in Prostate Cancer Metastasis

DTIC Science & Technology

2008-02-01

fluorescent probes for live cell imaging . PSMA distribution of cells grown on different extracellular matrices will be characterized to provide guidance...PCa migration, using in vitro cell model systems and live - cell imaging methods, we characterized the role of PSMA in cell motility and adhesion. Using...Generated fluorescently conjugated anti-PSMA antibodies for live cell imaging . 2. Optimized the siRNA-PSMA transfection and achieved an approximately

Detection of Bacteria Using Inkjet-Printed Enzymatic Test Strips

PubMed Central

2015-01-01

Low-cost diagnostics for drinking water contamination have the potential to save millions of lives. We report a method that uses inkjet printing to copattern an enzyme–nanoparticle sensor and substrate on a paper-based test strip for rapid detection of bacteria. A colorimetric response is generated on the paper substrate that allows visual detection of contamination without the need for expensive instrumentation. These strips demonstrate a viable nanomanufacturing strategy for low-cost bacterial detection. PMID:25318086

Enzymatic aspects in ENT cancer-Matrix metalloproteinases

PubMed Central

Zamfir Chiru, AA; Popescu, CR; Gheorghe, DC

2014-01-01

Abstract The study of ENT cancer allows the implementation of molecular biology methods in diagnosis, predicting the evolution of the disease and suggesting a certain treatment. MMPs are proteolytic enzymes, zinc dependent endopeptidases, secreted by tissues and proinflammatory cells that play a role in the clearance of cell surface receptors. They are expressed as zymogens (inactive forms). Proteolytic enzymes cleave zymogens generating active forms. They are involved in cell proliferation, adhesion, differentiation, migration, angiogenesis, apoptosis and host defense. PMID:25408759

PROCESS FOR CONTROLLING ANIMAL GROWTH RATE

DOEpatents

Visek, W.J.

1962-04-10

A method of injecting growing animals with the enzyme urease subcutaneously in increasing dosages is described; this generates within the blood anti-urease which enters the intestinal tract and inhibits the enzymatic decomposition of urea by urease in that location. Ammonia, one of the decomposition products, is thereby kept from diffusing through the intestinal walls into the blood, and this greatly reduces the energy requirements of the liver for removing the ammonia, thereby increasing the feeding efficiency of the animals. (AEC)

Molecular MR Imaging of CD44 in Breast Cancer with Hyaluronan-Based Contrast Agents

DTIC Science & Technology

2009-09-01

linear polysaccharide composed of alternating (β-1,4)-linked d- glucuronic acid and (β-1,3) N-acetyl-d-glucosamine residues with molecular weights as...enzymatic reactions in-vivo that generate polysaccharides of decreasing sizes, which in principle may facilitate the timely excretion of HA based...14CO2) or in urine (as low molecular weight HA or monosaccharide fragments). The same authors also reported that the total amount of excretion into

Engineering multifunctional protein nanoparticles by in vitro disassembling and reassembling of heterologous building blocks

NASA Astrophysics Data System (ADS)

Unzueta, Ugutz; Serna, Naroa; Sánchez-García, Laura; Roldán, Mónica; Sánchez-Chardi, Alejandro; Mangues, Ramón; Villaverde, Antonio; Vázquez, Esther

2017-12-01

The engineering of protein self-assembling at the nanoscale allows the generation of functional and biocompatible materials, which can be produced by easy biological fabrication. The combination of cationic and histidine-rich stretches in fusion proteins promotes oligomerization as stable protein-only regular nanoparticles that are composed by a moderate number of building blocks. Among other applications, these materials are highly appealing as tools in targeted drug delivery once empowered with peptidic ligands of cell surface receptors. In this context, we have dissected here this simple technological platform regarding the controlled disassembling and reassembling of the composing building blocks. By applying high salt and imidazole in combination, nanoparticles are disassembled in a process that is fully reversible upon removal of the disrupting agents. By taking this approach, we accomplish here the in vitro generation of hybrid nanoparticles formed by heterologous building blocks. This fact demonstrates the capability to generate multifunctional and/or multiparatopic or multispecific materials usable in nanomedical applications.

Ring system-based chemical graph generation for de novo molecular design

NASA Astrophysics Data System (ADS)

Miyao, Tomoyuki; Kaneko, Hiromasa; Funatsu, Kimito

2016-05-01

Generating chemical graphs in silico by combining building blocks is important and fundamental in virtual combinatorial chemistry. A premise in this area is that generated structures should be irredundant as well as exhaustive. In this study, we develop structure generation algorithms regarding combining ring systems as well as atom fragments. The proposed algorithms consist of three parts. First, chemical structures are generated through a canonical construction path. During structure generation, ring systems can be treated as reduced graphs having fewer vertices than those in the original ones. Second, diversified structures are generated by a simple rule-based generation algorithm. Third, the number of structures to be generated can be estimated with adequate accuracy without actual exhaustive generation. The proposed algorithms were implemented in structure generator Molgilla. As a practical application, Molgilla generated chemical structures mimicking rosiglitazone in terms of a two dimensional pharmacophore pattern. The strength of the algorithms lies in simplicity and flexibility. Therefore, they may be applied to various computer programs regarding structure generation by combining building blocks.

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

Elsasser, Brigitta M.; Schoenen, Iris; Fels, Gregor

Candida antarctica lipase B (CALB) efficiently catalyzes the ring-opening polymerization of lactones to high molecular weight products in good yield. In contrast, an efficient enzymatic synthesis of polyamides has so far not been described in the literature. This obvious difference in enzyme catalysis is the subject of our comparative study of the initial steps of a CALB catalyzed ring-opening polymerization of ε- caprolactone and ε-caprolactam. We have applied docking tools to generate the reactant state complex and performed quantum mechanical/molecular mechanical (QM/MM) calculations at the density functional theory (DFT) PBE0 level of theory to simulate the acylation of Ser105 bymore » the lactone and the lactam, respectively, via the corresponding first tetrahedral intermediates. We could identify a decisive difference in the accessibility of the two substrates in the ring-opening to the respective acyl enzyme complex as the attack of ε-caprolactam is hindered because of an energetically disfavored proton transfer during this part of the catalytic reaction while ε-caprolactone is perfectly processed along the widely accepted pathway using the catalytic triade of Ser105, His224, and Asp187. Since the generation of an acylated Ser105 species is the crucial step of the polymerization procedure, our results give an explanation for the unsatisfactory enzymatic polyamide formation and opens up new possibilities for targeted rational catalyst redesign in hope of an experimentally useful CALB catalyzed polyamide synthesis.« less

Establishing MALDI-TOF as Versatile Drug Discovery Readout to Dissect the PTP1B Enzymatic Reaction.

PubMed

Winter, Martin; Bretschneider, Tom; Kleiner, Carola; Ries, Robert; Hehn, Jörg P; Redemann, Norbert; Luippold, Andreas H; Bischoff, Daniel; Büttner, Frank H

2018-07-01

Label-free, mass spectrometric (MS) detection is an emerging technology in the field of drug discovery. Unbiased deciphering of enzymatic reactions is a proficient advantage over conventional label-based readouts suffering from compound interference and intricate generation of tailored signal mediators. Significant evolvements of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, as well as associated liquid handling instrumentation, triggered extensive efforts in the drug discovery community to integrate the comprehensive MS readout into the high-throughput screening (HTS) portfolio. Providing speed, sensitivity, and accuracy comparable to those of conventional, label-based readouts, combined with merits of MS-based technologies, such as label-free parallelized measurement of multiple physiological components, emphasizes the advantages of MALDI-TOF for HTS approaches. Here we describe the assay development for the identification of protein tyrosine phosphatase 1B (PTP1B) inhibitors. In the context of this precious drug target, MALDI-TOF was integrated into the HTS environment and cross-compared with the well-established AlphaScreen technology. We demonstrate robust and accurate IC 50 determination with high accordance to data generated by AlphaScreen. Additionally, a tailored MALDI-TOF assay was developed to monitor compound-dependent, irreversible modification of the active cysteine of PTP1B. Overall, the presented data proves the promising perspective for the integration of MALDI-TOF into drug discovery campaigns.

Highly sensitive glucose biosensor based on the effective immobilization of glucose oxidase/carbon-nanotube and gold nanoparticle in nafion film and peroxyoxalate chemiluminescence reaction of a new fluorophore.

PubMed

Zargoosh, Kiomars; Chaichi, Mohammad Javad; Shamsipur, Mojtaba; Hossienkhani, Saman; Asghari, Sakineh; Qandalee, Mohammad

2012-05-15

A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated H(2)O(2) was constructed by the effective immobilization of glucose oxidase (GOD)/carbon-nanotubes (CNTs)/gold nanoparticles (GNPs) in nafion film on graphite support. The influences of various experimental parameters such as solution pH, the action time of the enzyme, interferents and the concentration of CL reagents were investigated. Carbon nanotubes and gold nanoparticles offer excellent catalytic activity toward hydrogen peroxide generation in enzymatic reaction between glucose oxidase and glucose, which would enable sensitive determination of glucose. Under the optimum condition, the linear response range of glucose was found to be 2.25 × 10(-6) to 1.75 × 10(-4 ) mol L(-1), and the detection limit (defined as the concentration that could be detected at the signal-to-noise ratio of 3) was 1.00 × 10(-6) mol L(-1). The CL biosensor exhibited good storage stability, i.e., 80% of its initial response was retained after 10 days storage at pH 7.0. The present CL biosensor has been used to determine the glucose concentrations in real serum and urine samples with satisfactory results. Copyright © 2011 Elsevier B.V. All rights reserved.

A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis.

PubMed

Herget, Meike; Scheibinger, Mirko; Guo, Zhaohua; Jan, Taha A; Adams, Christopher M; Cheng, Alan G; Heller, Stefan

2013-01-01

Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

Reticulated vitreous carbon as a scaffold for enzymatic fuel cell designing.

PubMed

Kizling, Michal; Dzwonek, Maciej; Olszewski, Bartłomiej; Bącal, Paweł; Tymecki, Łukasz; Więckowska, Agnieszka; Stolarczyk, Krzysztof; Bilewicz, Renata

2017-09-15

Three - dimensional (3D) electrodes are successfully used to overcome the limitations of the low space - time yield and low normalized space velocity obtained in electrochemical processes with two - dimensional electrodes. In this study, we developed a three - dimensional reticulated vitreous carbon - gold (RVC-Au) sponge as a scaffold for enzymatic fuel cells (EFC). The structure of gold and the real electrode surface area can be controlled by the parameters of metal electrodeposition. In particular, a 3D RVC-Au sponge provides a large accessible surface area for immobilization of enzyme and electron mediators, moreover, effective mass diffusion can also take place through the uniform macro - porous scaffold. To efficiently bind the enzyme to the electrode and enhance electron transfer parameters the gold surface was modified with ultrasmall gold nanoparticles stabilized with glutathione. These quantum sized nanoparticles exhibit specific electronic properties and also expand the working surface of the electrode. Significantly, at the steady state of power generation, the EFC device with RVC-Au electrodes provided high volumetric power density of 1.18±0.14mWcm -3 (41.3±3.8µWcm -2 ) calculated based on the volume of electrode material with OCV 0.741±0.021V. These new 3D RVC-Au electrodes showed great promise for improving the power generation of EFC devices. Copyright © 2017 Elsevier B.V. All rights reserved.

An Overview of the Chemistry and Biology of Reactive Aldehydes

PubMed Central

Fritz, Kristofer S.; Petersen, Dennis R.

2012-01-01

The non-enzymatic free radical generation of reactive aldehydes is known to contribute to diseases of sustained oxidative stress including rheumatoid arthritis, atherosclerosis, neurodegenerative and a number of liver diseases. At the same time, the accumulation of lipid electrophiles has been demonstrated to play a role in cell signaling events through modification of proteins critical for cellular homeostasis. Given the broad scope of reactivity profiles and the ability to modify numerous proteomic and genomic processes, new emphasis is being placed on a systems-based analysis of the consequences of electrophilic adduction. This review focuses on the generation and chemical reactivity of lipid-derived aldehydes with a special focus on the homeostatic responses to electrophilic stress. PMID:22750507

Aging management program of the reactor building concrete at Point Lepreau Generating Station

NASA Astrophysics Data System (ADS)

Aldea, C.-M.; Shenton, B.; Demerchant, M. M.; Gendron, T.

2011-04-01

In order for New Brunswick Power Nuclear (NBPN) to control the risks of degradation of the concrete reactor building at the Point Lepreau Generating Station (PLGS) the development of an aging management plan (AMP) was initiated. The intention of this plan was to determine the requirements for specific structural components of concrete of the reactor building that require regular inspection and maintenance to ensure the safe and reliable operation of the plant. The document is currently in draft form and presents an integrated methodology for the application of an AMP for the concrete of the reactor building. The current AMP addresses the reactor building structure and various components, such as joint sealant and liners that are integral to the structure. It does not include internal components housed within the structure. This paper provides background information regarding the document developed and the strategy developed to manage potential degradation of the concrete of the reactor building, as well as specific programs and preventive and corrective maintenance activities initiated.

Using Openstreetmap Data to Generate Building Models with Their Inner Structures for 3d Maps

NASA Astrophysics Data System (ADS)

Wang, Z.; Zipf, A.

2017-09-01

With the development of Web 2.0, more and more data related to indoor environments has been collected within the volunteered geographic information (VGI) framework, which creates a need for construction of indoor environments from VGI. In this study, we focus on generating 3D building models from OpenStreetMap (OSM) data, and provide an approach to support construction and visualization of indoor environments on 3D maps. In this paper, we present an algorithm which can extract building information from OSM data, and can construct building structures as well as inner building components (e.g., doors, rooms, and windows). A web application is built to support the processing and visualization of the building models on a 3D map. We test our approach with an indoor dataset collected from the field. The results show the feasibility of our approach and its potentials to provide support for a wide range of applications, such as indoor and outdoor navigation, urban planning, and incident management.

Demand controlled ventilating systems: Sensor market survey. Energy conservation in buildings and community systems programme, annex 18, December 1991

NASA Astrophysics Data System (ADS)

Raatschen, W.; Sjoegren, M.

The subject of indoor and outdoor air quality has generated a great deal of attention in many countries. Areas of concern include outgassing of building materials as well as occupant-generated pollutants such as carbon dioxide, moisture, and odors. Progress has also been made towards addressing issues relating to the air tightness of the building envelope. Indoor air quality studies indicate that better control of supply flow rates as well as the air distribution pattern within buildings are necessary. One method of maintaining good indoor air quality without extensive energy consumption is to control the ventilation rate according to the needs and demands of the occupants, or to preserve the building envelope. This is accomplished through the use of demand controlled ventilating (DCV) systems. The specific objective of Annex 18 is to develop guidelines for demand controlled ventilating systems based on state of the art analyses, case studies on ventilation effectiveness, and proposed ventilation rates for different users in domestic, office, and school buildings.

Joint Real-Time Energy and Demand-Response Management using a Hybrid Coalitional-Noncooperative Game

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

He, Fulin; Gu, Yi; Hao, Jun

In order to model the interactions among utility companies, building demands and renewable energy generators (REGs), a hybrid coalitional-noncooperative game framework has been proposed. We formulate a dynamic non-cooperative game to study the energy dispatch within multiple utility companies, while we take a coalitional perspective on REGs and buildings demands through a hedonic coalition formation game approach. In this case, building demands request different power supply from REGs, then the building demands can be organized into an ultimate coalition structure through a distributed hedonic shift algorithm. At the same time, utility companies can also obtain a stable power generation profile.more » In addition, the interactive progress among the utility companies and building demands which cannot be supplied by REGs is implemented by distributed game theoretic algorithms. Numerical results illustrate that the proposed hybrid coalitional-noncooperative game scheme reduces the cost of both building demands and utility companies compared with the initial scene.« less

Urban Heat Island Effect on the Energy Consumption of Institutional Buildings in Rome

NASA Astrophysics Data System (ADS)

Calice, Claudia; Clemente, Carola; Salvati, Agnese; Palme, Massimo; Inostroza, Luis

2017-10-01

The urban heat island (UHI) effect is constantly increasing the energy consumption of buildings, especially in summer periods. The energy gap between the estimated energy performance - often simulated without considering UHI - and the real operational consumption is especially relevant for institutional buildings, where the cooling needs are in general higher than in other kind of buildings, due to more internal gains (people, appliances) and different architectural design (more transparent façades and light walls). This paper presents a calculation of the energy penalty due to UHI in two institutional buildings in Rome. Urban Weather Generator (UWG) is used to generate a modified weather file, taking into account the UHI phenomenon. Then, two building performance simulations are done for each case: the first simulation uses a standard weather file and the second uses the modified one. Results shows how is it necessary to re-develop mitigation strategies and a new energy retrofit approach, in order to include urbanization ad UHI effect, especially in this kind of buildings, characterized by very poor conditions of comfort during summer, taking into account users and occupant-driven demand.

Dynamic peptide libraries for the discovery of supramolecular nanomaterials

NASA Astrophysics Data System (ADS)

Pappas, Charalampos G.; Shafi, Ramim; Sasselli, Ivan R.; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V.

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

Sender–receiver systems and applying information theory for quantitative synthetic biology

PubMed Central

Barcena Menendez, Diego; Senthivel, Vivek Raj; Isalan, Mark

2015-01-01

Sender–receiver (S–R) systems abound in biology, with communication systems sending information in various forms. Information theory provides a quantitative basis for analysing these processes and is being applied to study natural genetic, enzymatic and neural networks. Recent advances in synthetic biology are providing us with a wealth of artificial S–R systems, giving us quantitative control over networks with a finite number of well-characterised components. Combining the two approaches can help to predict how to maximise signalling robustness, and will allow us to make increasingly complex biological computers. Ultimately, pushing the boundaries of synthetic biology will require moving beyond engineering the flow of information and towards building more sophisticated circuits that interpret biological meaning. PMID:25282688

Dynamic peptide libraries for the discovery of supramolecular nanomaterials.

PubMed

Pappas, Charalampos G; Shafi, Ramim; Sasselli, Ivan R; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

First Prismatic Building Model Reconstruction from Tomosar Point Clouds

NASA Astrophysics Data System (ADS)

Sun, Y.; Shahzad, M.; Zhu, X.

2016-06-01

This paper demonstrates for the first time the potential of explicitly modelling the individual roof surfaces to reconstruct 3-D prismatic building models using spaceborne tomographic synthetic aperture radar (TomoSAR) point clouds. The proposed approach is modular and works as follows: it first extracts the buildings via DSM generation and cutting-off the ground terrain. The DSM is smoothed using BM3D denoising method proposed in (Dabov et al., 2007) and a gradient map of the smoothed DSM is generated based on height jumps. Watershed segmentation is then adopted to oversegment the DSM into different regions. Subsequently, height and polygon complexity constrained merging is employed to refine (i.e., to reduce) the retrieved number of roof segments. Coarse outline of each roof segment is then reconstructed and later refined using quadtree based regularization plus zig-zag line simplification scheme. Finally, height is associated to each refined roof segment to obtain the 3-D prismatic model of the building. The proposed approach is illustrated and validated over a large building (convention center) in the city of Las Vegas using TomoSAR point clouds generated from a stack of 25 images using Tomo-GENESIS software developed at DLR.

Quantifying the waste reduction potential of using prefabrication in building construction in Hong Kong.

PubMed

Jaillon, L; Poon, C S; Chiang, Y H

2009-01-01

As Hong Kong is a compact city with limited available land and high land prices, the construction of high-rise buildings is prevalent. The construction industry produces a significant amount of building waste. In 2005, about 21.5 million tonnes of construction waste were generated, of which 11% was disposed of in landfills and 89% in public filling areas. At the present rate, Hong Kong will run out of both public filling areas and landfill space within the next decade. The government is taking action to tackle the problem, such as by introducing a construction waste landfill charge, and promoting prefabrication to reduce on-site waste generation. This paper reports an ongoing study on the use of prefabrication in buildings and its impact on waste reduction in Hong Kong. A questionnaire survey was administered to experienced professionals, and case studies of recently completed building projects were conducted. The results revealed that construction waste reduction is one of the major benefits when using prefabrication compared with conventional construction. The average wastage reduction level was about 52%. This implies that a wider use of prefabrication could considerably reduce construction waste generation in Hong Kong and alleviate the burdens associated with its management.

ENERGY STAR and Green Buildings--Using ENERGY STAR Resources for Green Building Rating Systems: LEED[R], Green Globes[R] and CHPS

ERIC Educational Resources Information Center

Utebay, Kudret

2011-01-01

Every building, from the smallest school to the tallest skyscraper, uses energy. This energy is most often generated by burning fossil fuels, which releases greenhouse gases into the atmosphere and contributes to climate change. Existing commercial buildings offer a significant opportunity for low-cost, immediate emissions and energy cost…

Improving the Quality of School Facilities through Building Performance Assessment: Educational Reform and School Building Quality in Sao Paulo, Brazil

ERIC Educational Resources Information Center

Ornstein, Sheila Walbe; Moreira, Nanci Saraiva; Ono, Rosaria; Limongi Franca, Ana J. G.; Nogueira, Roselene A. M. F.

2009-01-01

Purpose: The paper describes the purpose of and strategies for conducting post-occupancy evaluations (POEs) as a method for assessing school building performance. Set within the larger context of global efforts to develop and apply common indicators of school building quality, the authors describe research conducted within the newest generation of…

Evaluation of physical structural features on influencing enzymatic hydrolysis efficiency of micronized wood

Treesearch

Jinxue Jiang; Jinwu Wang; Xiao Zhang; Michael Wolcott

2016-01-01

Enzymatic hydrolysis of lignocellulosic biomass is highly dependent on the changes in structural features after pretreatment. Mechanical milling pretreatment is an effective approach to alter the physical structure of biomass and thus improve enzymatic hydrolysis. This study examined the influence of structural characteristics on the enzymatic hydrolysis of micronized...

Bill Livingood | NREL

Science.gov Websites

Bill Livingood Photo of Bill Livingood Bill Livingood Group Manager, Commercial Buildings manager of the commercial buildings research team, he oversees the development of next generation

The near-source impacts of diesel backup generators in urban environments

NASA Astrophysics Data System (ADS)

Tong, Zheming; Zhang, K. Max

2015-05-01

Distributed power generation, located close to consumers, plays an important role in the current and future power systems. However, its near-source impacts in complex urban environments are not well understood. In this paper, we focused on diesel backup generators that participate in demand response (DR) programs. We first improved the micro-environmental air quality simulations by employing a meteorology processor, AERMET, to generate site-specific boundary layer parameters for the Large Eddy Simulation (LES) modeling. The modeling structure was then incorporated into the CTAG model to evaluate the environmental impacts of diesel backup generators in near-source microenvironments. We found that the presence of either tall upwind or downwind building can deteriorate the air quality in the near-stack street canyons, largely due to the recirculation zones generated by the tall buildings, reducing the near-stack dispersion. Decreasing exhaust momentum ratio (stack exit velocity/ambient wind velocity) draws more exhaust into the recirculation zone, and reduces the effective stack height, which results in elevated near-ground concentrations inside downwind street canyons. The near-ground PM2.5 concentration for the worst scenarios could well exceed 100 μg m-3, posing potential health risk to people living and working nearby. In general, older diesel backup generators (i.e., Tier 1, 2 or older) without the up-to-date emission control may significantly increase the pollutant concentration in the near-source street canyons if participating in DR programs. Even generators that comply with Tier-4 standards could lead to PM hotspots if their stacks are next to tall buildings. Our study implies that the siting of diesel backup generators stacks should consider not only the interactions of fresh air intake and exhaust outlet for the building housing the backup generators, but also the dispersion of exhaust plumes in the surrounding environment.

Metals, toxicity and oxidative stress.

PubMed

Valko, M; Morris, H; Cronin, M T D

2005-01-01

Metal-induced toxicity and carcinogenicity, with an emphasis on the generation and role of reactive oxygen and nitrogen species, is reviewed. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). Whilst iron (Fe), copper (Cu), chromium (Cr), vanadium (V) and cobalt (Co) undergo redox-cycling reactions, for a second group of metals, mercury (Hg), cadmium (Cd) and nickel (Ni), the primary route for their toxicity is depletion of glutathione and bonding to sulfhydryl groups of proteins. Arsenic (As) is thought to bind directly to critical thiols, however, other mechanisms, involving formation of hydrogen peroxide under physiological conditions, have been proposed. The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Common mechanisms involving the Fenton reaction, generation of the superoxide radical and the hydroxyl radical appear to be involved for iron, copper, chromium, vanadium and cobalt primarily associated with mitochondria, microsomes and peroxisomes. However, a recent discovery that the upper limit of "free pools" of copper is far less than a single atom per cell casts serious doubt on the in vivo role of copper in Fenton-like generation of free radicals. Nitric oxide (NO) seems to be involved in arsenite-induced DNA damage and pyrimidine excision inhibition. Various studies have confirmed that metals activate signalling pathways and the carcinogenic effect of metals has been related to activation of mainly redox-sensitive transcription factors, involving NF-kappaB, AP-1 and p53. Antioxidants (both enzymatic and non-enzymatic) provide protection against deleterious metal-mediated free radical attacks. Vitamin E and melatonin can prevent the majority of metal-mediated (iron, copper, cadmium) damage both in vitro systems and in metal-loaded animals. Toxicity studies involving chromium have shown that the protective effect of vitamin E against lipid peroxidation may be associated rather with the level of non-enzymatic antioxidants than the activity of enzymatic antioxidants. However, a very recent epidemiological study has shown that a daily intake of vitamin E of more than 400 IU increases the risk of death and should be avoided. While previous studies have proposed a deleterious pro-oxidant effect of vitamin C (ascorbate) in the presence of iron (or copper), recent results have shown that even in the presence of redox-active iron (or copper) and hydrogen peroxide, ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in humans in vitro. Experimental results have also shown a link between vanadium and oxidative stress in the etiology of diabetes. The impact of zinc (Zn) on the immune system, the ability of zinc to act as an antioxidant in order to reduce oxidative stress and the neuroprotective and neurodegenerative role of zinc (and copper) in the etiology of Alzheimer's disease is also discussed. This review summarizes recent findings in the metal-induced formation of free radicals and the role of oxidative stress in the carcinogenicity and toxicity of metals.

Home | Simulation Research

Science.gov Websites

Group specializes in the research, development and deployment of software that support the design and controls design, the Spawn of EnergyPlus next-generation simulation engine, for building and control energy systems tools for OpenBuildingControl to support control design, deployment and verification of building

Center for the Built Environment: Research on Controls and Information

Science.gov Websites

and Control Sustainability and Whole Building Energy Publications Research Area : Research on Human Interactions Contributing to the next generation of high-performance building control systems. New information for sensing and control of buildings using wireless communications technology, micro-electromechancial

A S-Layer Protein of Bacillus anthracis as a Building Block for Functional Protein Arrays by In Vitro Self-Assembly.

PubMed

Wang, Xu-Ying; Wang, Dian-Bing; Zhang, Zhi-Ping; Bi, Li-Jun; Zhang, Ji-Bin; Ding, Wei; Zhang, Xian-En

2015-11-18

S-layer proteins create a cell-surface layer architecture in both bacteria and archaea. Because S-layer proteins self-assemble into a native-like S-layer crystalline structure in vitro, they are attractive building blocks in nanotechnology. Here, the potential use of the S-layer protein EA1 from Bacillus anthracis in constructing a functional nanostructure is investigated, and apply this nanostructure in a proof-of-principle study for serological diagnosis of anthrax. EA1 is genetically fused with methyl parathion hydrolase (MPH), to degrade methyl parathion and provide a label for signal amplification. EA1 not only serves as a nanocarrier, but also as a specific antigen to capture anthrax-specific antibodies. As results, purified EA1-MPH forms a single layer of crystalline nanostructure through self-assembly. Our chimeric nanocatalyst greatly improves enzymatic stability of MPH. When applied to the detection of anthrax-specific antibodies in serum samples, the detection of our EA1-MPH nanostructure is nearly 300 times more sensitive than that of the unassembled complex. Together, it is shown that it is possible to build a functional and highly sensitive nanosensor based on S-layer protein. In conclusion, our present study should serve as a model for the development of other multifunctional nanomaterials using S-layer proteins. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Coordination and Control of Flexible Building Loads for Renewable Integration; Demonstrations using VOLTTRON

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

Hao, He; Liu, Guopeng; Huang, Sen

Renewable energy resources such as wind and solar power have a high degree of uncertainty. Large-scale integration of these variable generation sources into the grid is a big challenge for power system operators. Buildings, in which we live and work, consume about 75% of the total electricity in the United States. They also have a large capacity of power flexibility due to their massive thermal capacitance. Therefore, they present a great opportunity to help the grid to manage power balance. In this report, we study coordination and control of flexible building loads for renewable integration. We first present the motivationmore » and background, and conduct a literature review on building-to-grid integration. We also compile a catalog of flexible building loads that have great potential for renewable integration, and discuss their characteristics. We next collect solar generation data from a photovoltaic panel on Pacific Northwest National Laboratory campus, and conduct data analysis to study their characteristics. We find that solar generation output has a strong uncertainty, and the uncertainty occurs at almost all time scales. Additional data from other sources are also used to verify our study. We propose two transactive coordination strategies to manage flexible building loads for renewable integration. We prove the theories that support the two transactive coordination strategies and discuss their pros and cons. In this report, we select three types of flexible building loads—air-handling unit, rooftop unit, and a population of WHs—for which we demonstrate control of the flexible load to track a dispatch signal (e.g., renewable generation fluctuation) using experiment, simulation, or hardware-in-the-loop study. More specifically, we present the system description, model identification, controller design, test bed setup, and experiment results for each demonstration. We show that coordination and control of flexible loads has a great potential to integrate variable generation sources. The flexible loads can successfully track a power dispatch signal from the coordinator, while having little impact on the quality of service to the end-users.« less

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

Lindsay, I.

The author limits his remarks to a discussion of the international generator`s marketplace, especially aimed at the developing countries. He discusses future global electricity demand, generating capacity build, its financing issues, and to the commercial generating opportunities which now abound outside the US.

Enzymatic membrane reactors for biodegradation of recalcitrant compounds. Application to dye decolourisation.

PubMed

López, C; Mielgo, I; Moreira, M T; Feijoo, G; Lema, J M

2002-11-13

Membrane bioreactors are being increasingly used in enzymatic catalysed transformations. However, the application of enzymatic-based treatment systems in the environmental field is rather unusual. The aim of this paper is to overview the application of enzymatic membrane reactors to wastewater treatment, more specifically to dye decolourisation. Firstly, the basic aspects such as different configurations of enzymatic reactors, advantages and disadvantages associated to their utilisation are revised as well as the application of this technology to wastewater treatment. Secondly, dye decolourisation by white-rot fungi and their oxidative enzymes are discussed, presenting an overall view from for in vivo and in vitro systems. Finally, dye decolourisation by manganese peroxidase in an enzymatic membrane reactor in continuous operation is presented.

Selective classification and quantification model of C&D waste from material resources consumed in residential building construction.

PubMed

Mercader-Moyano, Pilar; Ramírez-de-Arellano-Agudo, Antonio

2013-05-01

The unfortunate economic situation involving Spain and the European Union is, among other factors, the result of intensive construction activity over recent years. The excessive consumption of natural resources, together with the impact caused by the uncontrolled dumping of untreated C&D waste in illegal landfills have caused environmental pollution and a deterioration of the landscape. The objective of this research was to generate a selective classification and quantification model of C&D waste based on the material resources consumed in the construction of residential buildings, either new or renovated, namely the Conventional Constructive Model (CCM). A practical example carried out on ten residential buildings in Seville, Spain, enabled the identification and quantification of the C&D waste generated in their construction and the origin of the waste, in terms of the building material from which it originated and its impact for every m(2) constructed. This model enables other researchers to establish comparisons between the various improvements proposed for the minimization of the environmental impact produced by building a CCM, new corrective measures to be proposed in future policies that regulate the production and management of C&D waste generated in construction from the design stage to the completion of the construction process, and the establishment of sustainable management for C&D waste and for the selection of materials for the construction on projected or renovated buildings.

A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein--Destabilase-Lysozyme from medicinal leech.

PubMed

Kurdyumov, Alexey S; Manuvera, Valentin A; Baskova, Isolda P; Lazarev, Vassili N

2015-11-21

Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified. The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult. In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature. The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.

Modeling of uncertainties in biochemical reactions.

PubMed

Mišković, Ljubiša; Hatzimanikatis, Vassily

2011-02-01

Mathematical modeling is an indispensable tool for research and development in biotechnology and bioengineering. The formulation of kinetic models of biochemical networks depends on knowledge of the kinetic properties of the enzymes of the individual reactions. However, kinetic data acquired from experimental observations bring along uncertainties due to various experimental conditions and measurement methods. In this contribution, we propose a novel way to model the uncertainty in the enzyme kinetics and to predict quantitatively the responses of metabolic reactions to the changes in enzyme activities under uncertainty. The proposed methodology accounts explicitly for mechanistic properties of enzymes and physico-chemical and thermodynamic constraints, and is based on formalism from systems theory and metabolic control analysis. We achieve this by observing that kinetic responses of metabolic reactions depend: (i) on the distribution of the enzymes among their free form and all reactive states; (ii) on the equilibrium displacements of the overall reaction and that of the individual enzymatic steps; and (iii) on the net fluxes through the enzyme. Relying on this observation, we develop a novel, efficient Monte Carlo sampling procedure to generate all states within a metabolic reaction that satisfy imposed constrains. Thus, we derive the statistics of the expected responses of the metabolic reactions to changes in enzyme levels and activities, in the levels of metabolites, and in the values of the kinetic parameters. We present aspects of the proposed framework through an example of the fundamental three-step reversible enzymatic reaction mechanism. We demonstrate that the equilibrium displacements of the individual enzymatic steps have an important influence on kinetic responses of the enzyme. Furthermore, we derive the conditions that must be satisfied by a reversible three-step enzymatic reaction operating far away from the equilibrium in order to respond to changes in metabolite levels according to the irreversible Michelis-Menten kinetics. The efficient sampling procedure allows easy, scalable, implementation of this methodology to modeling of large-scale biochemical networks. © 2010 Wiley Periodicals, Inc.

Improving chocolate flavor in poor-quality cocoa almonds by enzymatic treatment.

PubMed

Oliveira, Hilana Salete Silva; Mamede, Maria Eugênia Oliveira; Góes-Neto, Aristóteles; Koblitz, Maria Gabriela Bello

2011-01-01

This paper proposes a method to enzymatically treat poor-quality cocoa almonds (known as "slate") to ensure the formation of chocolate flavor precursors. The production of flavor precursors improves the quality of these almonds, which are usually responsible for the low quality of the liquor produced. Proteases and carboxypeptidases from different sources were tested under various conditions. The different treatments were evaluated by chemical analysis (hydrolysis efficiency) and sensory analysis of the treated material compared to good-quality cocoa almonds. The results show that it is possible, through the use of microbial enzymes, to generate the mixture of compounds that will release, after roasting, the characteristic chocolate flavor in poor-quality almonds. However, it is necessary to optimize the conditions of enzymatic treatment to obtain better results and thus establish a process that can be used for industrial purposes for manufacturing cocoa and chocolate. The basidiomycete Moniliophtora perniciosa is the causative agent of witches' broom disease (WBD) of the cocoa tree, whose seeds are the source of chocolate. It is the most important phytopathological problem of cocoa-producing areas of the American continent, and has decimated the Brazilian cocoa industry. In Bahia (Brazil), M. perniciosa was identified in 1989 and, as a consequence of its spreading, the annual production of cocoa almonds dropped from 450,000 to 90,000 tons within 12 y, reducing export values from an all-time high of about US$ 1 billion to 110 million. The high incidence of WBD incapacitates Brazil to produce enough cocoa almonds even for the internal market, leading the country to import low-quality cocoa almonds mainly from African countries. Our work proposes an enzymatic treatment to increase the quality of that cocoa almonds and, consequently, to improve the quality of the chocolate produced and consumed in the country. © 2011 Institute of Food Technologists®

Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

PubMed

Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

2016-11-14

This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

Estimation of regional building-related C&D debris generation and composition: case study for Florida, US.

PubMed

Cochran, Kimberly; Townsend, Timothy; Reinhart, Debra; Heck, Howell

2007-01-01

Methodology for the accounting, generation, and composition of building-related construction and demolition (C&D) at a regional level was explored. Six specific categories of debris were examined: residential construction, nonresidential construction, residential demolition, nonresidential demolition, residential renovation, and nonresidential renovation. Debris produced from each activity was calculated as the product of the total area of activity and waste generated per unit area of activity. Similarly, composition was estimated as the product of the total area of activity and the amount of each waste component generated per unit area. The area of activity was calculated using statistical data, and individual site studies were used to assess the average amount of waste generated per unit area. The application of the methodology was illustrated using Florida, US approximately 3,750,000 metric tons of building-related C&D debris were estimated as generated in Florida in 2000. Of that amount, concrete represented 56%, wood 13%, drywall 11%, miscellaneous debris 8%, asphalt roofing materials 7%, metal 3%, cardboard 1%, and plastic 1%. This model differs from others because it accommodates regional construction styles and available data. The resulting generation amount per capita is less than the US estimate - attributable to the high construction, low demolition activity seen in Florida.

Linking wine lactic acid bacteria diversity with wine aroma and flavour.

PubMed

Cappello, Maria Stella; Zapparoli, Giacomo; Logrieco, Antonio; Bartowsky, Eveline J

2017-02-21

In the last two decades knowledge on lactic acid bacteria (LAB) associated with wine has increased considerably. Investigations on genetic and biochemistry of species involved in malolactic fermentation, such as Oenococcus oeni and of Lactobacillus have enabled a better understand of their role in aroma modification and microbial stability of wine. In particular, the use of molecular techniques has provided evidence on the high diversity at species and strain level, thus improving the knowledge on wine LAB taxonomy and ecology. These tools demonstrated to also be useful to detect strains with potential desirable or undesirable traits for winemaking purposes. At the same time, advances on the enzymatic properties of wine LAB responsible for the development of wine aroma molecules have been undertaken. Interestingly, it has highlighted the high intraspecific variability of enzymatic activities such as glucosidase, esterase, proteases and those related to citrate metabolism within the wine LAB species. This genetic and biochemistry diversity that characterizes wine LAB populations can generate a wide spectrum of wine sensory outcomes. This review examines some of these interesting aspects as a way to elucidate the link between LAB diversity with wine aroma and flavour. In particular, the correlation between inter- and intra-species diversity and bacterial metabolic traits that affect the organoleptic properties of wines is highlighted with emphasis on the importance of enzymatic potential of bacteria for the selection of starter cultures to control MLF and to enhance wine aroma. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficient chemical and enzymatic saccharification of the lignocellulosic residue from Agave tequilana bagasse to produce ethanol by Pichia caribbica.

PubMed

Saucedo-Luna, Jaime; Castro-Montoya, Agustin Jaime; Martinez-Pacheco, Mauro Manuel; Sosa-Aguirre, Carlos Ruben; Campos-Garcia, Jesus

2011-06-01

Bagasse of Agave tequilana (BAT) is the residual lignocellulosic waste that remains from tequila production. In this study we characterized the chemical composition of BAT, which was further saccharified and fermented to produce ethanol. BAT was constituted by cellulose (42%), hemicellulose (20%), lignin (15%), and other (23%). Saccharification of BAT was carried out at 147 °C with 2% sulfuric acid for 15 min, yielding 25.8 g/l of fermentable sugars, corresponding to 36.1% of saccharificable material (cellulose and hemicellulose contents, w/w). The remaining lignocellulosic material was further hydrolyzed by commercial enzymes, ~8.2% of BAT load was incubated for 72 h at 40 °C rendering 41 g/l of fermentable sugars corresponding to 73.6% of the saccharificable material (w/w). Mathematic surface response analysis of the acid and enzymatic BAT hydrolysis was used for process optimization. The results showed a satisfactory correlation (R (2) = 0.90) between the obtained and predicted responses. The native yeast Pichia caribbica UM-5 was used to ferment sugar liquors from both acid and enzymatic hydrolysis to ethanol yielding 50 and 87%, respectively. The final optimized process generated 8.99 g ethanol/50 g of BAT, corresponding to an overall 56.75% of theoretical ethanol (w/w). Thus, BAT may be employed as a lignocellulosic raw material for bioethanol production and can contribute to BAT residue elimination from environment.

Stochastic amplification and signaling in enzymatic futile cycles through noise-induced bistability with oscillations

NASA Astrophysics Data System (ADS)

Samoilov, Michael; Plyasunov, Sergey; Arkin, Adam P.

2005-02-01

Stochastic effects in biomolecular systems have now been recognized as a major physiologically and evolutionarily important factor in the development and function of many living organisms. Nevertheless, they are often thought of as providing only moderate refinements to the behaviors otherwise predicted by the classical deterministic system description. In this work we show by using both analytical and numerical investigation that at least in one ubiquitous class of (bio)chemical-reaction mechanisms, enzymatic futile cycles, the external noise may induce a bistable oscillatory (dynamic switching) behavior that is both quantitatively and qualitatively different from what is predicted or possible deterministically. We further demonstrate that the noise required to produce these distinct properties can itself be caused by a set of auxiliary chemical reactions, making it feasible for biological systems of sufficient complexity to generate such behavior internally. This new stochastic dynamics then serves to confer additional functional modalities on the enzymatic futile cycle mechanism that include stochastic amplification and signaling, the characteristics of which could be controlled by both the type and parameters of the driving noise. Hence, such noise-induced phenomena may, among other roles, potentially offer a novel type of control mechanism in pathways that contain these cycles and the like units. In particular, observations of endogenous or externally driven noise-induced dynamics in regulatory networks may thus provide additional insight into their topology, structure, and kinetics. network motif | signal transduction | chemical reaction | synthetic biology | systems biology

Characterization of four arginine kinases in the ciliate Paramecium tetraurelia: Investigation on the substrate inhibition mechanism.

PubMed

Yano, Daichi; Suzuki, Takaya; Hirokawa, Saki; Fuke, Kyoko; Suzuki, Tomohiko

2017-08-01

The ciliate Paramecium tetraurelia contains four arginine kinase genes (AK1-4). We detected cDNA for only three of the AKs (AK1-3) via PCR. Recombinant AK1-4 were expressed in Escherichia coli and their kinetics parameters determined. AK3 showed typical substrate inhibition toward arginine, and enzymatic activity markedly decreased when arginine concentration increased. This is the first example of substrate inhibition in wild-type phosphagen kinases. To explore the substrate inhibition mechanism, site-directed mutations were generated, targeting the amino acid sequence D-D-S-Q-V at positions 77-81 in P. tetraurelia AK3. Among the mutants, substrate inhibition was lost remarkably in the S79A mutant. In spite of high amino acid sequence identity (91%) between P. tetraurelia AK3 and AK4, the enzymatic activity of AK4 was less by 3% than that of AK3. We noticed that the conservative G298 was unusually replaced by R in P. tetraurelia AK4, and we constructed two mutants, R298G/AK4 and G298R/AK3. Enzymatic activity of the former mutant was comparable with that of the wild-type AK3, whereas that of the latter mutant was dramatically reduced. Thus, we concluded that the significantly low activity of P. tetraurelia AK4 is due to the residue R298. Copyright © 2017 Elsevier B.V. All rights reserved.

Activity of Uncleaved Caspase-8 Controls Anti-bacterial Immune Defense and TLR-Induced Cytokine Production Independent of Cell Death.

PubMed

Philip, Naomi H; DeLaney, Alexandra; Peterson, Lance W; Santos-Marrero, Melanie; Grier, Jennifer T; Sun, Yan; Wynosky-Dolfi, Meghan A; Zwack, Erin E; Hu, Baofeng; Olsen, Tayla M; Rongvaux, Anthony; Pope, Scott D; López, Carolina B; Oberst, Andrew; Beiting, Daniel P; Henao-Mejia, Jorge; Brodsky, Igor E

2016-10-01

Caspases regulate cell death programs in response to environmental stresses, including infection and inflammation, and are therefore critical for the proper operation of the mammalian immune system. Caspase-8 is necessary for optimal production of inflammatory cytokines and host defense against infection by multiple pathogens including Yersinia, but whether this is due to death of infected cells or an intrinsic role of caspase-8 in TLR-induced gene expression is unknown. Caspase-8 activation at death signaling complexes results in its autoprocessing and subsequent cleavage and activation of its downstream apoptotic targets. Whether caspase-8 activity is also important for inflammatory gene expression during bacterial infection has not been investigated. Here, we report that caspase-8 plays an essential cell-intrinsic role in innate inflammatory cytokine production in vivo during Yersinia infection. Unexpectedly, we found that caspase-8 enzymatic activity regulates gene expression in response to bacterial infection as well as TLR signaling independently of apoptosis. Using newly-generated mice in which caspase-8 autoprocessing is ablated (Casp8DA/DA), we now demonstrate that caspase-8 enzymatic activity, but not autoprocessing, mediates induction of inflammatory cytokines by bacterial infection and a wide variety of TLR stimuli. Because unprocessed caspase-8 functions in an enzymatic complex with its homolog cFLIP, our findings implicate the caspase-8/cFLIP heterodimer in control of inflammatory cytokines during microbial infection, and provide new insight into regulation of antibacterial immune defense.

Steric analysis of epoxyalcohol and trihydroxy derivatives of 9-hydroperoxy-linoleic acid from hematin and enzymatic synthesis

PubMed Central

Thomas, Christopher P.; Boeglin, William E.; Garcia-Diaz, Yoel; O’Donnell, Valerie B.; Brash, Alan R.

2013-01-01

We characterize the allylic epoxyalcohols and their trihydroxy hydrolysis products generated from 9R- and 9S-hydroperoxy-octadecenoic acid (HPODE) under non-enzymatic conditions, reaction with hematin and subsequent acid hydrolysis, and enzymatic conditions, incubation with Beta vulgaris containing a hydroperoxide isomerase and epoxide hydrolase. The products were resolved by HPLC and the regio and stereo-chemistry of the transformations were determined through a combination of 1H NMR and GC-MS analysis of dimethoxypropane derivatives. Four trihydroxy isomers were identified upon mild acid hydrolysis of 9S,10S-trans-epoxy-11E-13S-hydroxyoctadecenoate: 9S,10R,13S, 9S,12R,13S, 9S,10S,13S and 9S,12S,13S-trihydroxy-octadecenoic acids, in the ratio 40:26:22:12. We also identified a prominent -ketol rearrangement product from the hydrolysis as mainly the 9-hydroxy-10E-13-oxo isomer. Short incubation (5 min) of 9R- and 9S-HPODE with Beta vulgaris extract yielded the 9R- and 9S-hydroxy-10E-12R,13S-cis-epoxy products respectively. Longer incubation (60 min) gave one specific hydrolysis product via epoxide hydrolase, the 9R/S,12S,13S-trihydroxyoctadecenoate. These studies provide a practical approach for the isolation and characterization of allylic epoxy alcohol and trihydroxy products using a combination of HPLC, GC-MS and 1H NMR. PMID:23352713

Kinetics of enzymatic high-solid hydrolysis of lignocellulosic biomass studied by calorimetry.

PubMed

Olsen, Søren N; Lumby, Erik; McFarland, Kc; Borch, Kim; Westh, Peter

2011-03-01

Enzymatic hydrolysis of high-solid biomass (>10% w/w dry mass) has become increasingly important as a key step in the production of second-generation bioethanol. To this end, development of quantitative real-time assays is desirable both for empirical optimization and for detailed kinetic analysis. In the current work, we have investigated the application of isothermal calorimetry to study the kinetics of enzymatic hydrolysis of two substrates (pretreated corn stover and Avicel) at high-solid contents (up to 29% w/w). It was found that the calorimetric heat flow provided a true measure of the hydrolysis rate with a detection limit of about 500 pmol glucose s(-1). Hence, calorimetry is shown to be a highly sensitive real-time method, applicable for high solids, and independent on the complexity of the substrate. Dose-response experiments with a typical cellulase cocktail enabled a multidimensional analysis of the interrelationships of enzyme load and the rate, time, and extent of the reaction. The results suggest that the hydrolysis rate of pretreated corn stover is limited initially by available attack points on the substrate surface (<10% conversion) but becomes proportional to enzyme dosage (excess of attack points) at later stages (>10% conversion). This kinetic profile is interpreted as an increase in polymer end concentration (substrate for CBH) as the hydrolysis progresses, probably due to EG activity in the enzyme cocktail. Finally, irreversible enzyme inactivation did not appear to be the source of reduced hydrolysis rate over time.

Guidelines for Building Science Education

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

Metzger, Cheryn E.; Rashkin, Samuel; Huelman, Pat

The U.S. Department of Energy’s (DOE) residential research and demonstration program, Building America, has triumphed through 20 years of innovation. Partnering with researchers, builders, remodelers, and manufacturers to develop innovative processes like advanced framing and ventilation standards, Building America has proven an energy efficient design can be more cost effective, healthy, and durable than a standard house. As Building America partners continue to achieve their stretch goals, they have found that the barrier to true market transformation for high performance homes is the limited knowledge-base of the professionals working in the building industry. With dozens of professionals taking part inmore » the design and execution of building and selling homes, each person should have basic building science knowledge relevant to their role, and an understanding of how various home components interface with each other. Instead, our industry typically experiences a fragmented approach to home building and design. After obtaining important input from stakeholders at the Building Science Education Kick-Off Meeting, DOE created a building science education strategy addressing education issues preventing the widespread adoption of high performance homes. This strategy targets the next generation and provides valuable guidance for the current workforce. The initiative includes: • Race to Zero Student Design Competition: Engages universities and provides students who will be the next generation of architects, engineers, construction managers and entrepreneurs with the necessary skills and experience they need to begin careers in clean energy and generate creative solutions to real world problems. • Building Science to Sales Translator: Simplifies building science into compelling sales language and tools to sell high performance homes to their customers. • Building Science Education Guidance: Brings together industry and academia to solve problems related to building science education. This report summarizes the steps DOE has taken to develop guidance for building science education and outlines a path forward towards creating real change for an industry in need. The Guidelines for Building Science Education outlined in Appendix A of this report have been developed for external stakeholders to use to certify that their programs are incorporating the most important aspects of building science at the most appropriate proficiency level for their role. The guidelines are intended to be used primarily by training organizations, universities, and certification bodies. Each guideline can be printed or saved as a stand-alone document for ease-of-use by the respective stakeholder group. In 2015, DOE, with leadership from Pacific Northwest National Laboratory (PNNL), is launching a multi-year campaign to promote the adoption of the Guidelines for Building Science Education in a variety of training settings.« less

Citral, A Monoterpene Protect Against High Glucose Induced Oxidative Injury in HepG2 Cell In Vitro-An Experimental Study.

PubMed

Subramaniyan, Sri Devi; Natarajan, Ashok Kumar

2017-08-01

Diabetes mellitus, a major metabolic disorder associated with hyperglycaemia is one of the leading cause of death in many developed countries. However, use of natural phytochemicals have been proved to have a protective effect against oxidative damage. To investigate the effect of citral, a monoterpene on high glucose induced cytotoxicity and oxidative stress in human hepatocellular liver carcinoma (Hep G2) cell line. Cells were treated with 50 mM concentration of glucose for 24 hours incubation following citral (30 μM) was added to confluent HepG2 cells. Cell viability, Reactive Oxygen Species (ROS) generation, DNA damage, lipid peroxidation, antioxidants and Mitogen Activated Protein Kinases (MAPKs) signaling were assessed in citral and/or high glucose induced HepG2 cells. Cells treated with glucose (50 mM), resulted in increased cytotoxicity, ROS generation, DNA damage, lipid peroxidation and depletion of enzymatic and non enzymatic antioxidants. In contrast, treatment with citral (30 μM) significantly decreased cell cytotoxicity, ROS generation, DNA damage, lipid peroxidation and increased antioxidants enzymes in high glucose induced HepG2 cells. In addition, the present study highlighted that high glucose treated cells showed increased expression of Extracellular Signal Regulated Protein Kinase-1 (ERK-1), c-Jun N-terminal Kinase (JNK) and p38 in HepG2 cells. On the other hand treatment with citral significantly suppressed the expression of ERK-1, JNK and p38 in high glucose induced HepG2 cells. Citral protects against high glucose induced oxidative stress through inhibiting ROS activated MAPK signaling pathway in HepG2 cells.

Chemical diversity of labdane-type bicyclic diterpene biosynthesis in Actinomycetales microorganisms.

PubMed

Yamada, Yuuki; Komatsu, Mamoru; Ikeda, Haruo

2016-07-01

Five pairs of bacterial type-A and type-B diterpene synthases have been characterized: BAD86798/BAD86797, AHK61133/AHK61132, BAB39207/BAB39206, CldD/CldB and RmnD/RmnB, and are involved in the formation of pimara-9(11),15-diene, terpente-3,13,15-triene and labda-8(17),12(E),14-triene. Mining of bacterial genome data revealed an additional four pairs of type-A and type-B diterpene synthases: Sros_3191/Sros_3192 of Streptosporangium roseum DSM 43021, Sare_1287/Sare_1288 of Salinispora arenicola CNS-205, SCLAV_5671/SCLAV_5672 and SCLAV_p0491/SCLAV_p0490 of Streptomyces clavuligerus ATCC 27064. Since SCLAV_p0491/SCLAV_p0490 is similar to the labdane-type diterpene synthase pairs, CldD/CldB and RmnD/RmnB based on the alignment of the deduced amino acid sequences and phylogenetic analyses of the aligned sequences, these predicted diterpene synthases were characterized by an enzymatic reaction using a pair of recombinant type-A and type-B diterpene synthases prepared in Escherichia coli and the heterologous expression of two genes encoding type-A and type-B diterpene synthases in an engineered Streptomyces host. The generation of labda-8(17),12(E),14-triene (1) by CldB and CldD was reconfirmed by enzymatic synthesis. Furthermore, labda-8(17),13(16),14-triene (2) was generated by SCLAV_p0491 and CldB, and ladba-7,12(E),14-triene (3) by CldD and SCLAV_p0490. SCLAV_p0491 and SCLAV_p0490 catalyzed the generation of the novel diterpene hydrocarbon, labda-7,13(16),14-triene (4).

Benchmark analysis of native and artificial NAD+-dependent enzymes generated by a sequence based design method with or without phylogenetic data.

PubMed

Nakano, Shogo; Motoyama, Tomoharu; Miyashita, Yurina; Ishizuka, Yuki; Matsuo, Naoya; Tokiwa, Hiroaki; Shinoda, Suguru; Asano, Yasuhisa; Ito, Sohei

2018-05-22

The expansion of protein sequence databases has enabled us to design artificial proteins by sequence-based design methods, such as full consensus design (FCD) and ancestral sequence reconstruction (ASR). Artificial proteins with enhanced activity levels compared with native ones can potentially be generated by such methods, but successful design is rare because preparing a sequence library by curating the database and selecting a method is difficult. Utilizing a curated library prepared by reducing conservation energies, we successfully designed two artificial L-threonine 3-dehydrogenase (SDR-TDH) with higher activity levels than native SDR-TDH, FcTDH-N1 and AncTDH, using FCD and ASR, respectively. The artificial SDR-TDHs had excellent thermal stability and NAD+ recognition compared to native SDR-TDH from Cupriavidus necator (CnTDH): the melting temperatures of FcTDH-N1 and AncTDH were about 10 and 5°C higher than CnTDH, respectively, and the dissociation constants toward NAD+ of FcTDH-N1 and AncTDH were two- and seven-fold lower than that of CnTDH, respectively. Enzymatic efficiency of the artificial SDR-TDHs were comparable to that of CnTDH. Crystal structures of FcTDH-N1 and AncTDH were determined at 2.8 and 2.1 Å resolution, respectively. Structural and MD simulation analysis of the SDR-TDHs indicated that only the flexibility at specific regions was changed, suggesting that multiple mutations introduced in the artificial SDR-TDHs altered their flexibility and thereby affected their enzymatic properties. Benchmark analysis of the SDR-TDHs indicated that both FCD and ASR can generate highly functional proteins if a curated library is prepared appropriately.

Molecular mechanism of cell death induced by king cobra (Ophiophagus hannah) venom l-amino acid oxidase.

PubMed

Fung, Shin Yee; Lee, Mui Li; Tan, Nget Hong

2015-03-01

Snake venom LAAOs have been reported to exhibit a wide range of pharmacological activities, including cytotoxic, edema-inducing, platelet aggregation-inducing/platelet aggregation-inhibiting, bactericidal and antiviral activities. A heat-stable form of l-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom (OH-LAAO) has been shown to exhibit very potent cytotoxicity against human tumorigenic cells but not in their non-tumorigenic counterparts, and the cytotoxicity was due to the apoptosis-inducing effect of the enzyme. In this work, the molecular mechanism of cell death induced by OH-LAAO was investigated. The enzyme exerts its apoptosis-inducing effect presumably via both intrinsic and extrinsic pathways as suggested by the increase in caspase-8 and -9 activities. Oligonucleotide microarray analysis showed that the expression of a total of 178 genes was significantly altered as a result of oxidative stress induced by the hydrogen peroxide generated by the enzyme. Of the 178 genes, at least 27 genes are involved in apoptosis and cell death. These alterations of gene expression was presumably caused by the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidative modifications of signaling molecules that eventually lead to apoptosis and cell death. The very substantial up-regulation of cytochrome P450 genes may also contribute to the potent cytotoxic action of OH-LAAO by producing excessive reactive oxygen species (ROS). In conclusion, the potent apoptosis inducing activity of OH-LAAO was likely due to the direct cytotoxic effect of H2O2 generated during the enzymatic reaction, as well as the non-specific oxidation of signalling molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

Selective enzymatic cleavage and labeling for sensitive capillary electrophoresis laser-induced fluorescence analysis of oxidized DNA bases.

PubMed

Li, Cuiping; Wang, Hailin

2015-08-07

Oxidatively generated DNA damage is considered to be a significant contributing factor to cancer, aging, and age-related human diseases. It is important to detect oxidatively generated DNA damage to understand and clinically diagnosis diseases caused by oxidative damage. In this study, using selective enzymatic cleavage and quantum dot (QD) labeling, we developed a novel capillary electrophoresis-laser induced fluorescence method for the sensitive detection of oxidized DNA bases. First, oxidized DNA bases are recognized and removed by one DNA base excision repair glycosylase, leaving apurinic and apyrimidinic sites (AP sites) at the oxidized positions. The AP sites are further excised by the AP nicking activity of the chosen glycosylase, generating a nucleotide gap with 5'- and 3'- phosphate groups. After dephosphorylation with one alkaline phosphatase, a biotinylated ddNTP is introduced into the nucleotide space within the DNA strand by DNA polymerase I. The biotin-tagged DNA is further labeled with a QD-streptavidin conjugate via non-covalent interactions. The DNA-bound QD is well-separated from excess DNA-unbound QD by highly efficient capillary electrophoresis and is sensitively detected by online coupled laser-induced fluorescence analysis. Using this method, we can assess the trace levels of oxidized DNA bases induced by the Fenton reaction and UV irradiation. Interestingly, the use of the formamidopyrimidine glycosylase (FPG) protein and endonuclease VIII enables the detection of oxidized purine and pyrimidine bases, respectively. Using the synthesized standard DNA, the approach has low limits of detection of 1.1×10(-19)mol in mass and 2.9pM in concentration. Copyright © 2015 Elsevier B.V. All rights reserved.

School Building Design: The Building as an Instructional Tool.

ERIC Educational Resources Information Center

Rakestraw, William E.

1979-01-01

Concepts used in the design of a Dallas school make the building an integral part of the instructional program. These concepts include instrumented resource consumption, wind powered electrical generating capabilities, solar powered domestic hot water system, grey water cycling and sampling capabilities, and mechanical systems monitoring.…

Buildings That Think Green (LBNL Science at the Theater)

ScienceCinema

Majumdar, Arun [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

2018-05-23

Buildings are the SUVs of U.S. energy consumption, gobbling up 71 percent of the nation's electricity. In this Sept. 22, 2008 talk, Arun Majumdar, Director of Berkeley Lab's Environmental Energy Technologies Division, discusses how scientists are creating a new generation of net-zero energy, carbon-neutral buildings.

Adaptive Reuse: Reusing Buildings for Future Generations while Maintaining Connections to the Past.

ERIC Educational Resources Information Center

Rossi, John M.

2003-01-01

Describes adaptive reuse of college buildings, which involves reconfiguring existing buildings for entirely new functions, including its benefits. Examples include Bartlett Hall at the University of Chicago, Annenberg Hall and Locker Chambers at Harvard University, Goodrich Hall at Williams College, and Sarratt Student Center at Vanderbilt…

Energy and IAQ Implications of Alternative Minimum Ventilation Rates in California Retail and School Buildings

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

Dutton, Spencer M.; Fisk, William J.

For a stand-alone retail building, a primary school, and a secondary school in each of the 16 California climate zones, the EnergyPlus building energy simulation model was used to estimate how minimum mechanical ventilation rates (VRs) affect energy use and indoor air concentrations of an indoor-generated contaminant. The modeling indicates large changes in heating energy use, but only moderate changes in total building energy use, as minimum VRs in the retail building are changed. For example, predicted state-wide heating energy consumption in the retail building decreases by more than 50% and total building energy consumption decreases by approximately 10% asmore » the minimum VR decreases from the Title 24 requirement to no mechanical ventilation. The primary and secondary schools have notably higher internal heat gains than in the retail building models, resulting in significantly reduced demand for heating. The school heating energy use was correspondingly less sensitive to changes in the minimum VR. The modeling indicates that minimum VRs influence HVAC energy and total energy use in schools by only a few percent. For both the retail building and the school buildings, minimum VRs substantially affected the predicted annual-average indoor concentrations of an indoor generated contaminant, with larger effects in schools. The shape of the curves relating contaminant concentrations with VRs illustrate the importance of avoiding particularly low VRs.« less

Integration of Point Clouds Dataset from Different Sensors

NASA Astrophysics Data System (ADS)

Abdullah, C. K. A. F. Che Ku; Baharuddin, N. Z. S.; Ariff, M. F. M.; Majid, Z.; Lau, C. L.; Yusoff, A. R.; Idris, K. M.; Aspuri, A.

2017-02-01

Laser Scanner technology become an option in the process of collecting data nowadays. It is composed of Airborne Laser Scanner (ALS) and Terrestrial Laser Scanner (TLS). ALS like Phoenix AL3-32 can provide accurate information from the viewpoint of rooftop while TLS as Leica C10 can provide complete data for building facade. However if both are integrated, it is able to produce more accurate data. The focus of this study is to integrate both types of data acquisition of ALS and TLS and determine the accuracy of the data obtained. The final results acquired will be used to generate models of three-dimensional (3D) buildings. The scope of this study is focusing on data acquisition of UTM Eco-home through laser scanning methods such as ALS which scanning on the roof and the TLS which scanning on building façade. Both device is used to ensure that no part of the building that are not scanned. In data integration process, both are registered by the selected points among the manmade features which are clearly visible in Cyclone 7.3 software. The accuracy of integrated data is determined based on the accuracy assessment which is carried out using man-made registration methods. The result of integration process can achieve below 0.04m. This integrated data then are used to generate a 3D model of UTM Eco-home building using SketchUp software. In conclusion, the combination of the data acquisition integration between ALS and TLS would produce the accurate integrated data and able to use for generate a 3D model of UTM eco-home. For visualization purposes, the 3D building model which generated is prepared in Level of Detail 3 (LOD3) which recommended by City Geographic Mark-Up Language (CityGML).

Building Energy Asset Score for Architects

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

Building Technologies Office

2015-01-01

The Building Energy Asset Score is a national standardized tool for evaluating the physical and structural energy efficiency of commercial and multifamily residential buildings. The Asset Score generates a simple energy efficiency rating that enables comparison among buildings, and identifies opportunities for users to invest in energy efficiency upgrades. It is web-based and free to use. This fact sheet discusses the value of the score for architects.

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

Building Technologies Office

The Building Energy Asset Score is a national standardized tool for evaluating the physical and structural energy efficiency of commercial and multifamily residential buildings. The Asset Score generates a simple energy efficiency rating that enables comparison among buildings, and identifies opportunities for users to invest in energy efficiency upgrades. It is web-based and free to use. This fact sheet discusses the value of the score for energy services companies, engineers and green building consultants.

MultiGeMS: detection of SNVs from multiple samples using model selection on high-throughput sequencing data.

PubMed

Murillo, Gabriel H; You, Na; Su, Xiaoquan; Cui, Wei; Reilly, Muredach P; Li, Mingyao; Ning, Kang; Cui, Xinping

2016-05-15

Single nucleotide variant (SNV) detection procedures are being utilized as never before to analyze the recent abundance of high-throughput DNA sequencing data, both on single and multiple sample datasets. Building on previously published work with the single sample SNV caller genotype model selection (GeMS), a multiple sample version of GeMS (MultiGeMS) is introduced. Unlike other popular multiple sample SNV callers, the MultiGeMS statistical model accounts for enzymatic substitution sequencing errors. It also addresses the multiple testing problem endemic to multiple sample SNV calling and utilizes high performance computing (HPC) techniques. A simulation study demonstrates that MultiGeMS ranks highest in precision among a selection of popular multiple sample SNV callers, while showing exceptional recall in calling common SNVs. Further, both simulation studies and real data analyses indicate that MultiGeMS is robust to low-quality data. We also demonstrate that accounting for enzymatic substitution sequencing errors not only improves SNV call precision at low mapping quality regions, but also improves recall at reference allele-dominated sites with high mapping quality. The MultiGeMS package can be downloaded from https://github.com/cui-lab/multigems xinping.cui@ucr.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis.

PubMed

Braga, Daniel; Hoffmeister, Dirk; Nett, Markus

2016-01-01

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

Development of an On-Demand, Generic, Drug-Delivery System

DTIC Science & Technology

1985-08-06

systems Two systems were evaluated for CO2 evolution. The first of these was an enzymatic system based on urea and urease . The second system was based...PHM 84 Research pH Meter was used te monitor pH. Solutions of various buffer concen- trations and pHs were prepared for each buffer system. One urease ...Measurement of carbon dio~ide production was accomplished using the apparatus shown in Figure 2. Carbon dioxide was generated by putting a urease tablet in the

Environmental performance of green building code and certification systems.

PubMed

Suh, Sangwon; Tomar, Shivira; Leighton, Matthew; Kneifel, Joshua

2014-01-01

We examined the potential life-cycle environmental impact reduction of three green building code and certification (GBCC) systems: LEED, ASHRAE 189.1, and IgCC. A recently completed whole-building life cycle assessment (LCA) database of NIST was applied to a prototype building model specification by NREL. TRACI 2.0 of EPA was used for life cycle impact assessment (LCIA). The results showed that the baseline building model generates about 18 thousand metric tons CO2-equiv. of greenhouse gases (GHGs) and consumes 6 terajoule (TJ) of primary energy and 328 million liter of water over its life-cycle. Overall, GBCC-compliant building models generated 0% to 25% less environmental impacts than the baseline case (average 14% reduction). The largest reductions were associated with acidification (25%), human health-respiratory (24%), and global warming (GW) (22%), while no reductions were observed for ozone layer depletion (OD) and land use (LU). The performances of the three GBCC-compliant building models measured in life-cycle impact reduction were comparable. A sensitivity analysis showed that the comparative results were reasonably robust, although some results were relatively sensitive to the behavioral parameters, including employee transportation and purchased electricity during the occupancy phase (average sensitivity coefficients 0.26-0.29).

Estimating the Additional Greenhouse Gas Emissions in Korea: Focused on Demolition of Asbestos Containing Materials in Building

PubMed Central

Kim, Young-Chan; Hong, Won-Hwa; Zhang, Yuan-Long; Son, Byeung-Hun; Seo, Youn-Kyu; Choi, Jun-Ho

2016-01-01

When asbestos containing materials (ACM) must be removed from the building before demolition, additional greenhouse gas (GHG) emissions are generated. However, precedent studies have not considered the removal of ACM from the building. The present study aimed to develop a model for estimating GHG emissions created by the ACM removal processes, specifically the removal of asbestos cement slates (ACS). The second objective was to use the new model to predict the total GHG emission produced by ACM removal in the entire country of Korea. First, an input-equipment inventory was established for each step of the ACS removal process. Second, an energy consumption database for each equipment type was established. Third, the total GHG emission contributed by each step of the process was calculated. The GHG emissions generated from the 1,142,688 ACS-containing buildings in Korea was estimated to total 23,778 tonCO2eq to 132,141 tonCO2eq. This study was meaningful in that the emissions generated by ACS removal have not been studied before. Furthermore, the study deals with additional problems that can be triggered by the presence of asbestos in building materials. The method provided in this study is expected to contribute greatly to the calculation of GHG emissions caused by ACM worldwide. PMID:27626433

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging.

PubMed

Schmidt, F N; Zimmermann, E A; Campbell, G M; Sroga, G E; Püschel, K; Amling, M; Tang, S Y; Vashishth, D; Busse, B

2017-04-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic cross-links, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692cm -1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p<0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high-performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging

PubMed Central

Schmidt, F.N.; Zimmermann, E.A.; Campbell, G.M.; Sroga, G.E.; Püschel, K.; Amling, M.; Tang, S. Y.; Vashishth, D.; Busse, B.

2017-01-01

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic crosslinks, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692 cm−1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p< 0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture. PMID:28109917

Building Energy Asset Score for Real Estate Managers

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

Building Technologies Office

2015-01-01

The Building Energy Asset Score is a national standardized tool for evaluating the physical and structural energy efficiency of commercial and multifamily residential buildings. The Asset Score generates a simple energy efficiency rating that enables comparison among buildings, and identifies opportunities for users to invest in energy efficiency upgrades. It is web-based and free to use. This fact sheet discusses the value of the score for real estate managers.

Method for Evaluating Energy Use of Dishwashers, Clothes Washers, and Clothes Dryers: Preprint

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

Eastment, M.; Hendron, R.

Building America teams are researching opportunities to improve energy efficiency for some of the more challenging end-uses, such as lighting (both fixed and occupant-provided), appliances (clothes washer, dishwasher, clothes dryer, refrigerator, and range), and miscellaneous electric loads, which are all heavily dependent on occupant behavior and product choices. These end-uses have grown to be a much more significant fraction of total household energy use (as much as 50% for very efficient homes) as energy efficient homes have become more commonplace through programs such as ENERGY STAR and Building America. As modern appliances become more sophisticated the residential energy analyst ismore » faced with a daunting task in trying to calculate the energy savings of high efficiency appliances. Unfortunately, most whole-building simulation tools do not allow the input of detailed appliance specifications. Using DOE test procedures the method outlined in this paper presents a reasonable way to generate inputs for whole-building energy-simulation tools. The information necessary to generate these inputs is available on Energy-Guide labels, the ENERGY-STAR website, California Energy Commission's Appliance website and manufacturer's literature. Building America has developed a standard method for analyzing the effect of high efficiency appliances on whole-building energy consumption when compared to the Building America's Research Benchmark building.« less

Green Schools for Everyone within This Generation

ERIC Educational Resources Information Center

Van Mourik, Jaime

2011-01-01

At the U.S. Green Building Council (USGBC), people see the profound, positive impact that green buildings have on people's lives and the innovation they have poured into the marketplace--from office and retail buildings to government facilities and individual homes. However, none of these markets speaks more powerfully to the benefits and…

Macromolecular structure of cellulose studied by second-harmonic generation imaging microscopy

NASA Astrophysics Data System (ADS)

Brown, R. Malcom; Millard, Andrew C.; Campagnola, Paul J.

2003-11-01

The macromolecular structure of purified cellulose samples is studied by second-harmonic generation (SHG) imaging microscopy. We show that the SHG contrast in both Valonia and Acetobacter cellulose strongly resembles that of collagen from animal tissues, both in terms of morphology and polarization anisotropy. Polarization analysis shows that microfibrils in each lamella are highly aligned and ordered and change directions by 90° in adjacent lamellae. The angular dependence of the SHG intensity fits well to a cos2 θ distribution, which is characteristic of the electric dipole interaction. Enzymatic degradation of Valonia fibers by cellulase is followed in real time by SHG imaging and results in exponential decay kinetics, showing that SHG imaging microscopy is ideal for monitoring dynamics in biological systems.

Generation of lycopene-overproducing strains of the fungus Mucor circinelloides reveals important aspects of lycopene formation and accumulation.

PubMed

Zhang, Yingtong; Chen, Haiqin; Navarro, Eusebio; López-García, Sergio; Chen, Yong Q; Zhang, Hao; Chen, Wei; Garre, Victoriano

2017-03-01

To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation. Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains. A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

Lightweight application for generating clinical research information systems: MAGIC.

PubMed

Leskošek, Brane; Pajntar, Marjan

2015-12-01

Our purpose was to build and test a lightweight solution for generating clinical research information systems (CRIS) that would allow non-IT professionals with basic knowledge of computer usage to quickly define and build a ready-to-use, safe and secure web-based clinical research system for data management. We use the acronym MAGIC (Medical Application Generator InteraCtive) for the system. The generated CRIS should be very easy to build and use, so a common LAMP (Linux Apache MySQL Perl) platform was used, which also enables short development cycles. The application was built and tested using eXtreme Programming (XP) principles by a small development team consisting of one informatics specialist, one physician and one graphical designer/programmer. The parameter and graphical user interface (GUI) definitions for the CRIS can be made by non-IT professionals using an intuitive English-language-like formalism called application definition language (ADL). From these definitions, the MAGIC builds an end-user CRIS that can be used on a wide variety of platforms (from standard workstations to hand-held devices). A working example of a national health-care-quality assessment program is presented to illustrate this process. The lightweight application for generating CRIS (MAGIC) has proven to be useful for both clinical and analytical users in real working environment. To achieve better performance and interoperability, we are planning to recompile the application using XML schemas (XSD) in HL7 CDA or openEHR archetypes formats used for parameters definition and for data interchange between different information systems.

Building Extraction from Remote Sensing Data Using Fully Convolutional Networks

NASA Astrophysics Data System (ADS)

Bittner, K.; Cui, S.; Reinartz, P.

2017-05-01

Building detection and footprint extraction are highly demanded for many remote sensing applications. Though most previous works have shown promising results, the automatic extraction of building footprints still remains a nontrivial topic, especially in complex urban areas. Recently developed extensions of the CNN framework made it possible to perform dense pixel-wise classification of input images. Based on these abilities we propose a methodology, which automatically generates a full resolution binary building mask out of a Digital Surface Model (DSM) using a Fully Convolution Network (FCN) architecture. The advantage of using the depth information is that it provides geometrical silhouettes and allows a better separation of buildings from background as well as through its invariance to illumination and color variations. The proposed framework has mainly two steps. Firstly, the FCN is trained on a large set of patches consisting of normalized DSM (nDSM) as inputs and available ground truth building mask as target outputs. Secondly, the generated predictions from FCN are viewed as unary terms for a Fully connected Conditional Random Fields (FCRF), which enables us to create a final binary building mask. A series of experiments demonstrate that our methodology is able to extract accurate building footprints which are close to the buildings original shapes to a high degree. The quantitative and qualitative analysis show the significant improvements of the results in contrast to the multy-layer fully connected network from our previous work.

Catalyst-controlled oligomerization for the collective synthesis of polypyrroloindoline natural products.

PubMed

Jamison, Christopher R; Badillo, Joseph J; Lipshultz, Jeffrey M; Comito, Robert J; MacMillan, David W C

2017-12-01

In nature, many organisms generate large families of natural product metabolites that have related molecular structures as a means to increase functional diversity and gain an evolutionary advantage against competing systems within the same environment. One pathway commonly employed by living systems to generate these large classes of structurally related families is oligomerization, wherein a series of enzymatically catalysed reactions is employed to generate secondary metabolites by iteratively appending monomers to a growing serial oligomer chain. The polypyrroloindolines are an interesting class of oligomeric natural products that consist of multiple cyclotryptamine subunits. Herein we describe an iterative application of asymmetric copper catalysis towards the synthesis of six distinct oligomeric polypyrroloindoline natural products: hodgkinsine, hodgkinsine B, idiospermuline, quadrigemine H and isopsychotridine B and C. Given the customizable nature of the small-molecule catalysts employed, we demonstrate that this strategy is further amenable to the construction of quadrigemine H-type alkaloids not isolated previously from natural sources.

The antioxidant activity of allylpyrocatechol is mediated via decreased generation of free radicals along with escalation of antioxidant mechanisms.

PubMed

Sarkar, Debjani; Kundu, Sunanda; De, Soumita; Hariharan, Chellaram; Saha, Piu; Manna, Alak; Chattopadhyay, Subrata; Chatterjee, Mitali

2013-03-01

Allylpyrocatechol (APC) is responsible for the antiinflammatory activity exhibited by the methanolic extract of leaves of Piper betle. As antiinflammatory compounds may display antioxidant properties and vice versa, we investigated the antioxidant effect of APC. APC effectively reduced phorbol-myristate-acetate-induced generation of reactive oxygen species and superoxide in murine peritoneal macrophages as well as inhibited Escherichia-coli-induced phagocytic activity of macrophages. Furthermore, pBluescript SK(+) plasmid DNA damage induced by addition of sodium ascorbate was attenuated by APC as it inhibited transformation of the supercoiled form to a relaxed form. In addition, APC increased the enzymatic (catalase) and nonenzymatic (GSH) antioxidant components of murine macrophages. Taken together, APC exhibited an antioxidant activity which was mediated both via decreased generation of free radicals along with increase in cellular antioxidants. Copyright © 2012 John Wiley & Sons, Ltd.

Catalyst-controlled oligomerization for the collective synthesis of polypyrroloindoline natural products

NASA Astrophysics Data System (ADS)

Jamison, Christopher R.; Badillo, Joseph J.; Lipshultz, Jeffrey M.; Comito, Robert J.; MacMillan, David W. C.

2017-12-01

In nature, many organisms generate large families of natural product metabolites that have related molecular structures as a means to increase functional diversity and gain an evolutionary advantage against competing systems within the same environment. One pathway commonly employed by living systems to generate these large classes of structurally related families is oligomerization, wherein a series of enzymatically catalysed reactions is employed to generate secondary metabolites by iteratively appending monomers to a growing serial oligomer chain. The polypyrroloindolines are an interesting class of oligomeric natural products that consist of multiple cyclotryptamine subunits. Herein we describe an iterative application of asymmetric copper catalysis towards the synthesis of six distinct oligomeric polypyrroloindoline natural products: hodgkinsine, hodgkinsine B, idiospermuline, quadrigemine H and isopsychotridine B and C. Given the customizable nature of the small-molecule catalysts employed, we demonstrate that this strategy is further amenable to the construction of quadrigemine H-type alkaloids not isolated previously from natural sources.

Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures.

PubMed

Cerdan, Chantal; Hong, Seok Ho; Bhatia, Mickie

2007-10-01

The in vitro aggregation of human embryonic stem cells (hESCs) into clusters termed embryoid bodies (EBs) allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. This stochastic process results however, in the generation of low numbers of differentiated cells, and can be enhanced to some extent by the addition of exogenous growth factors or overexpression of regulatory genes. In the authors' laboratory, the use of hematopoietic cytokines in combination with the mesoderm inducer bone morphogenetic protein-4 (BMP-4) was able to generate up to 90% of CD45(+) hematopoietic cells with colony-forming unit (CFU) activity. This unit describes two protocols that have been successfully applied in the authors' laboratory for the generation of EBs in (1) suspension and (2) hanging drop (HD) cultures from enzymatically digested clumps of undifferentiated hESC colonies.

Enzymatic hydrolysis of sodium dodecyl sulphate (SDS)-pretreated newspaper for cellulosic ethanol production by Saccharomyces cerevisiae and Pichia stipitis.

PubMed

Xin, Fengxue; Geng, Anli; Chen, Ming Li; Gum, Ming Jun Marcus

2010-10-01

Fermentation of enzymatic hydrolysate of waste newspaper was investigated for cellulosic ethanol production in this study. Various nonionic and ionic surfactants were applied for waste newspaper pretreatment to increase the enzymatic digestibility. The surfactant-pretreated newspaper was enzymatically digested in 0.05 M sodium citrate buffer (pH 4.8) with varying solid content, filter paper unit loading (FPU/g newspaper), and ratio of filter paper unit/beta-glucosidase unit (FPU/CBU). Newspaper pretreated with the anionic surfactant sodium dodecyl sulphate (SDS) demonstrated the highest sugar yield. The addition of Tween-80 in the enzymatic hydrolysis process enhanced the enzymatic digestibility of newspaper pretreated with all of the surfactants. Enzymatic hydrolysis of SDS-pretreated newspaper with 15% solid content, 15 FPU/g newspaper, and FPU/CBU of 1:4 resulted in a newspaper hydrolysate conditioning 29.07 g/L glucose and 4.08 g/L xylose after 72 h of incubation at 50 degrees C. The fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae, Pichia stipitis, and their co-culture produced 14.29, 13.45, and 14.03 g/L of ethanol, respectively. Their corresponding ethanol yields were 0.43, 0.41, and 0.42 g/g.

Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.

[Enzymatic analysis of the quality of foodstuffs].

PubMed

Kolesnov, A Iu

1997-01-01

Enzymatic analysis is an independent and separate branch of enzymology and analytical chemistry. It has become one of the most important methodologies used in food analysis. Enzymatic analysis allows the quick, reliable determination of many food ingredients. Often these contents cannot be determined by conventional methods, or if methods are available, they are determined only with limited accuracy. Today, methods of enzymatic analysis are being increasingly used in the investigation of foodstuffs. Enzymatic measurement techniques are used in industry, scientific and food inspection laboratories for quality analysis. This article describes the requirements of an optimal analytical method: specificity, sample preparation, assay performance, precision, sensitivity, time requirement, analysis cost, safety of reagents.

Kinetics and stereochemistry of LinB-catalyzed δ-HBCD transformation: Comparison of in vitro and in silico results.

PubMed

Heeb, Norbert V; Mazenauer, Manuel; Wyss, Simon; Geueke, Birgit; Kohler, Hans-Peter E; Lienemann, Peter

2018-05-12

LinB is a haloalkane dehalogenase found in Sphingobium indicum B90A, an aerobic bacterium isolated from contaminated soils of hexachlorocyclohexane (HCH) dumpsites. We showed that this enzyme also converts hexabromocyclododecanes (HBCDs). Here we give new insights in the kinetics and stereochemistry of the enzymatic transformation of δ-HBCD, which resulted in the formation of two pentabromocyclododecanols (PBCDols) as first- (P 1δ , P 2δ ) and two tetrabromocyclododecadiols (TBCDdiols) as second-generation products (T 1δ , T 2δ ). Enzymatic transformations of δ-HBCD, α 1 -PBCDol, one of the transformation products, and α 2 -PBCDol, its enantiomer, were studied and modeled with Michaelis-Menten (MM) kinetics. Respective MM-parameters K M , v max , k cat /K M indicated that δ-HBCD is the best LinB substrate followed by α 2 - and α 1 -PBCDol. The stereochemistry of these transformations was modeled in silico, investigating respective enzyme-substrate (ES) and enzyme-product (EP) complexes. One of the four predicted ES-complexes led to the PBCDol product P 1δ , identical to α 2 -PBCDol with the 1R,2R,5S,6R,9R,10S-configuration. An S N 2-like substitution of bromine at C6 of δ-HBCD by Asp-108 of LinB and subsequent hydrolysis of the alkyl-enzyme led to α 2 -PBCDol. Modeling results further indicate that backside attacks at C1, C9 and C10 are reasonable too, selectively binding leaving bromide ions in a halide pocket found in LinB. Docking with α 2 -PBCDol, also allowed productive enzyme binding. A TBCD-1,5-diol with the 1S,2S,5R,6R,9S,10R-configuration is the predicted second-generation product T 1δ . In conclusion, in vitro- and in silico findings now allow a detailed description of step-wise enzymatic dehalohydroxylation reactions of δ-HBCD to specific PBCDols and TBCDdiols at Å-resolution and predictions of their stereochemistry. Copyright © 2018 Elsevier Ltd. All rights reserved.

2G ethanol from the whole sugarcane lignocellulosic biomass.

PubMed

Pereira, Sandra Cerqueira; Maehara, Larissa; Machado, Cristina Maria Monteiro; Farinas, Cristiane Sanchez

2015-01-01

In the sugarcane industry, large amounts of lignocellulosic residues are generated, which includes bagasse, straw, and tops. The use of the whole sugarcane lignocellulosic biomass for the production of second-generation (2G) ethanol can be a potential alternative to contribute to the economic viability of this process. Here, we conducted a systematic comparative study of the use of the lignocellulosic residues from the whole sugarcane lignocellulosic biomass (bagasse, straw, and tops) from commercial sugarcane varieties for the production of 2G ethanol. In addition, the feasibility of using a mixture of these residues from a selected variety was also investigated. The materials were pretreated with dilute acid and hydrolyzed with a commercial enzymatic preparation, after which the hydrolysates were fermented using an industrial strain of Saccharomyces cerevisiae. The susceptibility to enzymatic saccharification was higher for the tops, followed by straw and bagasse. Interestingly, the fermentability of the hydrolysates showed a different profile, with straw achieving the highest ethanol yields, followed by tops and bagasse. Using a mixture of the different sugarcane parts (bagasse-straw-tops, 1:1:1, in a dry-weight basis), it was possible to achieve a 55% higher enzymatic conversion and a 25% higher ethanol yield, compared to use of the bagasse alone. For the four commercial sugarcane varieties evaluated using the same experimental set of conditions, it was found that the variety of sugarcane was not a significant factor in the 2G ethanol production process. Assessment of use of the whole lignocellulosic sugarcane biomass clearly showed that 2G ethanol production could be significantly improved by the combined use of bagasse, straw, and tops, when compared to the use of bagasse alone. The lower susceptibility to saccharification of sugarcane bagasse, as well as the lower fermentability of its hydrolysates, can be compensated by using it in combination with straw and tops (sugarcane trash). Furthermore, given that the variety was not a significant factor for the 2G ethanol production process within the four commercial sugarcane varieties evaluated here, agronomic features such as higher productivity and tolerance of soil and climate variations can be used as the criteria for variety selection.

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

PubMed Central

Rodriguez-Teja, Mercedes; Breit, Claudia; Clarke, Mitchell; Talar, Kamil; Wang, Kai; Mohammad, Mohammad A.; Pickwell, Sage; Etchandy, Guillermina; Stasiuk, Graeme J.; Sturge, Justin

2016-01-01

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed. PMID:27684203

Identification and Characterization of Novel Matrix-Derived Bioactive Peptides: A Role for Collagenase from Santyl® Ointment in Post-Debridement Wound Healing?

PubMed

Sheets, Anthony R; Demidova-Rice, Tatiana N; Shi, Lei; Ronfard, Vincent; Grover, Komel V; Herman, Ira M

2016-01-01

Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFβ-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50-100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60-100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement.

Identification and Characterization of Novel Matrix-Derived Bioactive Peptides: A Role for Collagenase from Santyl® Ointment in Post-Debridement Wound Healing?

PubMed Central

Shi, Lei; Ronfard, Vincent; Grover, Komel V.; Herman, Ira M.

2016-01-01

Debridement, the removal of diseased, nonviable tissue, is critical for clinicians to readily assess wound status and prepare the wound bed for advanced therapeutics or downstream active healing. Removing necrotic slough and eschar through surgical or mechanical methods is less specific and may be painful for patients. Enzymatic debridement agents, such as Clostridial collagenase, selectively and painlessly degrade devitalized tissue. In addition to its debriding activities, highly-purified Clostridial collagenase actively promotes healing, and our past studies reveal that extracellular matrices digested with this enzyme yield peptides that activate cellular migratory, proliferative and angiogenic responses to injury in vitro, and promote wound closure in vivo. Intriguingly, while collagenase Santyl® ointment, a sterile preparation containing Clostridial collagenases and other non-specific proteases, is a well-accepted enzymatic debridement agent, its role as an active healing entity has never been established. Based on our previous studies of pure Clostridial collagenase, we now ask whether the mixture of enzymes contained within Santyl® produces matrix-derived peptides that promote cellular injury responses in vitro and stimulate wound closure in vivo. Here, we identify novel collagen fragments, along with collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, TGFβ-induced protein ig-h3 and tenascin-C, generated from Santyl® collagenase-digested human dermal capillary endothelial and fibroblastic matrices, which increase cell proliferation and angiogenic remodeling in vitro by 50–100% over controls. Using an established model of impaired healing, we further demonstrate a specific dose of collagenase from Santyl® ointment, as well as the newly-identified and chemically-synthesized ECM-derived peptides significantly increase wound re-epithelialization by 60–100% over saline-treated controls. These results not only confirm and extend our earlier studies using purified collagenase- and matrix-derived peptides to stimulate healing in vitro and in vivo, but these Santyl®-generated, matrix-derived peptides may also represent exciting new opportunities for creating advanced wound healing therapies that are enabled by enzymatic debridement and potentially go beyond debridement. PMID:27459729

A biosensor platform for rapid detection of E. coli in drinking water.

PubMed

Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza

2016-02-01

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. Copyright © 2015 Elsevier Inc. All rights reserved.

Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler.

PubMed

Bindewald, Eckart; Grunewald, Calvin; Boyle, Brett; O'Connor, Mary; Shapiro, Bruce A

2008-10-01

One approach to designing RNA nanoscale structures is to use known RNA structural motifs such as junctions, kissing loops or bulges and to construct a molecular model by connecting these building blocks with helical struts. We previously developed an algorithm for detecting internal loops, junctions and kissing loops in RNA structures. Here we present algorithms for automating or assisting many of the steps that are involved in creating RNA structures from building blocks: (1) assembling building blocks into nanostructures using either a combinatorial search or constraint satisfaction; (2) optimizing RNA 3D ring structures to improve ring closure; (3) sequence optimisation; (4) creating a unique non-degenerate RNA topology descriptor. This effectively creates a computational pipeline for generating molecular models of RNA nanostructures and more specifically RNA ring structures with optimized sequences from RNA building blocks. We show several examples of how the algorithms can be utilized to generate RNA tecto-shapes.

Computational strategies for the automated design of RNA nanoscale structures from building blocks using NanoTiler☆

PubMed Central

Bindewald, Eckart; Grunewald, Calvin; Boyle, Brett; O’Connor, Mary; Shapiro, Bruce A.

2013-01-01

One approach to designing RNA nanoscale structures is to use known RNA structural motifs such as junctions, kissing loops or bulges and to construct a molecular model by connecting these building blocks with helical struts. We previously developed an algorithm for detecting internal loops, junctions and kissing loops in RNA structures. Here we present algorithms for automating or assisting many of the steps that are involved in creating RNA structures from building blocks: (1) assembling building blocks into nanostructures using either a combinatorial search or constraint satisfaction; (2) optimizing RNA 3D ring structures to improve ring closure; (3) sequence optimisation; (4) creating a unique non-degenerate RNA topology descriptor. This effectively creates a computational pipeline for generating molecular models of RNA nanostructures and more specifically RNA ring structures with optimized sequences from RNA building blocks. We show several examples of how the algorithms can be utilized to generate RNA tecto-shapes. PMID:18838281