Single molecule targeted sequencing for cancer gene mutation detection.

PubMed

Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W; He, Jiankui

2016-05-19

With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.

A cascade reaction network mimicking the basic functional steps of adaptive immune response

NASA Astrophysics Data System (ADS)

Han, Da; Wu, Cuichen; You, Mingxu; Zhang, Tao; Wan, Shuo; Chen, Tao; Qiu, Liping; Zheng, Zheng; Liang, Hao; Tan, Weihong

2015-10-01

Biological systems use complex ‘information-processing cores’ composed of molecular networks to coordinate their external environment and internal states. An example of this is the acquired, or adaptive, immune system (AIS), which is composed of both humoral and cell-mediated components. Here we report the step-by-step construction of a prototype mimic of the AIS that we call an adaptive immune response simulator (AIRS). DNA and enzymes are used as simple artificial analogues of the components of the AIS to create a system that responds to specific molecular stimuli in vitro. We show that this network of reactions can function in a manner that is superficially similar to the most basic responses of the vertebrate AIS, including reaction sequences that mimic both humoral and cellular responses. As such, AIRS provides guidelines for the design and engineering of artificial reaction networks and molecular devices.

Process for operating equilibrium controlled reactions

DOEpatents

Nataraj, Shankar; Carvill, Brian Thomas; Hufton, Jeffrey Raymond; Mayorga, Steven Gerard; Gaffney, Thomas Richard; Brzozowski, Jeffrey Richard

2001-01-01

A cyclic process for operating an equilibrium controlled reaction in a plurality of reactors containing an admixture of an adsorbent and a reaction catalyst suitable for performing the desired reaction which is operated in a predetermined timed sequence wherein the heating and cooling requirements in a moving reaction mass transfer zone within each reactor are provided by indirect heat exchange with a fluid capable of phase change at temperatures maintained in each reactor during sorpreaction, depressurization, purging and pressurization steps during each process cycle.

Total Synthesis of (+)- and (±)-Hosieine A.

PubMed

Huang, Yu-Wen; Kong, Ke; Wood, John

2018-05-03

Described here is a concise total synthesis of the highly potent nicotinic acetylcholine receptor antagonist hosieine A in racemic ((±)-2) and enantioenriched ((+)-2) forms. The synthesis requires only 7-steps and features a telescoped reaction sequence initiated by a gold-catalyzed Rautenstrauch reaction. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

Organic reactions for the electrochemical and photochemical production of chemical fuels from CO2--The reduction chemistry of carboxylic acids and derivatives as bent CO2 surrogates.

PubMed

Luca, Oana R; Fenwick, Aidan Q

2015-11-01

The present review covers organic transformations involved in the reduction of CO2 to chemical fuels. In particular, we focus on reactions of CO2 with organic molecules to yield carboxylic acid derivatives as a first step in CO2 reduction reaction sequences. These biomimetic initial steps create opportunities for tandem electrochemical/chemical reductions. We draw parallels between long-standing knowledge of CO2 reactivity from organic chemistry, organocatalysis, surface science and electrocatalysis. We point out some possible non-faradaic chemical reactions that may contribute to product distributions in the production of solar fuels from CO2. These reactions may be accelerated by thermal effects such as resistive heating and illumination. Copyright © 2015 Elsevier B.V. All rights reserved.

Five- and six-membered ring opening of pyroglutamic diketopiperazine.

PubMed

Parrish, Dennis A; Mathias, Lon J

2002-03-22

A variety of ring-opening reactions of pyroglutamic diketopiperazine at both the five-membered and six-membered rings is described. Mild, basic conditions facilitate nucleophilic attack by amines at the diketopiperazine carbonyls giving pyroglutamides in excellent yield. Reaction with nucleophiles under acidic conditions give bis-glutamate derivatives of 2,5-diketopiperazine (DKP). These reactions provide simple, two-step sequences to pyroglutamides and symmetrical diketopiperazines from commercial pyroglutamic acid with control of product dictated by reaction conditions, catalyst, and nucleophile.

Diastereoselective chain-elongation reactions using microreactors for applications in complex molecule assembly.

PubMed

Carter, Catherine F; Lange, Heiko; Sakai, Daiki; Baxendale, Ian R; Ley, Steven V

2011-03-14

Diastereoselective chain-elongation reactions are important transformations for the assembly of complex molecular structures, such as those present in polyketide natural products. Here we report new methods for performing crotylation reactions and homopropargylation reactions by using newly developed low-temperature flow-chemistry technology. In-line purification protocols are described, as well as the application of the crotylation protocol in an automated multi-step sequence. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequential allylic substitution/Pauson-Khand reaction: a strategy to bicyclic fused cyclopentenones from MBH-acetates of acetylenic aldehydes.

PubMed

Raji Reddy, Chada; Kumaraswamy, Paridala; Singarapu, Kiran K

2014-09-05

An efficient approach for the construction of novel bicyclic fused cyclopentenones starting from Morita-Baylis-Hillman (MBH) acetates of acetylenic aldehydes with flexible scaffold diversity has been achieved using a two-step reaction sequence involving allylic substitution and the Pauson-Khand reaction. This strategy provided a facile access to various bicyclic cyclopentenones fused with either a carbocyclic or a heterocyclic ring system in good yield.

Prebiotic synthesis of histidine

NASA Technical Reports Server (NTRS)

Shen, C.; Yang, L.; Miller, S. L.; Oro, J.

1990-01-01

The prebiotic formation of histidine (His) has been accomplished experimentally by the reaction of erythrose with formamidine followed by a Strecker synthesis. In the first step of this reaction sequence, the formation of imidazole-4-acetaldehyde took place by the condensation of erythrose and formamidine, two compounds that are known to be formed under prebiotic conditions. In a second step, the imidazole-4-acetaldehyde was converted to His, without isolation of the reaction products by adding HCN and ammonia to the reaction mixture. LC, HPLC, thermospray liquid chromatography-mass spectrometry, and tandem mass spectrometry were used to identify the product, which was obtained in a yield of 3.5% based on the ratio of His/erythrose. This is a new chemical synthesis of one of the basic amino acids which had not been synthesized prebiotically until now.

Natural product-inspired cascade synthesis yields modulators of centrosome integrity.

PubMed

Dückert, Heiko; Pries, Verena; Khedkar, Vivek; Menninger, Sascha; Bruss, Hanna; Bird, Alexander W; Maliga, Zoltan; Brockmeyer, Andreas; Janning, Petra; Hyman, Anthony; Grimme, Stefan; Schürmann, Markus; Preut, Hans; Hübel, Katja; Ziegler, Slava; Kumar, Kamal; Waldmann, Herbert

2011-12-25

In biology-oriented synthesis, the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is, in particular, met by the scaffolds of natural products selected in evolution. The synthesis of natural product-inspired compound collections calls for efficient reaction sequences that preferably combine multiple individual transformations in one operation. Here we report the development of a one-pot, twelve-step cascade reaction sequence that includes nine different reactions and two opposing kinds of organocatalysis. The cascade sequence proceeds within 10-30 min and transforms readily available substrates into complex indoloquinolizines that resemble the core tetracyclic scaffold of numerous polycyclic indole alkaloids. Biological investigation of a corresponding focused compound collection revealed modulators of centrosome integrity, termed centrocountins, which caused fragmented and supernumerary centrosomes, chromosome congression defects, multipolar mitotic spindles, acentrosomal spindle poles and multipolar cell division by targeting the centrosome-associated proteins nucleophosmin and Crm1.

Acid–base bifunctional shell cross-linked micelle nanoreactor for one-pot tandem reaction

DOE PAGES

Lee, Li -Chen; Lu, Jie; Weck, Marcus; ...

2015-12-29

In shell cross-linked micelles (SCMs) containing acid sites in the shell and base sites in the core are prepared from amphiphilic poly(2-oxazoline) triblock copolymers. These materials are utilized as two-chamber nanoreactors for a prototypical acid-base bifunctional tandem deacetalization-nitroaldol reaction. Furthermore, the acid and base sites are localized in different regions of the micelle, allowing the two steps in the reaction sequence to largely proceed in separate compartments, akin to the compartmentalization that occurs in biological systems.

Fragmentation of whole-transcriptome RNA using E. coli RNase III.

PubMed

Ares, Manuel

2013-05-01

High-throughput sequencing (HTS) methods can provide short sequence reads for many millions of individual molecules in a sample, allowing the use of sequencing to measure the abundance of RNA molecules. To quantify the amount of a particular sequence in a sample of large RNAs (e.g., mRNAs), it is important to fragment the RNA into short pieces that can be ligated to oligonucleotides that allow polymerase chain reaction (PCR) amplification and sequencing. The most desired end structure of RNA for such ligation steps is a 5' phosphate and a 3' OH. Thus, enzymes that leave these groups after cleavage are of particular utility, avoiding the need to dephosphorylate the 3' end with phosphatases or phosphorylate the 5' end with kinase before proceeding. One such enzyme, RNase III, is widely available. Although it primarily cuts duplex RNA, this specificity is salt- and concentration-dependent, and many RNAs that lack strong extended duplexes are nonetheless susceptible to cleavage at many spots. RNA fragmentation by RNase III does not seem to grossly affect the distribution of RNA sequencing reads. Thus, it has become a standard method for creating nominally representative pools of transcriptome sequences with 5' phosphates and 3' OH for library construction. Three steps in preparing fragmented transcriptome RNA for sequencing library construction are described here: (1) fragmenting the RNA with RNase III to the extent that ~60-100-nucleotide fragments are created, (2) purifying the RNA from the RNase III reaction, and (3) analyzing the digestion products for their suitability in library production.

What Is Happening when the Blue Bottle Bleaches: An Investigation of the Methylene Blue-Catalyzed Air Oxidation of Glucose

ERIC Educational Resources Information Center

Anderson, Laurens; Wittkopp, Stacy M.; Painter, Christopher J.; Liegel, Jessica J.; Schreiner, Rodney; Bell, Jerry A.; Shakhashiri, Bassam Z.

2012-01-01

An investigation of the Blue Bottle Experiment, a well-known lecture demonstration reaction involving the dye-catalyzed air oxidation of a reducing sugar in alkaline solution, has delineated the sequence of reactions leading to the bleaching of the dye, the regeneration of color, and so forth. Enolization of the sugar is proposed as a key step in…

Solid state synthesis of poly(dichlorophosphazene)

DOEpatents

Allen, Christopher W.; Hneihen, Azzam S.; Peterson, Eric S.

2001-01-01

A method for making poly(dichlorophosphazene) using solid state reactants is disclosed and described. The present invention improves upon previous methods by removing the need for chlorinated hydrocarbon solvents, eliminating complicated equipment and simplifying the overall process by providing a "single pot" two step reaction sequence. This may be accomplished by the condensation reaction of raw materials in the melt phase of the reactants and in the absence of an environmentally damaging solvent.

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

PubMed

Wang, Yi; Wang, Yan; Ma, Ai-Jing; Li, Dong-Xun; Luo, Li-Juan; Liu, Dong-Xin; Jin, Dong; Liu, Kai; Ye, Chang-Yun

2015-07-08

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61-65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Multistep divergent synthesis of benzimidazole linked benzoxazole/benzothiazole via copper catalyzed domino annulation.

PubMed

Liao, Jen-Yu; Selvaraju, Manikandan; Chen, Chih-Hau; Sun, Chung-Ming

2013-04-21

An efficient, facile synthesis of structurally diverse benzimidazole integrated benzoxazole and benzothiazoles has been developed. In a multi-step synthetic sequence, 4-fluoro-3-nitrobenzoic acid was converted into benzimidazole bis-heterocycles, via the intermediacy of benzimidazole linked ortho-chloro amines. The amphiphilic reactivity of this intermediate was designed to achieve the title compounds by the reaction of various acid chlorides and isothiocyanates in a single step through the in situ formation of ortho-chloro anilides and thioureas under microwave irradiation. A versatile one pot domino annulation reaction was developed to involve the reaction of benzimidazole linked ortho-chloro amines with acid chlorides and isothiocyanates. The initial acylation and urea formation followed by copper catalyzed intramolecular C-O and C-S cross coupling reactions furnished the angularly oriented bis-heterocycles which bear a close resemblance to the streptomyces antibiotic UK-1.

Catalytic reduction of dinitrogen to ammonia at a single molybdenum center

PubMed Central

Weare, Walter W.; Dai, Xuliang; Byrnes, Matthew J.; Chin, Jia Min; Schrock, Richard R.; Müller, Peter

2006-01-01

Since our discovery of the catalytic reduction of dinitrogen to ammonia at a single molybdenum center, we have embarked on a variety of studies designed to further understand this complex reaction cycle. These include studies of both individual reaction steps and of ligand variations. An important step in the reaction sequence is exchange of ammonia for dinitrogen in neutral molybdenum(III) compounds. We have found that this exchange reaction is first order in dinitrogen and relatively fast (complete in <1 h) at 1 atm of dinitrogen. Variations of the terphenyl substituents in the triamidoamine ligand demonstrate that the original ligand is not unique in its ability to yield successful catalysts. However, complexes that contain sterically less demanding ligands fail to catalyze formation of ammonia from dinitrogen; it is proposed as a consequence of a base-catalyzed decomposition of a diazenido (MoNNH) intermediate. PMID:17085586

USE OF TAQMAN TO ENUMERATE ENTEROCOCCUS FAECALIS IN WATER

EPA Science Inventory

The Polymerase Chain Reaction (PCR) has become a useful tool in the detection of microorganisms. However, conventional PCR is somewhat time-consuming considering that additional steps (e.g., gel electrophoresis and gene sequencing) are required to confirm the presence of the tar...

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences

PubMed Central

Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L.

2017-01-01

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. PMID:28628204

High-throughput automated microfluidic sample preparation for accurate microbial genomics

PubMed Central

Kim, Soohong; De Jonghe, Joachim; Kulesa, Anthony B.; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P.; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C.

2017-01-01

Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications. PMID:28128213

[Complete genome sequencing and sequence analysis of BCG Tice].

PubMed

Wang, Zhiming; Pan, Yuanlong; Wu, Jun; Zhu, Baoli

2012-10-04

The objective of this study is to obtain the complete genome sequence of Bacillus Calmette-Guerin Tice (BCG Tice), in order to provide more information about the molecular biology of BCG Tice and design more reasonable vaccines to prevent tuberculosis. We assembled the data from high-throughput sequencing with SOAPdenovo software, with many contigs and scaffolds obtained. There are many sequence gaps and physical gaps remained as a result of regional low coverage and low quality. We designed primers at the end of contigs and performed PCR amplification in order to link these contigs and scaffolds. With various enzymes to perform PCR amplification, adjustment of PCR reaction conditions, and combined with clone construction to sequence, all the gaps were finished. We obtained the complete genome sequence of BCG Tice and submitted it to GenBank of National Center for Biotechnology Information (NCBI). The genome of BCG Tice is 4334064 base pairs in length, with GC content 65.65%. The problems and strategies during the finishing step of BCG Tice sequencing are illuminated here, with the hope of affording some experience to those who are involved in the finishing step of genome sequencing. The microarray data were verified by our results.

First highly stereoselective synthesis of fungicide systhane.

PubMed

García Ruano, José L; Cifuentes García, Marta; Martín Castro, Ana M; Rodríguez Ramos, Jesús H

2002-01-10

[reaction: see text] Highly enantiopure (R)-2-p-chlorophenyl-2-(1H-1,2,4-triazol-1-ylmethyl)hexanenitrile 1 (myclobutanil or systhane) was obtained in six synthetic steps from commercially available 1-hexyne (35% yield, 92% ee). The sulfinyl group controls the two key steps of the synthetic sequence, the highly stereoselective hydrocyanation of vinyl sulfoxides with Et(2)AlCN and the further introduction of the proper functionality into the molecule.

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

Lee, Li -Chen; Lu, Jie; Weck, Marcus

In shell cross-linked micelles (SCMs) containing acid sites in the shell and base sites in the core are prepared from amphiphilic poly(2-oxazoline) triblock copolymers. These materials are utilized as two-chamber nanoreactors for a prototypical acid-base bifunctional tandem deacetalization-nitroaldol reaction. Furthermore, the acid and base sites are localized in different regions of the micelle, allowing the two steps in the reaction sequence to largely proceed in separate compartments, akin to the compartmentalization that occurs in biological systems.

Neutron Scattering for Materials Science. Materials Research Society Symposium Proceedings, Volume 166

DTIC Science & Technology

1990-01-01

NEUTRON SCATTERING STUDY OF SHORT-RANGE ORDER IN Fe 0 .8Al0.2 ALLOY 249 Werner Schweika *Invited Paper vN |- __-_ LATTICE MISFIT AND DECOMPOSITION IN...thermodifiractometric measurenmen of this sequence of dehydration and reconstructive phase transformation shows that the Initial dehydration transforrmton reaction...occurs In two steps (see figure 5). Firstly, the dehydration reaction occurs (note in figure 5 the decrease in the incoherent hydrogen background

Understanding the fate of the oxyallyl cation following Nazarov electrocyclization: sequential Wagner-Meerwein migrations and the synthesis of spirocyclic cyclopentenones

PubMed Central

Huang, Jie; Lebœuf, David; Frontier, Alison J.

2011-01-01

A general reaction sequence is described that involves Nazarov cyclization followed by two sequential Wagner Meerwein migrations, to afford spirocyclic compounds from divinyl ketones in the presence of one equivalent of copper(II) complexes. A detailed investigation of this sequence is described including a study of substrate scope and limitations. It was found that after 4π electrocyclization, two different pathways are available to the oxyallyl cation intermediate: elimination of a proton can give the usual Nazarov cycloadduct, or ring contraction can give an alternative tertiary carbocation. After ring contraction, either [1,2]-hydride or carbon migration can occur, depending upon the substitution pattern of the substrate, to furnish spirocyclic products. The rearrangement pathway is favored over the elimination pathway when catalyst loading was high and the copper(II) counterion is noncoordinating. Several ligands were found to be effective for the reaction. Thus, the reaction sequence can be controlled by judicious choice of reaction conditions to allow selective generation of richly functionalized spirocycles. The three steps of the sequence are stereospecific: electrocyclization followed by two [1,2]-suprafacial Wagner-Meerwein shifts: the ring contraction and then an hydride, alkenyl or aryl shift. The method allows stereospecific installation of adjacent stereocenters or adjacent quaternary centers arrayed around a cyclopentenone ring. PMID:21466152

Solution-Phase Synthesis of a Tricyclic Pyrrole-2-Carboxamide Discovery Library Applying a Stetter-Paal-Knorr Reaction Sequence

PubMed Central

Iyer, Pravin S.; Fodor, Matthew D.; Coleman, Claire M.; Twining, Leslie A.; Mitasev, Branko

2012-01-01

The solution phase synthesis of a discovery library of 178 tricyclic pyrrole-2-carboxamides was accomplished in nine steps and seven purifications starting with three benzoyl protected amino acid methyl esters. Further diversity was introduced by two glyoxaldehydes and forty-one primary amines. The combination of Pauson-Khand, Stetter and microwave assisted Paal Knorr reactions was applied as a key sequence. The discovery library was designed with the help of QikProp 2.1 and physicochemical data are presented for all pyrroles. Library members were synthesized and purified in parallel and analyzed by LC-MS. Selected compounds were fully characterized. PMID:16677007

Hybrid Organic-Inorganic Films Grown Using Molecular Layer Deposition

DTIC Science & Technology

2011-03-01

shown that zincone films based on DEZ and hydroquinone (HQ) have displayed some conductivity when alloyed with ZnO ALD films [35]. The schematic...11 Schematic showing the two-step reaction sequence for AB zincone MLD growth using diethylzinc (DEZ) and hydroquinone (HQ). The hybrid organic

High-throughput amplification of mature microRNAs in uncharacterized animal models using polyadenylated RNA and stem-loop reverse transcription polymerase chain reaction.

PubMed

Biggar, Kyle K; Wu, Cheng-Wei; Storey, Kenneth B

2014-10-01

This study makes a significant advancement on a microRNA amplification technique previously used for expression analysis and sequencing in animal models without annotated mature microRNA sequences. As research progresses into the post-genomic era of microRNA prediction and analysis, the need for a rapid and cost-effective method for microRNA amplification is critical to facilitate wide-scale analysis of microRNA expression. To facilitate this requirement, we have reoptimized the design of amplification primers and introduced a polyadenylation step to allow amplification of all mature microRNAs from a single RNA sample. Importantly, this method retains the ability to sequence reverse transcription polymerase chain reaction (RT-PCR) products, validating microRNA-specific amplification. Copyright © 2014 Elsevier Inc. All rights reserved.

The Domino Way to Heterocycles

PubMed Central

Padwa, Albert; Bur, Scott K.

2007-01-01

Sequential transformations enable the facile synthesis of complex target molecules from simple building blocks in a single preparative step. Their value is amplified if they also create multiple stereogenic centers. In the ongoing search for new domino processes, emphasis is usually placed on sequential reactions which occur cleanly and without forming by-products. As a prerequisite for an ideally proceeding one-pot sequential transformation, the reactivity pattern of all participating components has to be such that each building block gets involved in a reaction only when it is supposed to do so. The development of sequences that combine transformations of fundamentally different mechanisms broadens the scope of such procedures in synthetic chemistry. This mini review contains a representative sampling from the last 15 years on the kinds of reactions that have been sequenced into cascades to produce heterocyclic molecules. PMID:17940591

Gemi: PCR Primers Prediction from Multiple Alignments

PubMed Central

Sobhy, Haitham; Colson, Philippe

2012-01-01

Designing primers and probes for polymerase chain reaction (PCR) is a preliminary and critical step that requires the identification of highly conserved regions in a given set of sequences. This task can be challenging if the targeted sequences display a high level of diversity, as frequently encountered in microbiologic studies. We developed Gemi, an automated, fast, and easy-to-use bioinformatics tool with a user-friendly interface to design primers and probes based on multiple aligned sequences. This tool can be used for the purpose of real-time and conventional PCR and can deal efficiently with large sets of sequences of a large size. PMID:23316117

New redox-active layer create via epoxy-amine reaction - The base of genosensor for the detection of specific DNA and RNA sequences of avian influenza virus H5N1.

PubMed

Malecka, Kamila; Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka; Zagórski-Ostoja, Włodzimierz; Dehaen, Wim; Radecka, Hanna; Radecki, Jerzy

2015-03-15

This paper concerns the development of a redox-active monolayer and its application for the construction of an electrochemical genosensor designed for the detection of specific DNA and RNA oligonucleotide sequences related to the avian influenza virus (AIV) type H5N1. This new redox layer was created on a gold electrode surface step by step. Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry and Differential Pulse Voltammetry were used for its characterization. This new redox-active layer was applied for the construction of the DNA biosensor. The NH2-NC3 probe (20-mer) was covalently attached to the gold electrode surface via a "click" reaction between the amine and an epoxide group. The hybridization process was monitored using the Osteryoung Square-Wave Voltammetry. The 20-mer DNA and ca. 280-mer RNA oligonucleotides were used as the targets. The constructed genosensor was capable to determine complementary oligonucleotide sequences with a detection limit in the pM range. It is able to distinguish the different position of the part RNA complementary to the DNA probe. The genosensor was very selective. The 20-mer DNA as well as the 280-mer RNA oligonucleotides without a complementary sequence generated a weak signal. Copyright © 2014 Elsevier B.V. All rights reserved.

Reaction of azides and enolisable aldehydes under the catalysis of organic bases and Cinchona based quaternary ammonium salts.

PubMed

Destro, Dario; Sanchez, Sandra; Cortigiani, Mauro; Adamo, Mauro F A

2017-06-21

Herein we report a two-step sequence for the preparation of amides starting from azides and enolisable aldehydes. The reaction proceeded via the formation of triazoline intermediates that were converted into amides via Lewis acid catalysis. Preliminary studies on the preparation of triazolines under chiral phase transfer catalysis are also presented, demonstrating that enantioenriched amides could be prepared from achiral aldehydes in moderate to low enantioselectivity.

Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

PubMed

V, Pavana Jyothi; S, Akila; Selvan, Malini K; Naidu, Hariprasad; Raghunathan, Shwethaa; Kota, Sathish; Sundaram, R C Raja; Rana, Samir Kumar; Raj, G Dhinakar; Srinivasan, V A; Mohana Subramanian, B

2016-12-01

Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing. Copyright © 2016. Published by Elsevier B.V.

A universal pathway for kinesin stepping.

PubMed

Clancy, Bason E; Behnke-Parks, William M; Andreasson, Johan O L; Rosenfeld, Steven S; Block, Steven M

2011-08-14

Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.

Mechanism of atomic layer deposition of SiO2 on the silicon (100)-2×1 surface using SiCl4 and H2O as precursors

NASA Astrophysics Data System (ADS)

Kang, Jeung Ku; Musgrave, Charles B.

2002-03-01

We use density functional theory to investigate atomic layer deposition (ALD) mechanism of silicon dioxide on the Si(100)-2×1 surface from the precursors SiCl4 and H2O. First, we explore the reaction mechanism of water with the bare Si(100)-2×1 surface to produce surface hydroxyl groups. We find that this reaction proceeds through a two-step pathway with an overall barrier of 33.3 kcal/mol. Next, we investigate the ALD mechanism for the binary reaction sequence: the SiCl4 half reaction and the H2O half reaction. For the SiCl4 half reaction, SiCl4 first forms a σ-bond with the oxygen of the surface OH group and then releases an HCl molecule. The predicted barrier for this process is 15.8 kcal/mol. Next, adsorbed SiCl3 reacts with a neighboring OH group to form bridged SiCl2 with a barrier of 22.6 kcal/mol. The H2O half reaction also proceeds through two sequential steps with an overall barrier of 19.1 kcal/mol for the reaction of H2O with bridged SiCl2 to form bridged Si(OH)2. The predicted barrier of 22.6 kcal/mol for the rate-limiting step of the ALD binary reaction mechanism is consistent with the experimental value of 22.0 kcal/mol. In addition, we find that the calculated frequencies are in good agreement with the experimentally measured IR spectra.

Synthesis of Polyimides Curable by Intramolecular Cycloaddition

DTIC Science & Technology

1977-03-01

5.17; N, 6.90; mol. wt., 384 (by mass spectrometry). c. 2’-Iodo-4! nitroacetanilide 2’-Iodo-4-acetanilide has been previously prepared in two steps...material) and was used without further purification or drying for the next step in the reaction sequence. (2) 2’-Iodo-4’- nitroacetanilide The crude 2...194 g (95% based upon p-nitroaniline) of 2’-iodo-4’- nitroacetanilide . Elemental analysis, I.R. and m.p. (120 0 C) were in agreement with the literature

Chemoenzymatic Synthesis of an Enantiomerically Pure Lactone: A Three-Step Synthesis for Undergraduate Organic Chemistry Laboratory

NASA Astrophysics Data System (ADS)

McClure, Cynthia K.; Chenault, H. Keith

1996-05-01

A three-step laboratory sequence for the undergraduate organic laboratory is described. This series of experiments requires a student to use the product from one reaction as the starting material for a subsequent reaction, and thus the affords the student a "real world" experience of multistep synthesis. Thermal extrusion of sulfur dioxide from sulfolene is used to generate 1,3-butadiene in situ for a Diels-Alder cyclization with maleic anhydride. The anhydride is then reduced to the diol with lithium aluminum hydride. Oxidation of the diol to the chiral lactone is catalyzed by horse-liver alcohol dehydrogenase. This enzymatic oxidation illustrates in situ cofactor regeneration and allows students to measure simple enzyme kinetics.

Substituted dipyrromethanes and their preparation

DOEpatents

Wijesekera, T.

1997-02-04

New chemical compounds, bis(pyrrol-2-yl)halocarbylmethanes, also known as meso-halocarbyl dipyrromethanes, are made by reacting pyrrole in either of two reaction schemes. One such scheme converts pyrrole through an intermediate, a halocarbyl carbonyl pyrrole, to a [2-(1-hydroxyl-1-hydro1-halocarbyl)pyrrole], and then converts the latter to the desired halocarbyldipyrromethane; the last step in this scheme is a novel and useful method in itself. The other such sequence converts pyrrole, by reaction with a halocarbyl aldehyde, directly to the desired halocarbyl dipyrromethane.

Substituted dipyrromethanes and their preparation

DOEpatents

Wijesekera, T.

1996-03-26

New chemical compounds, bis(pyrrol-2-yl)halocarbylmethanes, also known as meso-halocarbyl dipyrromethanes, are made by reacting pyrrole in either of two reaction schemes. One such scheme converts pyrrole through an intermediate, a halocarbyl carbonyl pyrrole, to a [2-(1-hydroxyl-1-hydro-1-halocarbyl)pyrrole], and then converts the latter to the desired halocarbyldipyrromethane; the last step in this scheme is a novel and useful method in itself. The other such sequence converts pyrrole, by reaction with a halocarbyl aldehyde, directly to the desired halocarbyl dipyrromethane.

Substituted dipyrromethanes and their preparation

DOEpatents

Wijesekera, T.

1996-07-16

New chemical compounds, bis(pyrrol-2-yl)halocarbylmethanes, also known as meso-halocarbyl dipyrromethanes, are made by reacting pyrrole in either of two reaction schemes. One such scheme converts pyrrole through an intermediate, a halocarbyl carbonyl pyrrole, to a [2-(1-hydroxyl-1-hydro1-halocarbyl)pyrrole], and then converts the latter to the desired halocarbyldipyrromethane; the last step in this scheme is a novel and useful method in itself. The other such sequence converts pyrrole, by reaction with a halocarbyl aldehyde, directly to the desired halocarbyl dipyrromethane.

Substituted dipyrromethanes and their preparation

DOEpatents

Wijesekera, Tilak

1996-01-01

New chemical compounds, bis(pyrrol-2-yl)halocarbylmethanes, also known as meso-halocarbyl dipyrromethanes, are made by recting pyrrole in either of two reaction schemes. Once such scheme converts pyrrole through an intermediate, a halocarbyl carbonyl pyrrole, to a [2-(1-hydroxyl-1-hydro-1-halocarbyl)pyrrole], and then converts the latter to the desired halocarbyldipyrromethane; the last step in this scheme is a novel and useful method in itself. The other such sequence converts pyrrole, by reaction with a halocarbyl aldehyde, directly to the desired halocarbyl dipyrromethane.

Substituted dipyrromethanes and their preparation

DOEpatents

Wijesekera, Tilak

1996-01-01

New chemical compounds, bis(pyrrol-2-yl)halocarbylmethanes, also known as meso-halocarbyl dipyrromethanes, are made by recting pyrrole in either of two reaction schemes. Once such scheme converts pyrrole through an intermediate, a halocarbyl carbonyl pyrrole, to a 2-(1-hydroxyl-1-hydro-1-halocarbyl)pyrrole!, and then converts the latter to the desired halocarbyldipyrromethane; the last step in this scheme is a novel and useful method in itself. The other such sequence converts pyrrole, by reaction with a halocarbyl aldehyde, directly to the desired halocarbyl dipyrromethane.

Substituted dipyrromethanes and their preparation

DOEpatents

Wijesekera, Tilak

1997-01-01

New chemical compounds, bis(pyrrol-2-yl)halocarbylmethanes, also known as meso-halocarbyl dipyrromethanes, are made by recting pyrrole in either of two reaction schemes. Once such scheme converts pyrrole through an intermediate, a halocarbyl carbonyl pyrrole, to a [2-(1-hydroxyl-1-hydro-1-halocarbyl)pyrrole], and then converts the latter to the desired halocarbyldipyrromethane; the last step in this scheme is a novel and useful method in itself. The other such sequence converts pyrrole, by reaction with a halocarbyl aldehyde, directly to the desired halocarbyl dipyrromethane.

Hydrogen Generation from Ammonia Borane and Water Through the Combustion Reactions with Mechanically Alloyed Al/Mg Powder

DTIC Science & Technology

2014-08-11

generated through a self-sustained propagation of the reaction wave over the generator’s chemical core. The project objective is to determine the...iodine (I2, chips, Sigma Aldrich, 99% pure), at 4 wt% of the initial powder load. The 9.5 mm balls were removed and replaced with the same mass of...of boron (nominal size 0.7 Engineers). The nanocomposite powders were prepared in this project using a sequence of two milling steps. In the first

Carboxylic acid reductase enzymes (CARs).

PubMed

Winkler, Margit

2018-04-01

Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

DOEpatents

Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

2000-01-01

A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

A Simple Method for Amplifying RNA Targets (SMART)

PubMed Central

McCalla, Stephanie E.; Ong, Carmichael; Sarma, Aartik; Opal, Steven M.; Artenstein, Andrew W.; Tripathi, Anubhav

2012-01-01

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl2 are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem. PMID:22691910

Synthesis of a Fluorescent Acridone Using a Grignard Addition, Oxidation, and Nucleophilic Aromatic Substitution Reaction Sequence

ERIC Educational Resources Information Center

Goodrich, Samuel; Patel, Miloni; Woydziak, Zachary R.

2015-01-01

A three-pot synthesis oriented for an undergraduate organic chemistry laboratory was developed to construct a fluorescent acridone molecule. This laboratory experiment utilizes Grignard addition to an aldehyde, alcohol oxidation, and iterative nucleophilic aromatic substitution steps to produce the final product. Each of the intermediates and the…

A Method for Preparing DNA Sequencing Templates Using a DNA-Binding Microplate

PubMed Central

Yang, Yu; Hebron, Haroun R.; Hang, Jun

2009-01-01

A DNA-binding matrix was immobilized on the surface of a 96-well microplate and used for plasmid DNA preparation for DNA sequencing. The same DNA-binding plate was used for bacterial growth, cell lysis, DNA purification, and storage. In a single step using one buffer, bacterial cells were lysed by enzymes, and released DNA was captured on the plate simultaneously. After two wash steps, DNA was eluted and stored in the same plate. Inclusion of phosphates in the culture medium was found to enhance the yield of plasmid significantly. Purified DNA samples were used successfully in DNA sequencing with high consistency and reproducibility. Eleven vectors and nine libraries were tested using this method. In 10 μl sequencing reactions using 3 μl sample and 0.25 μl BigDye Terminator v3.1, the results from a 3730xl sequencer gave a success rate of 90–95% and read-lengths of 700 bases or more. The method is fully automatable and convenient for manual operation as well. It enables reproducible, high-throughput, rapid production of DNA with purity and yields sufficient for high-quality DNA sequencing at a substantially reduced cost. PMID:19568455

Mini-midi-mito: adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples.

PubMed

Berger, Cordula; Parson, Walther

2009-06-01

The degradation state of some biological traces recovered from the crime scene requires the amplification of very short fragments to attain a useful mitochondrial (mt)DNA sequence. We have previously introduced two mini-multiplex assays that amplify 10 overlapping control region (CR) fragments in two separate multiplex PCRs, which brought successful CR consensus sequences from even highly degraded DNA extracts. This procedure requires a total of 20 sequencing reactions per sample, which is laborious and cost intensive. For only moderately degraded samples that we encounter more frequently with typical mtDNA casework material, we developed two new multiplex assays that use a subset of the mini-amplicon primers but embrace larger fragments (midis) and require only 10 sequencing reactions to build a double-stranded CR consensus sequence. We used a preceding mtDNA quantitation step by real-time PCR with two different target fragments (143 and 283 bp) that roughly correspond to the average fragment sizes of the different multiplex approaches to estimate size-dependent mtDNA quantities and to aid the choice of the appropriate PCR multiplexes with respect to quality of the results and required costs.

The synthesis of highly functionalised pyridines using Ghosez-type reactions of dihydropyrazoles.

PubMed

Catti, Federica; Kiuru, Paula S; Slawin, Alexandra M Z; Westwood, Nicholas J

2008-09-29

The aza-Diels-Alder reaction of αβ-unsaturated hydrazones is a general methodology that has been applied both to the synthesis of natural products and in the development of multicomponent reactions. Trends have emerged as to the effect of substituents on the efficiency of this reaction with substituents at the C2 and C4-positions of the aza-diene in general suppressing the reaction. Here we report that 4,5-dihydropyrazoles can function as substrates in this process despite the presence of substituents at both of these positions. A one pot, four chemical step sequence carried out under standard thermal or microwave conditions results in the formation of the corresponding pyridine-containing compounds. The scope of the reaction is explored and additional insights into the proposed mechanism of this reaction are provided.

Mechanism of chimera formation during the Multiple Displacement Amplification reaction.

PubMed

Lasken, Roger S; Stockwell, Timothy B

2007-04-12

Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2-21 nucleotides (nts) in the new templates. Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.

Mechanism of chimera formation during the Multiple Displacement Amplification reaction

PubMed Central

Lasken, Roger S; Stockwell, Timothy B

2007-01-01

Background Multiple Displacement Amplification (MDA) is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Results Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts) in the new templates. Conclusion Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications. PMID:17430586

Gold Nanoparticles Supported on a Layered Double Hydroxide as Efficient Catalysts for the One-Pot Synthesis of Flavones.

PubMed

Yatabe, Takafumi; Jin, Xiongjie; Yamaguchi, Kazuya; Mizuno, Noritaka

2015-11-02

Flavones are a class of natural products with diverse biological activities and have frequently been synthesized by step-by-step procedures using stoichiometric amounts of reagents. Herein, a catalytic one-pot procedure for the synthesis of flavone and its derivatives is developed. In the presence of gold nanoparticles supported on a Mg-Al layered double hydroxide (Au/LDH), various kinds of flavones can be synthesized starting from 2'-hydroxyacetophenones and benzaldehydes (or benzyl alcohols). The present one-pot procedure consists of a sequence of several reactions, and Au/LDH can catalyze all these different types of reactions. The catalysis is shown to be truly heterogeneous, and Au/LDH can be readily recovered and reused. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Composition for nucleic acid sequencing

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-08-26

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Method for sequencing nucleic acid molecules

DOEpatents

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

2006-06-06

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Method for sequencing nucleic acid molecules

DOEpatents

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

2006-05-30

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

A Facile Synthesis of Blue Luminescent [7]Helicenocarbazoles Based on Gold-Catalyzed Rearrangement-Iodonium Migration and Suzuki-Miyaura Benzannulation Reactions.

PubMed

Alcaide, Benito; Almendros, Pedro; Aragoncillo, Cristina; Busto, Eduardo; López-Calixto, Carmen G; Liras, Marta; de la Peña O'Shea, Víctor A; García-Sánchez, Alba; Stone, Hannah V

2018-05-28

New azahelicenes having interesting photophysical properties have been prepared in a four-step sequence. These [7]helicenocarbazoles are efficient blue luminophores, demonstrating the utility of gold catalysis in the preparation of advanced materials. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multi-Step Lithiation of Tin Sulfide: An Investigation Using In Situ Electron Microscopy

DOE PAGES

Hwang, Sooyeon; Yao, Zhenpeng; Zhang, Lei; ...

2018-04-03

Two-dimensional metal sulfides have been widely explored as promising electrodes for lithium ion batteries since their two-dimensional layered structure allows lithium ions to intercalate between layers. For tin disulfide, the lithiation process proceeds via a sequence of three different types of reactions: intercalation, conversion, and alloying but the full scenario of reaction dynamics remains nebulous. In this paper, we investigate the dynamical process of the multi-step reactions using in situ electron microscopy and discover an intermediate rock-salt phase with the disordering of Li and Sn cations, after the initial 2-dimensional intercalation. The disordered cations occupy all the octahedral sites andmore » block the channels for intercalation, which alter the reaction pathways during further lithiation. Our first principles calculations of the non-equilibrium lithiation of SnS2 corroborate the energetic preference of the disordered rock-salt structure over known layered polymorphs. The in situ observations and calculations suggest a two-phase reaction nature for intercalation, disordering, and following conversion reactions. In addition, in situ de-lithiation observation confirms that the alloying reaction is reversible while the conversion reaction is not, which is consistent to the ex situ analysis. This work reveals the full lithiation characteristic of SnS2 and sheds light on the understanding of complex multistep reactions in two-dimensional materials.« less

Multi-Step Lithiation of Tin Sulfide: An Investigation Using In Situ Electron Microscopy

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

Hwang, Sooyeon; Yao, Zhenpeng; Zhang, Lei

Two-dimensional metal sulfides have been widely explored as promising electrodes for lithium ion batteries since their two-dimensional layered structure allows lithium ions to intercalate between layers. For tin disulfide, the lithiation process proceeds via a sequence of three different types of reactions: intercalation, conversion, and alloying but the full scenario of reaction dynamics remains nebulous. In this paper, we investigate the dynamical process of the multi-step reactions using in situ electron microscopy and discover an intermediate rock-salt phase with the disordering of Li and Sn cations, after the initial 2-dimensional intercalation. The disordered cations occupy all the octahedral sites andmore » block the channels for intercalation, which alter the reaction pathways during further lithiation. Our first principles calculations of the non-equilibrium lithiation of SnS2 corroborate the energetic preference of the disordered rock-salt structure over known layered polymorphs. The in situ observations and calculations suggest a two-phase reaction nature for intercalation, disordering, and following conversion reactions. In addition, in situ de-lithiation observation confirms that the alloying reaction is reversible while the conversion reaction is not, which is consistent to the ex situ analysis. This work reveals the full lithiation characteristic of SnS2 and sheds light on the understanding of complex multistep reactions in two-dimensional materials.« less

Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

DOEpatents

Gardner, Shea N [San Leandro, CA; Mariella, Jr., Raymond P.; Christian, Allen T [Tracy, CA; Young, Jennifer A [Berkeley, CA; Clague, David S [Livermore, CA

2011-01-18

A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

Enhanced sampling simulations of DNA step parameters.

PubMed

Karolak, Aleksandra; van der Vaart, Arjan

2014-12-15

A novel approach for the selection of step parameters as reaction coordinates in enhanced sampling simulations of DNA is presented. The method uses three atoms per base and does not require coordinate overlays or idealized base pairs. This allowed for a highly efficient implementation of the calculation of all step parameters and their Cartesian derivatives in molecular dynamics simulations. Good correlation between the calculated and actual twist, roll, tilt, shift, and slide parameters is obtained, while the correlation with rise is modest. The method is illustrated by its application to the methylated and unmethylated 5'-CATGTGACGTCACATG-3' double stranded DNA sequence. One-dimensional umbrella simulations indicate that the flexibility of the central CG step is only marginally affected by methylation. © 2014 Wiley Periodicals, Inc.

II. Inhibited Diffusion Driven Surface Transmutations

NASA Astrophysics Data System (ADS)

Chubb, Talbot A.

2006-02-01

This paper is the second of a set of three papers dealing with the role of coherent partitioning as a common element in Low Energy Nuclear Reactions (LENR), by which is meant cold-fusion related processes. This paper discusses the first step in a sequence of four steps that seem to be necessary to explain Iwamura 2-α-addition surface transmutations. Three concepts are examined: salt-metal interface states, sequential tunneling that transitions D+ ions from localized interstitial to Bloch form, and the general applicability of 2-dimensional vs. 3-dimensional symmetry hosting networks.

Enantioselective syntheses of carbanucleosides from the Pauson-Khand adduct of trimethylsilylacetylene and norbornadiene.

PubMed

Vázquez-Romero, Ana; Rodríguez, Julia; Lledó, Agustí; Verdaguer, Xavier; Riera, Antoni

2008-10-16

A new enantioselective approach to carbanucleosides from Pauson-Khand (PK) adduct 1 is disclosed. The chiral cyclopentenone 1 is readily accessible in enantiomerically pure form via PK reaction of trimethylsilylacetylene and norbornadiene using N-benzyl-N-diphenylphosphino-tert-butyl-sulfinamide as a chiral P,S ligand. (-)-Carbavir and (-)-Abacavir were enantioselectively synthesized starting from (-)-1. The key steps of the sequence are a photochemical conjugate addition of a hydroxymethyl radical, a retro-Diels-Alder reaction, and a palladium catalyzed allylic substitution to introduce the nucleobase.

Aromatic sulfonation with sulfur trioxide: mechanism and kinetic model.

PubMed

Moors, Samuel L C; Deraet, Xavier; Van Assche, Guy; Geerlings, Paul; De Proft, Frank

2017-01-01

Electrophilic aromatic sulfonation of benzene with sulfur trioxide is studied with ab initio molecular dynamics simulations in gas phase, and in explicit noncomplexing (CCl 3 F) and complexing (CH 3 NO 2 ) solvent models. We investigate different possible reaction pathways, the number of SO 3 molecules participating in the reaction, and the influence of the solvent. Our simulations confirm the existence of a low-energy concerted pathway with formation of a cyclic transition state with two SO 3 molecules. Based on the simulation results, we propose a sequence of elementary reaction steps and a kinetic model compatible with experimental data. Furthermore, a new alternative reaction pathway is proposed in complexing solvent, involving two SO 3 and one CH 3 NO 2 .

Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

PubMed Central

Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

2013-01-01

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

Mechanisms of the Knoevenagel hetero Diels-Alder sequence in multicomponent reactions to dihydropyrans: experimental and theoretical investigations into the role of water.

PubMed

Frapper, Gilles; Bachmann, Christian; Gu, Yanlong; Coval De Sousa, Rodolphe; Jérôme, François

2011-01-14

The role of water in a multicomponent domino reaction (MCR) involving styrene, 2,4-pentanedione, and formaldehyde was studied. Whereas anhydrous conditions produced no reaction, the MCR successfully proceeded in the presence of water, affording the targeted dihydropyran derivatives with good yield. The mechanism of this MCR (Knoevenagel hetero Diels-Alder sequence) was studied with and without explicit water molecules using the SMD continuum solvation model in combination with the B3LYP density functional and the 6-311++G** basis set to compute the water and acetone (aprotic organic solvent) solution Gibbs free energies. In the Knoevenagel step, we found that water acted as a proton relay to favor the formation of more flexible six-membered ring transition state structures both in concerted (direct H(2)O elimination) and stepwise (keto-enol tautomerization and dehydration) pathways. The inclusion of a water molecule in our model resulted in a significant decrease (-8.5 kcal mol(-1)ΔG(water)(‡)) of the direct water elimination activation barrier. Owing to the presence of water, all chemical steps involved in the MCR mechanism had activation free energies barriers lower than 39 kcal mol(-1) at 25 °C in aqueous solvent (<21 kcal mol(-1) ZPE corrected electronic energies barriers). Consequently, the MCR proceeded without the assistance of any catalyst.

Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation.

PubMed

Shore, Sabrina; Henderson, Jordana M; Lebedev, Alexandre; Salcedo, Michelle P; Zon, Gerald; McCaffrey, Anton P; Paul, Natasha; Hogrefe, Richard I

2016-01-01

For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in size to the tagged library. Most sRNA library preparation strategies thus employ a gel purification step to isolate tagged library from adapter dimer contaminants. At very low sample inputs, adapter dimer side products dominate the reaction and limit the sensitivity of this technique. Here we address the need for improved specificity of sRNA library preparation workflows with a novel library preparation approach that uses modified adapters to suppress adapter dimer formation. This workflow allows for lower sample inputs and elimination of the gel purification step, which in turn allows for an automatable sRNA library preparation protocol.

Mechanism insight into the cyanide-catalyzed benzoin condensation: a density functional theory study.

PubMed

He, Yunqing; Xue, Ying

2010-09-02

The reaction mechanism of the cyanide-catalyzed benzoin condensation without protonic solvent assistance has been studied computationally for the first time employing the density functional theory (B3LYP) method in conjunction with 6-31+G(d,p) basis set. Four possible pathways have been investigated. A new proposed pathway on the basis of the Lapworth mechanism is determined to be the dominant pathway in aprotic solvent, in which the formation of the Lapworth's cyanohydrin intermediate is a sequence including three steps assisted by benzaldehyde, clearly manifesting that the reaction can take place in aprotic solvents such as DMSO. In this favorable pathway with six possible transition states located along the potential energy surface, the reaction of the cyanide/benzaldehyde complex with another benzaldehyde to afford an alpha-hydroxy ether is the rate-determining dynamically with the activation free energy barrier of 26.9 kcal/mol, and the step to form cyanohydrin intermediate from alpha-hydroxy ether is partially rate-determining for its relatively significant barrier 20.0 kcal/mol.

Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

NASA Astrophysics Data System (ADS)

Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

1990-09-01

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

Picoliter DNA Sequencing Chemistry on an Electrowetting-based Digital Microfluidic Platform

PubMed Central

Ferguson Welch, Erin R.; Lin, Yan-You; Madison, Andrew; Fair, R.B.

2011-01-01

The results of investigations into performing DNA sequencing chemistry on a picoliter-scale electrowetting digital microfluidic platform are reported. Pyrosequencing utilizes pyrophosphate produced during nucleotide base addition to initiate a process ending with detection through a chemiluminescence reaction using firefly luciferase. The intensity of light produced during the reaction can be quantified to determine the number of bases added to the DNA strand. The logic-based control and discrete fluid droplets of a digital microfluidic device lend themselves well to the pyrosequencing process. Bead-bound DNA is magnetically held in a single location, and wash or reagent droplets added or split from it to circumvent product dilution. Here we discuss the dispensing, control, and magnetic manipulation of the paramagnetic beads used to hold target DNA. We also demonstrate and characterize the picoliter-scale reaction of luciferase with adenosine triphosphate to represent the detection steps of pyrosequencing and all necessary alterations for working on this scale. PMID:21298802

Novel, In-House, SYBR Green Based One-Step rRT-PCR: Rapid and Accurate Diagnosis of Crimean-Congo Hemorrhagic Fever Virus in Suspected Patients From Iran.

PubMed

Zahraei, Bentolhoda; Hashemzadeh, Mohammad Sadegh; Najarasl, Mohammad; Zahiriyeganeh, Samaneh; Tat, Mahdi; Metanat, Maliheh; Sepehri Rad, Nahid; Khansari-Nejad, Behzad; Zafari, Ehsan; Sharti, Mojtaba; Dorostkar, Ruhollah

2016-01-01

The Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans, with a high mortality rate. Since, there is no approved vaccine or specific treatment for CCHF, an early and accurate diagnosis, as well as reliable surveillance, is essential for case management and patient improvement. For this research, our aim was to evaluate the application of a novel SYBR Green based one-step real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay for the in-house diagnosis of the CCHF virus. In this experimental study, the highly conserved S-region sequence of the CCHF viral genome was first adapted from GenBank, and the specific primers targeting this region were designed. Then, the viral RNA was extracted from 75 serum samples from different patients in eastern Iran. The sensitivity and specificity of the primers were also evaluated in positive serum samples previously confirmed to have the CCHF virus, by this one-step rRT-PCR assay, as well as a DNA sequencing analysis. From a total of 75 suspected serum samples, 42 were confirmed to be positive for CCHF virus, with no false-positives detected by the sequencing results. After 40 amplification cycles, the melting curve analysis revealed a mean melting temperature (Tm) of 86.5 ± 0.6°C (quite different from those of the primer-dimers), and the positive samples showed only a small variation in the parameters. In all of the positive samples, the predicted length of 420 bp was confirmed by electrophoresis. Moreover, the sensitivity test showed that this assay can detect less than 20 copies of viral RNA per reaction. This study showed that this novel one-step rRT-PCR assay is a rapid, reliable, repeatable, specific, sensitive, and simple tool for the detection of the CCHF virus.

Effect of pyrimido[1,6-a]benzimidazoles, quinolones, and Ca2+ on the DNA gyrase-mediated cleavage reaction.

PubMed Central

Gmünder, H; Kuratli, K; Keck, W

1995-01-01

The quinolones inhibit the A subunit of DNA gyrase in the presence of Mg2+ by interrupting the DNA breakage and resealing steps, and the latter step is also retarded without quinolones if Mg2+ is replaced by Ca2+. Pyrimido[1,6-a]benzimidazoles have been found to represent a new class of potent DNA gyrase inhibitors which also act at the A subunit. To determine alterations in the DNA sequence specificity of DNA gyrase for cleavage sites in the presence of inhibitors of both classes or in the presence of Ca2+, we used DNA restriction fragments of 164, 85, and 71 bp from the pBR322 plasmid as model substrates. Each contained, at a different position, the 20-bp pBR322 sequence around position 990, where DNA gyrase preferentially cleaves in the presence of quinolones. Our results show that pyrimido[1,6-a]benzimidazoles have a mode of action similar to that of quinolones; they inhibit the resealing step and influence the DNA sequence specificity of DNA gyrase in the same way. Differences between inhibitors of both classes could be observed only in the preferences of DNA gyrase for these cleavage sites. The 20-bp sequence appeared to have some properties that induced DNA gyrase to cleave all three DNA fragments in the presence of inhibitors within this sequence, whereas cleavage in the presence of Ca2+ was in addition dependent on the length of the DNA fragments. PMID:7695300

Labeled nucleotide phosphate (NP) probes

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2009-02-03

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Sugars: their origin in photosynthesis and subsequent biological interconversions.

PubMed

Nevins, D J

1995-04-01

Sugar has been valued as a commodity for thousands of years. Despite its long history in commerce, the biological mechanisms accounting for the production of sugar are rather recent discoveries. The reactions are remarkable. Sugar is produced by all green plants and photosynthetic bacteria in a reaction sequence capable of forming carbon-carbon bonds. The very first steps occur independently of solar energy input, but to sustain the reaction, the products of initial fixation are phosphorylated and undergo a reduction in oxidation state. These steps responsible for phosphorylation and reduction are driven by products generated in the chloroplast upon the absorption of light. At this point, after just a few reactions, the products of photosynthesis have already acquired the attributes characteristic of sugars. Once carbon is stabilized as simple sugars in the chloroplast, the products undergo a sequence of rearrangements to sustain a cycle leading to new carbon dioxide acceptor molecules, and with each turn of the cycle a new carbon atom is introduced into the pool. As the process continues some of the carbon is diverted to synthesize starch within the chloroplast. Sucrose is synthesized in the cytoplasm adjacent to the chloroplast from exported carbohydrate as a diversion from the formation of starch. Sucrose represents the principal transport substance in most plants. Storage starch, cellulose, and other complex cell wall polysaccharides are typically derived from the sugar monomers found in sucrose. Sugars supply all the fixed carbon for synthesis of biological compounds and are fundamental for sustaining the energy flow to all food systems.

Biogenetically inspired approach to the Strychnos alkaloids. Concise syntheses of (+/-)-akuammicine and (+/-)-strychnine.

PubMed

Ito, M; Clark, C W; Mortimore, M; Goh, J B; Martin, S F

2001-08-22

A linear synthesis of the indole alkaloid (+/-)-akuammicine (2) was completed by a novel sequence of reactions requiring only 10 steps from commercially available starting materials. The approach features a tandem vinylogous Mannich addition and an intramolecular hetero Diels-Alder reaction to rapidly assemble the pentacyclic heteroyohimboid derivative 8 from the readily available hydrocarboline 6. Oxidation of the E ring of 8 gave the lactone 9 that was converted into deformylgeissoschizine (11). The subsequent elaboration of 11 into 2 was effected by a biomimetically patterned transformation that involved sequential oxidation and base-induced skeletal reorganization. A variation of these tactics was then applied to the synthesis of the C(18) hydroxylated akuammicine derivative 36. Because 36 had previously been converted into strychnine (1) in four steps, its preparation constitutes a concise, formal synthesis of this complex alkaloid.

Total Synthesis of Spirotenuipesines A and B

PubMed Central

2008-01-01

Spirotenuipesines A and B, isolated from the entomopathogenic fungus Paecilomyces tenuipes by Oshima and co-workers, have been synthesized. The synthesis features the highly stereoselective construction of two vicinal all-carbon quaternary centers (C5 and C6) via an intramolecular cyclopropanation/radical initiated fragmentation sequence and a diastereoselective intermolecular Diels−Alder reaction between α-methylenelactone dienophile 20 and synergistic diene 6a. Installation of the C9 tertiary alcohol occurred via nucleophilic methylation. An RCM reaction to produce a tetrasubstituted double bond in the presence of free allylic alcohol and homoallylic oxygenated functional group is also described. This route shortened the synthesis of 11 from 9 steps to 3 steps. We have further developed a strategy to gain access to optically active spirotenuipesines A and B through the synthesis of enantioenriched 10 from commercially available R-(−)-epichlorohydrin. PMID:18973385

Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

PubMed

Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

2018-03-09

High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

Experimental study of electrochemical fluorination of trichloroethylene

NASA Technical Reports Server (NTRS)

Polisena, C.; Liu, C. C.; Savinell, R. F.

1982-01-01

The electrochemical fluorination of trichloroethylene in anhydrous hydrogen fluoride at 0 C and at constant cell potential was investigated. A microprocessor-aided electrochemical fluorination reactor system that yields highly reproducible results was utilized. The following major two-carbon-chain products were observed: CHCl2-CCl2F, CHCl2-CClF2, CHClF-CCl2F, and CCl2F-CClF2. The first step in the reaction sequence was determined to be fluorine addition to the double bond, followed by replacement of first hydrogen and then chlorine by fluorine. Polymerization reactions yielded higher molecular weight or possible ring-type chlorofluorohydrocarbons. A comparison of the reaction products of electrochemical and chemical fluorinations of trichloroethylene is also discussed.

Kinetically designed conditions for the catalytic formation of disfavored products. The reaction of ({eta}{sup 5}-C{sub 5}H{sub 5})Mo(CO){sub 3}* with N,N,N{prime},N{prime}-tetramethyl-1,4-phenylenediamine

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

Balla, J.; Espenson, J.H.; Bakac, A.

1995-03-16

In the absence of other reagents, the 17e molybdenum radical, ($eta{sup 5}-C{sub 5}H{sub 5})Mo(CO){sub 3}*, combines to form the stable dimer, [CpMo(CO){sub 3}]{sub 2}. In the presence of TMPD, however, an electron transfer process ensues, in which the normally persistent radical TMPD*{sup +} is produced. Under these conditions, the absorbance of the TMPD*{sup +} radical disappear shortly thereafter. Various kinetic tests have been applied to show that this is the result of a sequence of two electron transfer steps. One is the reaction between CpMo(CO){sub 3}* (Mo*) and TMPD, and the other is the reaction between Mo* and TMPD*{sup +}.more » The net result of the two reactions occurring in sequence is the disproportionation of the molybdenum radical, rather than the combination reaction that occurs in the absence of this redox-active amine. To the contrary, PhNMe{sub 2} shows no such effect, confirming that these observations are correctly attributed to electron transfer and not to ligand-catalyzed disproportionation. That the TMPD-catalyzed sequence really is disproportionation was confirmed by the chemical identification of the products, CpMo(CO){sub 3}{sup -} and CpMo(CO){sub 3}NCCH{sub 3}{sup +}. 40 refs., 8 figs., 1 tab.« less

A DFT Investigation of the Mechanism of Propene Ammoxidation over α-Bismuth Molybdate

DOE PAGES

Licht, Rachel B.; Bell, Alexis T.

2016-11-17

We investigated the mechanisms and energetics for the propene oxidation and ammoxidation occurring on the (010) surface of Bi 2 Mo 3 O 12 using density functional theory (DFT). An energetically feasible sequence of elementary steps for propene oxidation to acrolein, propene ammoxidation to acrylonitrile, and acrolein ammoxidation to acrylonitrile is proposed. Consistent with experimental findings, the rate-limiting step for both propene oxidation and ammoxidation is the initial hydrogen abstraction from the methyl group of propene, which is calculated to have an apparent activation energy of 27.3 kcal/mol. The allyl species produced in this reaction is stabilized as an allylmore » alkoxide, which can then undergo hydrogen abstraction to form acrolein or react with ammonia adsorbed on under-coordinated surface Bi 3+ cations to form allylamine. Dehydrogenation of allylamine is shown to produce acrylonitrile, whereas reaction with additional adsorbed ammonia leads to the formation of acetonitrile and hydrogen cyanide. The dehydrogenation of allyalkoxide species is found to have a significantly higher activation barrier than reaction with adsorbed ammonia, consistent with the observation that very little acrolein is produced when ammonia is present. Finally, we found that rapid reoxidation of the catalyst surface to release wate the driving force for all reactions involving the cleavage of C-H or N-H bonds, because practically all of these steps are endothermic. (Chemical Equation Presented).« less

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

PubMed

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Label-free technology for the amplified detection of microRNA based on the allosteric hairpin DNA switch and hybridization chain reaction.

PubMed

Cai, Sheng; Cao, Zhijuan; Lau, Choiwan; Lu, Jianzhong

2014-11-21

By using the allosteric hairpin DNA switch, a novel assay for the detection of microRNA (miRNA) let-7a via a hybridization chain reaction (HCR) was introduced. Briefly, the hairpin DNA switch probe is a single-stranded DNA consisting of a streptavidin (SA) aptamer sequence, a target binding sequence and a certain sequence that acts as a trigger of the HCR. In the presence of target let-7a, the hairpin DNA switch would open and expose the stem region sequences, where a part of this sequence acts as initiator sequence strands for the HCR and triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers, another part is the SA aptamer sequence which activates its binding affinity to SA on SA-coated magnetic particles. The hybridization event could be sensitively detected via an instantaneous derivatization reaction between a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG) and the guanine nucleotides within the target, the hairpin DNA switch probe, and HCR helices to form an unstable CL intermediate for the generation of light. Our results show that the coupling of the hairpin DNA switch probe and the HCR for the amplified detection of let-7a achieves a better performance (e.g. wide linear response range: 0.1-1000 fmol, low detection limit: 0.1 fmol, and high specificity). Furthermore, this approach could be easily applied to the detection of let-7a in human lung cells, and extended to detect other types of miRNA and proteins such as PDGF based on aptamers. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment.

Reaction cycle and thermodynamics in bacteriorhodopsin

NASA Technical Reports Server (NTRS)

Lanyi, J. K.

1992-01-01

Light causes the all-trans to 13-cis isomerization of the retinal in bacteriorhodopsin; the thermal relaxation leading back to the initial state drives proton transport first via proton transfer between the retinal Schiff base and D85 and then between the Schiff base and D96. The reaction sequence and thermodynamics of this photocycle are described by measuring time-resolved absorption changes with a gated multichannel analyzer between 100 ns and 100 ms, at six temperatures between 5 degrees C and 30 degrees C. Analysis of the energetics of the chromophore reaction sequence is on the basis of a recently proposed model (Varo & Lanyi, Biochemistry 30, 5016-5022, 1991) which consists of a single cycle and many reversible reactions: BR -hv-->K<==>L<==>M1-->M2<==>N<==>O-->BR. The existence of the M1-->M2 reaction, which functions as the switch in the proton transfer, is confirmed by spectroscopic evidence. The calculated thermodynamic parameters indicate that the exchange of free energy between the protein and the protons is at the switch step. Further, a large entropy decrease at this reaction suggests a protein conformation change which will conserve delta G for driving the completion of the reaction cycle. The results provide insights to mechanism and energy coupling in this system, with possible relevance to the general question of how ion pumps function.

Radical chemistry of artemisinin

NASA Astrophysics Data System (ADS)

Denisov, Evgenii T.; Solodova, S. L.; Denisova, Taisa G.

2010-12-01

The review summarizes physicochemical characteristics of the natural sesquiterpene peroxide artemisinin. The kinetic schemes of transformations of artemisinin radicals under anaerobic conditions are presented and analyzed. The sequence of radical reactions of artemisinin in the presence of oxygen is considered in detail. Special emphasis is given to the intramolecular chain oxidation resulting in the transformation of artemisinin into polyatomic hydroperoxide. The kinetic characteristics of elementary reaction steps involving alkyl, alkoxyl, and peroxyl radicals generated from artemisinin are discussed. The results of testing of artemisinin and its derivatives for the antimalarial activity and the scheme of the biochemical synthesis of artemisinin in nature are considered.

Molecular implementation of molecular shift register memories

NASA Technical Reports Server (NTRS)

Beratan, David N. (Inventor); Onuchic, Jose N. (Inventor)

1991-01-01

An electronic shift register memory (20) at the molecular level is described. The memory elements are based on a chain of electron transfer molecules (22) and the information is shifted by photoinduced (26) electron transfer reactions. Thus, multi-step sequences of charge transfer reactions are used to move charge with high efficiency down a molecular chain. The device integrates compositions of the invention onto a VLSI substrate (36), providing an example of a molecular electronic device which may be fabricated. Three energy level schemes, molecular implementation of these schemes, optical excitation strategies, charge amplification strategies, and error correction strategies are described.

Molecular Layer Deposition of Hybrid Organic-Inorganic Alucone Polymer Films Using a Three-Step ABC Reaction Sequence

DTIC Science & Technology

2009-11-02

versus TMA exposure at 150 C is shown in Figure 4a. The TMA exposure is defined by the number of TMA microdoses . EachTMAmicrodosewas a 0.5 s exposure...at 80mTorr of partial pressure. Figure 4a indicates that the TMA reac- tion is self-limiting and reaches completion after 10 TMA microdoses . The...of EA microdoses . Each EA microdose was a 0.5 s exposure at 20 mTorr of partial pressure. Figure 4b indicates that the EA reaction is self-limiting

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test.

PubMed

Suin, Vanessa; Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael; Van Gucht, Steven

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤ 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible.

A Two-Step Lyssavirus Real-Time Polymerase Chain Reaction Using Degenerate Primers with Superior Sensitivity to the Fluorescent Antigen Test

PubMed Central

Nazé, Florence; Francart, Aurélie; Lamoral, Sophie; De Craeye, Stéphane; Kalai, Michael

2014-01-01

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was ≤1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible. PMID:24822188

Computer Assisted Design, Prediction, and Execution of Economical Organic Syntheses

NASA Astrophysics Data System (ADS)

Gothard, Nosheen Akber

The synthesis of useful organic molecules via simple and cost-effective routes is a core challenge in organic chemistry. In industry or academia, organic chemists use their chemical intuition, technical expertise and published procedures to determine an optimal pathway. This approach, not only takes time and effort, but also is cost prohibitive. Many potential optimal routes scratched on paper fail to get experimentally tested. In addition, with new methods being discovered daily are often overlooked by established techniques. This thesis reports a computational technique that assist the discovery of economical synthetic routes to useful organic targets. Organic chemistry exists as a network where chemicals are connected by reactions, analogous to citied connected by roads in a geographic map. This network topology of organic reactions in the network of organic chemistry (NOC) allows the application of graph-theory to devise algorithms for synthetic optimization of organic targets. A computational approach comprised of customizable algorithms, pre-screening filters, and existing chemoinformatic techniques is capable of answering complex questions and perform mechanistic tasks desired by chemists such as optimization of organic syntheses. One-pot reactions are central to modern synthesis since they save resources and time by avoiding isolation, purification, characterization, and production of chemical waste after each synthetic step. Sometimes, such reactions are identified by chance or, more often, by careful inspection of individual steps that are to be wired together. Algorithms are used to discover one-pot reactions and validated experimentally. Which demonstrate that the computationally predicted sequences can indeed by carried out experimentally in good overall yields. The experimental examples are chosen to from small networks of reactions around useful chemicals such as quinoline scaffolds, quinoline-based inhibitors of phosphoinositide 3-kinase delta (PI3Kdelta) enzyme, and thiophene derivatives. In this way, we replace individual synthetic connections with two-, three-, or even four-step one-pot sequences. Lastly, the computational method is utilized to devise hypothetical synthetic route to popular pharmaceutical drugs like NaproxenRTM and TaxolRTM. The algorithmically generated optimal pathways are evaluated with chemistry logic. By applying labor/cost factor It was revealed that not all shorter synthesis routes are economical, sometimes "Longest way round is the shortest way home" lengthier routes are found to be more economical and environmentally friendly.

The mechanism of the formation of the hemiaminal and Schiff base from the benzaldehyde and triazole studied by means of the topological analysis of electron localisation function and catastrophe theory

NASA Astrophysics Data System (ADS)

Berski, Slawomir; Zbigniew Ciunik, Leszek

2015-04-01

The mechanisms of reaction of benzaldehyde (ald) with 4-amine-4H-1,2,4-triazole (4at), leading to Schiff base (Sch) and water, were investigated using topological analysis of the electron localisation function and catastrophe theory. Two reactions (synthesis of hemiaminal and synthesis of Schiff base) are represented by one catastrophe sequence: ald+4at: 1-14-[FF†F†FFTS1FF†F†FF†F†CF†]-2-9-[C†FFTS3F†F†FFF]-0:Sch+H2O with only fold (F) and cusp (C) catastrophes. The first reaction, in which a molecule of the hemiaminal is formed, consists of 14 steps separated by 13 catastrophes. The mechanism is non-concerted. The covalent bond C-N is formed after the formation of the O-H bond is terminated. The Schiff base formation through the water molecule elimination in the second reaction requires nine steps with eight catastrophes. The mechanism is non-concerted because first the C-O bond is broken and then the proton transfer occurs that results in the O-H bond creation.

Abbott prism: a multichannel heterogeneous chemiluminescence immunoassay analyzer.

PubMed

Khalil, O S; Zurek, T F; Tryba, J; Hanna, C F; Hollar, R; Pepe, C; Genger, K; Brentz, C; Murphy, B; Abunimeh, N

1991-09-01

We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.

A seminested PCR assay for detection and typing of human papillomavirus based on E1 gene sequences.

PubMed

Cavalcante, Gustavo Henrique O; de Araújo, Josélio M G; Fernandes, José Veríssimo; Lanza, Daniel C F

2018-05-01

HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

A simple and novel method for RNA-seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase.

PubMed

Brouilette, Scott; Kuersten, Scott; Mein, Charles; Bozek, Monika; Terry, Anna; Dias, Kerith-Rae; Bhaw-Rosun, Leena; Shintani, Yasunori; Coppen, Steven; Ikebe, Chiho; Sawhney, Vinit; Campbell, Niall; Kaneko, Masahiro; Tano, Nobuko; Ishida, Hidekazu; Suzuki, Ken; Yashiro, Kenta

2012-10-01

Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. Here, we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus, this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Copyright © 2012 Wiley Periodicals, Inc.

Control of DNA strand displacement kinetics using toehold exchange.

PubMed

Zhang, David Yu; Winfree, Erik

2009-12-02

DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

PubMed

Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

2017-02-23

Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

Nucleic acid analysis using terminal-phosphate-labeled nucleotides

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-04-22

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Translational resistivity/conductivity of coding sequences during exponential growth of Escherichia coli.

PubMed

Takai, Kazuyuki

2017-01-21

Codon adaptation index (CAI) has been widely used for prediction of expression of recombinant genes in Escherichia coli and other organisms. However, CAI has no mechanistic basis that rationalizes its application to estimation of translational efficiency. Here, I propose a model based on which we could consider how codon usage is related to the level of expression during exponential growth of bacteria. In this model, translation of a gene is considered as an analog of electric current, and an analog of electric resistance corresponding to each gene is considered. "Translational resistance" is dependent on the steady-state concentration and the sequence of the mRNA species, and "translational resistivity" is dependent only on the mRNA sequence. The latter is the sum of two parts: one is the resistivity for the elongation reaction (coding sequence resistivity), and the other comes from all of the other steps of the decoding reaction. This electric circuit model clearly shows that some conditions should be met for codon composition of a coding sequence to correlate well with its expression level. On the other hand, I calculated relative frequency of each of the 61 sense codon triplets translated during exponential growth of E. coli from a proteomic dataset covering over 2600 proteins. A tentative method for estimating relative coding sequence resistivity based on the data is presented. Copyright © 2016. Published by Elsevier Ltd.

Reaction-diffusion processes and metapopulation models on duplex networks

NASA Astrophysics Data System (ADS)

Xuan, Qi; Du, Fang; Yu, Li; Chen, Guanrong

2013-03-01

Reaction-diffusion processes, used to model various spatially distributed dynamics such as epidemics, have been studied mostly on regular lattices or complex networks with simplex links that are identical and invariant in transferring different kinds of particles. However, in many self-organized systems, different particles may have their own private channels to keep their purities. Such division of links often significantly influences the underlying reaction-diffusion dynamics and thus needs to be carefully investigated. This article studies a special reaction-diffusion process, named susceptible-infected-susceptible (SIS) dynamics, given by the reaction steps β→α and α+β→2β, on duplex networks where links are classified into two groups: α and β links used to transfer α and β particles, which, along with the corresponding nodes, consist of an α subnetwork and a β subnetwork, respectively. It is found that the critical point of particle density to sustain reaction activity is independent of the network topology if there is no correlation between the degree sequences of the two subnetworks, and this critical value is suppressed or extended if the two degree sequences are positively or negatively correlated, respectively. Based on the obtained results, it is predicted that epidemic spreading may be promoted on positive correlated traffic networks but may be suppressed on networks with modules composed of different types of diffusion links.

Investigations of Scope and Mechanism of Nickel-Catalyzed Transformations of Glycosyl Trichloroacetimidates to Glycosyl Trichloroacetamides and Subsequent, Atom-Economical, One-Step Conversion to α-Urea-Glycosides

PubMed Central

McKay, Matthew J.; Park, Nathaniel H.; Nguyen, Hien M.

2014-01-01

The development and mechanistic investigation of a highly stereoselective methodology for preparing α-linked-urea neo-glycoconjugates and pseudo-oligosaccharides is described. This two-step procedure begins with the selective nickel-catalyzed conversion of glycosyl trichloroacetimidates to the corresponding α-trichloroacetamides. The α-selective nature of the conversion is controlled with a cationic nickel(II) catalyst, Ni(dppe)(OTf)2. Mechanistic studies have identified the coordination of the nickel catalyst with the equatorial C2-ether functionality of the α-glycosyl trichloroacetimidate to be paramount for achieving an α-stereoselective transformation. A cross-over experiment has indicated that the reaction does not proceed in an exclusively-intramolecular fashion. The second step in this sequence is the direct conversion of α-glycosyl trichloroacetamide products into the corresponding α-urea glycosides by reacting them with a wide variety of amine nucleophiles in presence of cesium carbonate. Only α-urea-product formation is observed, as the reaction proceeds with complete retention of stereochemical integrity at the anomeric C-N bond. PMID:24905328

A combined mechanistic and computational study of the gold(I)-catalyzed formation of substituted indenes.

PubMed

Nun, Pierrick; Gaillard, Sylvain; Poater, Albert; Cavallo, Luigi; Nolan, Steven P

2011-01-07

Substituted indenes can be prepared after a sequence [1,3] O-acyl shift-hydroarylation-[1,3] O-acyl shift. Each step is catalyzed by a cationic NHC-Gold(I) species generated in situ after reaction between [(IPr)AuOH] and HBF(4)·OEt(2). This interesting silver-free way is fully supported by a computational study justifying the formation of each intermediate.

Formal Synthesis of (±)-Aplykurodinone-1 through a Hetero-Pauson-Khand Cycloaddition Approach.

PubMed

Tao, Cheng; Zhang, Jing; Chen, Xiaoming; Wang, Huifei; Li, Yun; Cheng, Bin; Zhai, Hongbin

2017-03-03

The tricyclic intermediate 2 has been synthesized in eight steps from known compound 6 in 20% overall yield. As such, this constitutes a highly efficient formal synthesis of (±)-aplykurodinone-1. This synthesis features a unique, one-pot, intramolecular hetero-Pauson-Khand reaction (h-PKR)/desilylation sequence to expeditiously construct the tricyclic framework, providing valuable insights for expanding the scope and boundaries of h-PKR.

Antimalarial peroxide dyads from natural artemisinin and hydroxyalkylated 1,2,4-trioxanes.

PubMed

Griesbeck, Axel G; Neudörfl, Jörg; Hörauf, Achim; Specht, Sabine; Raabe, Angela

2009-05-28

Three synthetic approaches to highly antimalarial peroxide dyads that are composed of the natural artemisinin part (either as dihydroartemisinin or artesunic acid components) and synthetic 1,2,4-trioxanes linked by ether or ester bridges are described. Photooxygenation is the key step to introduce the trioxane group initially or at the end of the reaction sequence, respectively. Dihydroartemisinin or artesunate coupling to hydroxyethyltrioxanes are the two processes that use intact peroxide units from the beginning, whereas the dihydroartemisinin-coupling to an allylic alcohol is a postphotooxygenation route, where the second trioxane ring is installed in the last step of the procedure.

Hierarchical self-assembly of actin in micro-confinements using microfluidics

PubMed Central

Deshpande, Siddharth; Pfohl, Thomas

2012-01-01

We present a straightforward microfluidics system to achieve step-by-step reaction sequences in a diffusion-controlled manner in quasi two-dimensional micro-confinements. We demonstrate the hierarchical self-organization of actin (actin monomers—entangled networks of filaments—networks of bundles) in a reversible fashion by tuning the Mg2+ ion concentration in the system. We show that actin can form networks of bundles in the presence of Mg2+ without any cross-linking proteins. The properties of these networks are influenced by the confinement geometry. In square microchambers we predominantly find rectangular networks, whereas triangular meshes are predominantly found in circular chambers. PMID:24032070

Total synthesis of rupestine G and its epimers

PubMed Central

Yusuf, Abdullah; Zhao, Jiangyu; Wang, Bianlin; Aibibula, Paruke; Aisa, Haji Akber

2018-01-01

Rupestine G is a guaipyridine sesquiterpene alkaloid isolated from Artemisia rupestris L. The total synthesis of rupestine G and its epimers was accomplished employing a Suzuki reaction to build a terminal diene moiety. The diene was further elaborated into the desired guaipyridine structure by a ring-closing metathesis reaction. Over all, rupestine G and its three epimers were obtained as a mixture in a sequence of nine linear steps with 18.9% yield. Rupestine G and its optically pure isomers were isolated by chiral preparative HPLC and fully characterized by 1H ,13C NMR, HRMS, optical rotation value, and experimental and calculated electronic circular dichroism spectroscopy. PMID:29657802

In-Gel Determination of L-Amino Acid Oxidase Activity Based on the Visualization of Prussian Blue-Forming Reaction

PubMed Central

Zhou, Ning; Zhao, Chuntian

2013-01-01

L-amino acid oxidase (LAAO) is attracting increasing attention due to its important functions. Diverse detection methods with their own properties have been developed for characterization of LAAO. In the present study, a simple, rapid, sensitive, cost-effective and reproducible method for quantitative in-gel determination of LAAO activity based on the visualization of Prussian blue-forming reaction is described. Coupled with SDS-PAGE, this Prussian blue agar assay can be directly used to determine the numbers and approximate molecular weights of LAAO in one step, allowing straightforward application for purification and sequence identification of LAAO from diverse samples. PMID:23383337

Design, development, mechanistic elucidation, and rational optimization of a tandem Ireland Claisen/Cope rearrangement reaction for rapid access to the (iso)cyclocitrinol core.

PubMed

Plummer, Christopher W; Wei, Carolyn S; Yozwiak, Carrie E; Soheili, Arash; Smithback, Sara O; Leighton, James L

2014-07-16

An approach to the synthesis of the (iso)cyclocitrinol core structure is described. The key step is a tandem Ireland Claisen/Cope rearrangement sequence, wherein the Ireland Claisen rearrangement effects ring contraction to a strained 10-membered ring, and that strain in turn drives the Cope rearrangement under unusually mild thermal conditions. A major side product was identified as resulting from an unexpected and remarkably facile [1,3]-sigmatropic rearrangement, and a tactic to disfavor the [1,3] pathway and increase the efficiency of the tandem reaction was rationally devised.

Iso-seco-tanapartholides: Isolation, Synthesis and Biological Evaluation

PubMed Central

Makiyi, Edward F; Frade, Raquel F M; Lebl, Tomas; Jaffray, Ellis G; Cobb, Susan E; Harvey, Alan L; Slawin, Alexandra M Z; Hay, Ronald T; Westwood, Nicholas J

2009-01-01

The isolation, identification and total synthesis of two plant-derived inhibitors of the NF-κB signaling pathway from the iso-seco-tanapartholide family of natural products is described. A key step in the efficient reaction sequence is a late-stage oxidative cleavage reaction that was carried out in the absence of protecting groups to give the natural products directly. A detailed comparison of the synthetic material with samples of the natural products proved informative. Biological studies on synthetic material confirmed that these compounds act late in the NF-κB signaling pathway. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) PMID:23606807

Visualizing non-equilibrium lithiation of spinel oxide via in situ transmission electron microscopy

PubMed Central

He, Kai; Zhang, Sen; Li, Jing; Yu, Xiqian; Meng, Qingping; Zhu, Yizhou; Hu, Enyuan; Sun, Ke; Yun, Hongseok; Yang, Xiao-Qing; Zhu, Yimei; Gan, Hong; Mo, Yifei; Stach, Eric A.; Murray, Christopher B.; Su, Dong

2016-01-01

Spinel transition metal oxides are important electrode materials for lithium-ion batteries, whose lithiation undergoes a two-step reaction, whereby intercalation and conversion occur in a sequential manner. These two reactions are known to have distinct reaction dynamics, but it is unclear how their kinetics affects the overall electrochemical response. Here we explore the lithiation of nanosized magnetite by employing a strain-sensitive, bright-field scanning transmission electron microscopy approach. This method allows direct, real-time, high-resolution visualization of how lithiation proceeds along specific reaction pathways. We find that the initial intercalation process follows a two-phase reaction sequence, whereas further lithiation leads to the coexistence of three distinct phases within single nanoparticles, which has not been previously reported to the best of our knowledge. We use phase-field theory to model and describe these non-equilibrium reaction pathways, and to directly correlate the observed phase evolution with the battery's discharge performance. PMID:27157119

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

PubMed

Choudhary, Nandlal; Wei, G; Govindarajulu, A; Roy, Avijit; Li, Wenbin; Picton, Deric D; Nakhla, M K; Levy, L; Brlansky, R H

2015-11-01

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence. Published by Elsevier B.V.

Rapid polymerase chain reaction-based screening assay for bacterial biothreat agents.

PubMed

Yang, Samuel; Rothman, Richard E; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A

2008-04-01

To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents.

Synthesis of (+)-dumetorine and congeners by using flow chemistry technologies.

PubMed

Riva, Elena; Rencurosi, Anna; Gagliardi, Stefania; Passarella, Daniele; Martinelli, Marisa

2011-05-23

An efficient total synthesis of the natural alkaloid (+)-dumetorine by using flow technology is described. The process entailed five separate steps starting from the enantiopure (S)-2-(piperidin-2-yl)ethanol 4 with 29% overall yield. Most of the reactions were carried out by exploiting solvent superheating and by using packed columns of immobilized reagents or scavengers to minimize handling. New protocols for performing classical reactions under continuous flow are disclosed: the ring-closing metathesis reaction with a novel polyethylene glycol-supported Hoveyda catalyst and the unprecedented flow deprotection/Eschweiler-Clarke methylation sequence. The new protocols developed for the synthesis of (+)-dumetorine were applied to the synthesis of its simplified natural congeners (-)-sedamine and (+)-sedridine. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

PubMed

Nafissi, Nafiseh; Sum, Chi Hong; Wettig, Shawn; Slavcev, Roderick A

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼ 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

Identification of the coupling step in Na(+)-translocating NADH:quinone oxidoreductase from real-time kinetics of electron transfer.

PubMed

Belevich, Nikolai P; Bertsova, Yulia V; Verkhovskaya, Marina L; Baykov, Alexander A; Bogachev, Alexander V

2016-02-01

Bacterial Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) uses a unique set of prosthetic redox groups-two covalently bound FMN residues, a [2Fe-2S] cluster, FAD, riboflavin and a Cys4[Fe] center-to catalyze electron transfer from NADH to ubiquinone in a reaction coupled with Na(+) translocation across the membrane. Here we used an ultra-fast microfluidic stopped-flow instrument to determine rate constants and the difference spectra for the six consecutive reaction steps of Vibrio harveyi Na(+)-NQR reduction by NADH. The instrument, with a dead time of 0.25 ms and optical path length of 1 cm allowed collection of visible spectra in 50-μs intervals. By comparing the spectra of reaction steps with the spectra of known redox transitions of individual enzyme cofactors, we were able to identify the chemical nature of most intermediates and the sequence of electron transfer events. A previously unknown spectral transition was detected and assigned to the Cys4[Fe] center reduction. Electron transfer from the [2Fe-2S] cluster to the Cys4[Fe] center and all subsequent steps were markedly accelerated when Na(+) concentration was increased from 20 μM to 25 mM, suggesting coupling of the former step with tight Na(+) binding to or occlusion by the enzyme. An alternating access mechanism was proposed to explain electron transfer between subunits NqrF and NqrC. According to the proposed mechanism, the Cys4[Fe] center is alternatively exposed to either side of the membrane, allowing the [2Fe-2S] cluster of NqrF and the FMN residue of NqrC to alternatively approach the Cys4[Fe] center from different sides of the membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

PubMed

Shen, Feng; Davydova, Elena K; Du, Wenbin; Kreutz, Jason E; Piepenburg, Olaf; Ismagilov, Rustem F

2011-05-01

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications.

Digital Isothermal Quantification of Nucleic Acids via Simultaneous Chemical Initiation of Recombinase Polymerase Amplification Reactions on SlipChip

PubMed Central

Shen, Feng; Davydova, Elena K.; Du, Wenbin; Kreutz, Jason E.; Piepenburg, Olaf; Ismagilov, Rustem F.

2011-01-01

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter, isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipette loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false positive results from pre-initiation of the RPA amplification reaction before incubation were eliminated. End-point fluorescence readout was used for “yes or no” digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, Methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37–42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications. PMID:21476587

A molecular dynamics study of the role of pressure on the response of reactive materials to thermal initiation

NASA Astrophysics Data System (ADS)

Weingarten, N. Scott; Mattson, William D.; Yau, Anthony D.; Weihs, Timothy P.; Rice, Betsy M.

2010-05-01

To elucidate the mechanisms of energy release in a reacting nickel/aluminum bilayer, we simulate the exothermic alloying reactions using both microcanonical and isoenthalpic-isobaric molecular dynamics simulations and an embedded-atom method type potential. The mechanism of the mixing consists of a sequence of steps in which mixing and reaction first occurs at the interface; the resulting heat generated from the mixing then melts the Al layer; subsequent mixing leads to further heat generation after which the Ni layer melts. The mixing continues until the alloying reactions are completed. The results indicate that pressure has a significant influence on the rates of atomic mixing and alloying reactions. Local pressures and temperatures within the individual layers at the time of melting are calculated, and these results are compared with the pressure-dependent melting curves determined for pure Al and pure Ni using this interaction potential.

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion.

PubMed

Santillán, Orlando; Ramírez-Romero, Miguel A; Dávila, Guillermo

2017-06-25

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction.

PubMed

Abdulmawjood, A; Roth, S; Bülte, M

2002-10-01

For the detection of food born bacteria by polymerase chain reaction (PCR) in food products, an internal amplification control (IAC) is required in order to prevent false negative results that might be caused by PCR inhibitors. In the present study, two IACs were constructed using two different methods. These IACs were designed in a way that the same primer pair can be used to amplify the target DNA and coamplify the IAC. The first IAC with a size of approximately 200 bp was constructed by deleting a part of the amplicon of the original target DNA (500 bp) between the two primer sites to produce an IAC smaller than the target DNA. The second IAC with a size of approximately 600 bp was synthesized in a one step PCR reaction. The primers used in this reaction possessed 5' over-hanging ends, which were identical to the primers used in the diagnostic reaction, whereas their 3' ends were complementary to the (pUC19) predetermined DNA sequence of defined length and sequence. The concentration of IACs appeared to be critical. Too much IAC DNA template would out-compete the target DNA template, thus giving a false negative result. However the use of an optimal IAC concentration increased the reliability of the PCR assays and appeared to be useful for food diagnostics.

SNAP Assay Technology.

PubMed

O'Connor, Thomas P

2015-12-01

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

PubMed

Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

2017-07-12

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

PubMed

Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

2016-12-01

Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular Identification of Fungal Contamination in Date Palm Tissue Cultures.

PubMed

Abass, Mohammed H

2017-01-01

Fungal contamination of in vitro cultures of date palm (Phoenix dactylifera L.) is the major constraint to their initiation and maintenance. Different molecular approaches have been applied successfully to analyze both inter- and intraspecific variation among fungal species as well as determine their identity. This chapter describes step-by-step procedures of molecular identification of fungal contaminants by internal transcribed spacer (ITS) products of the most common fungal contaminants of date palm tissue culture. To begin with, samples of genera Alternaria, Aspergillus, Cladosporium, Epicoccum, and Penicillium were collected to isolate each fungal genus and extraction of genomic DNA. Polymerase chain reactions were accomplished by ITS primers (ITS1 and ITS4) for each fungal contaminant as well as for sequencing. Subsequently, they are analyzed by Basic Local Alignment Search Tool (BLAST) search of ITS sequence to reveal the identity of each individual fungal contaminant species. The molecular identification herein is a rapid and reliable procedure to identify date palm fungal contaminants which is very important in their control and treatment.

Direct, enantioselective α-alkylation of aldehydes using simple olefins.

PubMed

Capacci, Andrew G; Malinowski, Justin T; McAlpine, Neil J; Kuhne, Jerome; MacMillan, David W C

2017-11-01

Although the α-alkylation of ketones has already been established, the analogous reaction using aldehyde substrates has proven surprisingly elusive. Despite the structural similarities between the two classes of compounds, the sensitivity and unique reactivity of the aldehyde functionality has typically required activated substrates or specialized additives. Here, we show that the synergistic merger of three catalytic processes-photoredox, enamine and hydrogen-atom transfer (HAT) catalysis-enables an enantioselective α-aldehyde alkylation reaction that employs simple olefins as coupling partners. Chiral imidazolidinones or prolinols, in combination with a thiophenol, iridium photoredox catalyst and visible light, have been successfully used in a triple catalytic process that is temporally sequenced to deliver a new hydrogen and electron-borrowing mechanism. This multicatalytic process enables both intra- and intermolecular aldehyde α-methylene coupling with olefins to construct both cyclic and acyclic products, respectively. With respect to atom and step-economy ideals, this stereoselective process allows the production of high-value molecules from feedstock chemicals in one step while consuming only photons.

Thieno[3,2-c]pyran-4-one based novel small molecules: their synthesis, crystal structure analysis and in vitro evaluation as potential anticancer agents.

PubMed

Nakhi, Ali; Adepu, Raju; Rambabu, D; Kishore, Ravada; Vanaja, G R; Kalle, Arunasree M; Pal, Manojit

2012-07-01

Novel thieno[3,2-c]pyran-4-one based small molecules were designed as potential anticancer agents. Expeditious synthesis of these compounds was carried out via a multi-step sequence consisting of few steps such as Gewald reaction, Sandmeyer type iodination, Sonogashira type coupling followed by iodocyclization and then Pd-mediated various C-C bond forming reactions. The overall strategy involved the construction of thiophene ring followed by the fused pyranone moiety and then functionalization at C-7 position of the resultant thieno[3,2-c]pyran-4-one framework. Some of the compounds synthesized showed selective growth inhibition of cancer cells in vitro among which two compounds for example, 5d and 6c showed IC(50) values in the range of 2.0-2.5 μM. The crystal structure analysis of an active compound along with hydrogen bonding patterns and molecular arrangement present within the molecule is described. Copyright © 2012 Elsevier Ltd. All rights reserved.

Integration of advanced preparation with coal liquefaction. Second quarterly technical progress report, January 1-March 31, 1984

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

Steedman, W.G.; Longanbach, J.R.; Muralidhara, H.S.

Standard reaction conditions of 427 C, 5 minutes reaction time, 2:1 solvent/coal ratio and 1000 psig (r.t.) hydrogen overpressure result in good, but not maximum, conversions to THF soluble with both Illinois No. 6 and Wyodak (upper seam) coals. The cumulative effects of the pretreatment steps were also examined using feedstocks which were dried in a vacuum oven at room temperature under nitrogen before liquefaction to remove the effects of moisture. Chloride removal followed by drying had a positive effect on liquefaction. Oil agglomeration followed by drying also improved liquefaction reactivity significantly. Solvent drying also resulted in a small increasemore » in liquefaction reactivity. The overall reactivity of coal treated in sequence with each pretreatment step was slightly less than that of the dry ground coal. Liquefaction under a high partial pressure of hydrogen sulfide in hydrogen also results in a significant increase in conversion to THF solubles. 1 reference, 12 figures, 7 tables.« less

Direct, enantioselective α-alkylation of aldehydes using simple olefins

PubMed Central

Capacci, Andrew G.; Malinowski, Justin T.; McAlpine, Neil J.; Kuhne, Jerome; MacMillan, David W. C.

2017-01-01

Although the α-alkylation of ketones has already been established, the analogous reaction using aldehyde substrates has proven surprisingly elusive. Despite the structural similarities between the two classes of compounds, the sensitivity and unique reactivity of the aldehyde functionality has typically required activated substrates or specialized additives. Here, we show that the synergistic merger of three catalytic processes—photoredox, enamine and hydrogen-atom transfer (HAT) catalysis—enables an enantioselective α-aldehyde alkylation reaction that employs simple olefins as coupling partners. Chiral imidazolidinones or prolinols, in combination with a thiophenol, iridium photoredox catalyst and visible light, have been successfully used in a triple catalytic process that is temporally sequenced to deliver a new hydrogen and electron-borrowing mechanism. This multicatalytic process enables both intra- and intermolecular aldehyde α-methylene coupling with olefins to construct both cyclic and acyclic products, respectively. With respect to atom and step-economy ideals, this stereoselective process allows the production of high-value molecules from feedstock chemicals in one step while consuming only photons. PMID:29064486

Direct, enantioselective α-alkylation of aldehydes using simple olefins

NASA Astrophysics Data System (ADS)

Capacci, Andrew G.; Malinowski, Justin T.; McAlpine, Neil J.; Kuhne, Jerome; MacMillan, David W. C.

2017-11-01

Although the α-alkylation of ketones has already been established, the analogous reaction using aldehyde substrates has proven surprisingly elusive. Despite the structural similarities between the two classes of compounds, the sensitivity and unique reactivity of the aldehyde functionality has typically required activated substrates or specialized additives. Here, we show that the synergistic merger of three catalytic processes—photoredox, enamine and hydrogen-atom transfer (HAT) catalysis—enables an enantioselective α-aldehyde alkylation reaction that employs simple olefins as coupling partners. Chiral imidazolidinones or prolinols, in combination with a thiophenol, iridium photoredox catalyst and visible light, have been successfully used in a triple catalytic process that is temporally sequenced to deliver a new hydrogen and electron-borrowing mechanism. This multicatalytic process enables both intra- and intermolecular aldehyde α-methylene coupling with olefins to construct both cyclic and acyclic products, respectively. With respect to atom and step-economy ideals, this stereoselective process allows the production of high-value molecules from feedstock chemicals in one step while consuming only photons.

Regular threshold-energy increase with charge for neutral-particle emission in collisions of electrons with oligonucleotide anions.

PubMed

Tanabe, T; Noda, K; Saito, M; Starikov, E B; Tateno, M

2004-07-23

Electron-DNA anion collisions were studied using an electrostatic storage ring with a merging electron-beam technique. The rate of neutral particles emitted in collisions started to increase from definite threshold energies, which increased regularly with ion charges in steps of about 10 eV. These threshold energies were almost independent of the length and sequence of DNA, but depended strongly on the ion charges. Neutral particles came from breaks of DNAs, rather than electron detachment. The step of the threshold energy increase approximately agreed with the plasmon excitation energy. It is deduced that plasmon excitation is closely related to the reaction mechanism. Copyright 2004 The American Physical Society

Mechanistic insights into the decomposition of fructose to hydroxy methyl furfural in neutral and acidic environments using high-level quantum chemical methods.

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

Assary, R. S.; Redfern, P. C.; Greeley, J.

2011-03-28

Efficient catalytic chemical transformation of fructose to hydroxy methyl furfural (HMF) is one of the key steps for attaining industrial level conversion of biomass to useful chemicals. We report an investigation of the reaction mechanisms for the decomposition of fructose to HMF in both neutral and acidic environments at the Gaussian-4 level of theory including calculation of enthalpies, free energies, and effective solvation interactions. In neutral water solvent, the transformation of fructose to HMF involves a four step reaction sequence with four transition states. The effective activation energy relative to fructose in neutral water at 298 K is very large,more » about 74 kcal/mol, so that transformation in neutral media around this temperature is unlikely. In contrast, the computed potential energy surface is much more favorable for the transformation in acidic media at 498 K, as the effective activation barrier is about 39 kcal/mol. The transformation in acidic media is a much more complex mechanism involving dehydration and hydrogen transfer steps, which are more favorable when protonated intermediates are involved.« less

Mechanistic Insights into the Decomposition of Fructose to Hydroxy Methyl Furfural in Neutral and Acidic Environments Using High-Level Quantum Chemical Methods

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

Assary, Rajeev S.; Redfern, Paul C.; Greeley, Jeffrey P.

2011-04-21

Efficient catalytic chemical transformation of fructose to hydroxy methyl furfural (HMF) is one of the key steps for attaining industrial level conversion of biomass to useful chemicals. We report an investigation of the reaction mechanisms for the decomposition of fructose to HMF in both neutral and acidic environments at the Gaussian-4 level of theory including calculation of enthalpies, free energies, and effective solvation interactions. In neutral water solvent, the transformation of fructose to HMF involves a four step reaction sequence with four transition states. The effective activation energy relative to fructose in neutral water at 298 K is very large,more » about 74 kcal/mol, so that transformation in neutral media around this temperature is unlikely. In contrast, the computed potential energy surface is much more favorable for the transformation in acidic media at 498 K, as the effective activation barrier is about 39 kcal/mol. The transformation in acidic media is a much more complex mechanism involving dehydration and hydrogen transfer steps, which are more favorable when protonated intermediates are involved.« less

Mechanistic Insights into the Decomposition of Fructose to Hydroxy Methyl Furfural in Neutral and Acidic Environments Using High-Level Quantum Chemical Methods

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

Assary, Rajeev S.; Redfern, Paul C.; Greeley, Jeffrey

2011-03-28

Efficient catalytic chemical transformation of fructose to hydroxy methyl furfural (HMF) is one of the key steps for attaining industrial level conversion of biomass to useful chemicals. We report an investigation of the reaction mechanisms for the decomposition of fructose to HMF in both neutral and acidic environments at the Gaussian-4 level of theory including calculation of enthalpies, free energies, and effective solvation interactions. In neutral water solvent, the transformation of fructose to HMF involves a four step reaction sequence with four transition states. The effective activation energy relative to fructose in neutral water at 298 K is very large,more » about 74 kcal/mol, so that transformation in neutral media around this temperature is unlikely. In contrast, the computed potential energy surface is much more favorable for the transformation in acidic media at 498 K, as the effective activation barrier is about 39 kcal/mol. The transformation in acidic media is a much more complex mechanism involving dehydration and hydrogen transfer steps, which are more favorable when protonated intermediates are involved.« less

Developmentally programmed DNA deletion in Tetrahymena thermophila by a transposition-like reaction pathway.

PubMed Central

Saveliev, S V; Cox, M M

1996-01-01

We provide a molecular description of key intermediates in the deletion of two internal eliminated sequences (IES elements), the M and R regions, during macronuclear development in Tetrahymena thermophila. Using a variety of PCR-based methods in vivo, double-strand breaks are detected that are generated by hydrolytic cleavage and correspond closely to the observed chromosomal junctions left behind in the macronuclei. The breaks exhibit a temporal and structural relationship to the deletion reaction that provides strong evidence that they are intermediates in the deletion pathway. Breaks in the individual strands are staggered by 4 bp, producing a four nucleotide 5' extension. Evidence is presented that breaks do not occur simultaneously at both ends. The results are most consistent with a deletion mechanism featuring initiation by double-strand cleavage at one end of the deleted element, followed by transesterification to generate the macronuclear junction on one DNA strand. An adenosine residue is found at all the nucleophilic 3' ends used in the postulated transesterification step. Evidence for the transesterification step is provided by detection of a 3' hydroxyl that would be liberated by such a step at a deletion boundary where no other DNA strand ends are detected. Images PMID:8654384

Isolation of a new dual-functional caffeine synthase gene encoding an enzyme for the conversion of 7-methylxanthine to caffeine from coffee (Coffea arabica L.).

PubMed

Mizuno, Kouichi; Okuda, Akira; Kato, Misako; Yoneyama, Naho; Tanaka, Hiromi; Ashihara, Hiroshi; Fujimura, Tatsuhito

2003-01-16

In coffee and tea plants, caffeine is synthesized from xanthosine via a pathway that includes three methylation steps. We report the isolation of a bifunctional coffee caffeine synthase (CCS1) clone from coffee endosperm by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique using previously reported sequence information for theobromine synthases (CTSs). The predicted amino acid sequences of CCS1 are more than 80% identical to CTSs and are about 40% similar to those of tea caffeine synthase (TCS1). Interestingly, CCS1 has dual methylation activity like tea TCS1.

Exploration of mild copper-mediated coupling of organotrifluoroborates in the synthesis of thiirane-based inhibitors of matrix metalloproteinases.

PubMed

Testero, Sebastian A; Bouley, Renee; Fisher, Jed F; Chang, Mayland; Mobashery, Shahriar

2011-05-01

The copper-mediated and non-basic oxidative cross-coupling of organotrifluoroborates with phenols was applied to elaboration of the structures of thiirane-based inhibitors of matrix metalloproteinases. By revision of the synthetic sequence to allow this cross-coupling as the final step, and taking advantage of the neutral nature of organotrifluoroborate cross-coupling, a focussed series of inhibitors showing aryloxy and alkenyloxy replacement of the phenoxy substituent was prepared. This reaction shows exceptional promise as an alternative to the classic copper-mediated but strongly basic Ullmann reaction, for the diversification of ether segments within base-labile lead structures. Copyright © 2010 Elsevier Ltd. All rights reserved.

Development of a ruthenium/phosphite catalyst system for domino hydroformylation-reduction of olefins with carbon dioxide.

PubMed

Liu, Qiang; Wu, Lipeng; Fleischer, Ivana; Selent, Detlef; Franke, Robert; Jackstell, Ralf; Beller, Matthias

2014-06-02

An efficient domino ruthenium-catalyzed reverse water-gas-shift (RWGS)-hydroformylation-reduction reaction of olefins to alcohols is reported. Key to success is the use of specific bulky phosphite ligands and triruthenium dodecacarbonyl as the catalyst. Compared to the known ruthenium/chloride system, the new catalyst allows for a more efficient hydrohydroxymethylation of terminal and internal olefins with carbon dioxide at lower temperature. Unwanted hydrogenation of the substrate is prevented. Preliminary mechanism investigations uncovered the homogeneous nature of the active catalyst and the influence of the ligand and additive in individual steps of the reaction sequence. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic Progress toward Azadirachtins. 1. Enantio- and Diastereoselective Synthesis of the Left-Wing Fragment of 11-epi-Azadirachtin I.

PubMed

Shi, Hang; Tan, Ceheng; Zhang, Weibin; Zhang, Zichun; Long, Rong; Luo, Tuoping; Yang, Zhen

2015-05-15

A highly enantio- and diastereoselective synthesis of the left-wing fragment of 11-epi-azadirachtin I characterized with the pairwise use of palladium- and gold-catalyzed cascade reactions is presented. By enlisting a sequence of stereocontrolled transformations, our 21-step route established the stereocenters of the left-wing fragment from one chiral starting material, (-)-carvone, which would significantly facilitate the synthetic studies of the azadirachtin-type limonoids.

Transient kinetics measured with force steps discriminate between double-stranded DNA elongation and melting and define the reaction energetics

PubMed Central

Bongini, Lorenzo; Melli, Luca; Lombardi, Vincenzo; Bianco, Pasquale

2014-01-01

Under a tension of ∼65 pN, double-stranded DNA undergoes an overstretching transition from its basic (B-form) conformation to a 1.7 times longer conformation whose nature is only recently starting to be understood. Here we provide a structural and thermodynamic characterization of the transition by recording the length transient following force steps imposed on the λ-phage DNA with different melting degrees and temperatures (10–25°C). The shortening transient following a 20–35 pN force drop from the overstretching force shows a sequence of fast shortenings of double-stranded extended (S-form) segments and pauses owing to reannealing of melted segments. The lengthening transients following a 2–35 pN stretch to the overstretching force show the kinetics of a two-state reaction and indicate that the whole 70% extension is a B-S transition that precedes and is independent of melting. The temperature dependence of the lengthening transient shows that the entropic contribution to the B-S transition is one-third of the entropy change of thermal melting, reinforcing the evidence for a double-stranded S-form that maintains a significant fraction of the interstrand bonds. The cooperativity of the unitary elongation (22 bp) is independent of temperature, suggesting that structural factors, such as the nucleic acid sequence, control the transition. PMID:24353317

Semisupervised Gaussian Process for Automated Enzyme Search.

PubMed

Mellor, Joseph; Grigoras, Ioana; Carbonell, Pablo; Faulon, Jean-Loup

2016-06-17

Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such design requirements in an automated way, we present here a tool for exploring the space of enzymatic reactions. Given a reaction and an enzyme the tool provides a probability estimate that the enzyme catalyzes the reaction. Our tool first considers the similarity of a reaction to known biochemical reactions with respect to signatures around their reaction centers. Signatures are defined based on chemical transformation rules by using extended connectivity fingerprint descriptors. A semisupervised Gaussian process model associated with the similar known reactions then provides the probability estimate. The Gaussian process model uses information about both the reaction and the enzyme in providing the estimate. These estimates were validated experimentally by the application of the Gaussian process model to a newly identified metabolite in Escherichia coli in order to search for the enzymes catalyzing its associated reactions. Furthermore, we show with several pathway design examples how such ability to assign probability estimates to enzymatic reactions provides the potential to assist in bioengineering applications, providing experimental validation to our proposed approach. To the best of our knowledge, the proposed approach is the first application of Gaussian processes dealing with biological sequences and chemicals, the use of a semisupervised Gaussian process framework is also novel in the context of machine learning applied to bioinformatics. However, the ability of an enzyme to catalyze a reaction depends on the affinity between the substrates of the reaction and the enzyme. This affinity is generally quantified by the Michaelis constant KM. Therefore, we also demonstrate using Gaussian process regression to predict KM given a substrate-enzyme pair.

Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing.

PubMed

Guo, Hongshan; Zhu, Ping; Guo, Fan; Li, Xianlong; Wu, Xinglong; Fan, Xiaoying; Wen, Lu; Tang, Fuchou

2015-05-01

The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These modifications enable scRRBS to provide digitized methylation information on ∼1 million CpG sites within an individual diploid mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements for DNA methylation. The entire procedure takes ∼3 weeks, and it requires strong molecular biology skills.

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

French, Jarrod B.; Ealick, Steven E.

The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight intomore » the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.« less

Ceramic surfaces, interfaces and solid-state reactions

NASA Astrophysics Data System (ADS)

Heffelfinger, Jason Roy

Faceting, the decomposition of a surface into two or more surfaces of different orientation, is studied as a function of annealing time for ceramic surfaces. Single-crystals of Alsb2Osb3\\ (alpha-Alsb2Osb3 or corundum structure) are carefully prepared and characterized by atomic force microscopy, scanning electron microscopy and transmission electron microscopy. The mechanisms by which the originally smooth vicinal surface transforms into either a hill-and-valley or a terrace-and-step structure have been characterized. The progression of faceting is found to have a series of stages: surface smoothing, nucleation and growth of individual facets, formation of facet domains, coalescence of individual and facet domains and facet coarsening. These stages provide a model for the mechanisms of how other ceramic surfaces may facet into hill-and-valley and terrace-and-step surface microstructures. The well characterized Alsb2Osb3 surfaces provide excellent substrates by which to study the effect of surface structure on thin-film growth. Pulsed-laser deposition was used to grow thin films of yttria stabilized zirconia (YSZ) and Ysb2Osb3 onto annealed Alsb2Osb3 substrates. The substrate surface structure, such as surface steps and terraces, was found to have several effects on thin-film growth. Thin-films grown onto single-crystal substrates serve as a model geometry for studying thin-film solid-state reactions. Here, the reaction sequence and orientation relationship between thin films of Ysb2Osb3 and an Alsb2Osb3 substrate were characterized for different reaction temperatures. In a system were multiple reaction phases can form, the yttria aluminum monoclinic phase (YAM) was found to form prior to formation of other phases in this system. In a second system, a titanium alloy was reacted with single crystal Alsb2Osb3 in order to study phase formation in an intermetallic system. Both Tisb3Al and TiAl were found to form as reaction products and their orientation relationships with the Alsb2Osb3 are discussed.

Unexpected Reaction Pathway for butyrylcholinesterase-catalyzed inactivation of “hunger hormone” ghrelin

NASA Astrophysics Data System (ADS)

Yao, Jianzhuang; Yuan, Yaxia; Zheng, Fang; Zhan, Chang-Guo

2016-02-01

Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway.

Microchemical Plant in a Liquid Droplet: Plasmonic Liquid Marble for Sequential Reactions and Attomole Detection of Toxin at Microliter Scale.

PubMed

Han, Xuemei; Koh, Charlynn Sher Lin; Lee, Hiang Kwee; Chew, Wee Shern; Ling, Xing Yi

2017-11-15

Miniaturizing the continuous multistep operations of a factory into a microchemical plant offers a safe and cost-effective approach to promote high-throughput screening in drug development and enforcement of industrial/environmental safety. While particle-assembled microdroplets in the form of liquid marble are ideal as microchemical plant, these platforms are mainly restricted to single-step reactions and limited to ex situ reaction monitoring. Herein, we utilize plasmonic liquid marble (PLM), formed by encapsulating liquid droplet with Ag nanocubes, to address these issues and demonstrate it as an ideal microchemical plant to conduct reaction-and-detection sequences on-demand in a nondisruptive manner. Utilizing a two-step azo-dye formation as our model reaction, our microchemical plant allows rapid and efficient diazotization of nitroaniline to form diazonium nitrobenzene, followed by the azo coupling of this intermediate with target aromatic compound to yield azo-dye. These molecular events are tracked in situ via SERS measurement through the plasmonic shell and further verified with in silico investigation. Furthermore, we apply our microchemical plant for ultrasensitive SERS detection and quantification of bisphenol A (BPA) with detection limit down to 10 amol, which is 50 000-fold lower than the BPA safety limit. Together with the protections offered by plasmonic shell against external environments, these collective advantages empower PLM as a multifunctional microchemical plant to facilitate small-volume testing and optimization of processes relevant in industrial and research contexts.

Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

PubMed

Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

2015-02-01

Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

Copper-catalyzed domino cycloaddition/C-N coupling/cyclization/(C-H arylation): an efficient three-component synthesis of nitrogen polyheterocycles.

PubMed

Qian, Wenyuan; Wang, Hao; Allen, Jennifer

2013-10-11

A cat of all trades: A single copper catalyst promoted up to three reaction steps with separate catalytic cycles in a domino sequence (azide-alkyne cycloaddition/Goldberg amidation/Camps cyclization/(CH arylation)) for the rapid construction of complex heterocycles from three simple components under mild conditions. Facile cleavage of the triazole ring enables further elaboration of the condensation products. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

One-Pot Isomerization–Cross Metathesis–Reduction (ICMR) Synthesis of Lipophilic Tetrapeptides

PubMed Central

2015-01-01

An efficient, versatile and rapid method toward homologue series of lipophilic tetrapeptide derivatives (herein, the opioid peptides H-TIPP-OH and H-DIPP-OH) is reported. High atom economy and a minimal number of synthetic steps resulted from a one-pot tandem isomerization-cross metathesis-reduction sequence (ICMR), applicable both in solution and solid phase methodology. The broadly applicable synthesis proceeds with short reaction times and simple work-up, as illustrated in this work for alkylated opioid tetrapeptides. PMID:24906051

Miniaturized isothermal nucleic acid amplification, a review.

PubMed

Asiello, Peter J; Baeumner, Antje J

2011-04-21

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

Alternating electron and proton transfer steps in photosynthetic water oxidation

PubMed Central

Klauss, André; Haumann, Michael; Dau, Holger

2012-01-01

Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel–production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese–calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S2 → S3 transition of the classical S-state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant (). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein–water interface is characterized by a high activation energy (Ea = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S0 → S1 transition are similar (τ, approximately 100 µs; Ea = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established. PMID:22988080

Alternating electron and proton transfer steps in photosynthetic water oxidation.

PubMed

Klauss, André; Haumann, Michael; Dau, Holger

2012-10-02

Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel-production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese-calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S(2) → S(3) transition of the classical S-state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant (Y Z OX). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein-water interface is characterized by a high activation energy (E(a) = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S(0) → S(1) transition are similar (τ, approximately 100 µs; E(a) = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.

Contributions to a neurophysiology of meaning: the interpretation of written messages could be an automatic stimulus-reaction mechanism before becoming conscious processing of information

PubMed Central

Convertini, Livia S.; Quatraro, Sabrina; Ressa, Stefania; Velasco, Annalisa

2015-01-01

Background. Even though the interpretation of natural language messages is generally conceived as the result of a conscious processing of the message content, the influence of unconscious factors is also well known. What is still insufficiently known is the way such factors work. We have tackled interpretation assuming it is a process, whose basic features are the same for the whole humankind, and employing a naturalistic approach (careful observation of phenomena in conditions the closest to “natural” ones, and precise description before and independently of data statistical analysis). Methodology. Our field research involved a random sample of 102 adults. We presented them with a complete real world-like case of written communication using unabridged message texts. We collected data (participants’ written reports on their interpretations) in controlled conditions through a specially designed questionnaire (closed and opened answers); then, we treated it through qualitative and quantitative methods. Principal Findings. We gathered some evidence that, in written message interpretation, between reading and the attribution of conscious meaning, an intermediate step could exist (we named it “disassembling”) which looks like an automatic reaction to the text words/expressions. Thus, the process of interpretation would be a discontinuous sequence of three steps having different natures: the initial “decoding” step (i.e., reading, which requires technical abilities), disassembling (the automatic reaction, an unconscious passage) and the final conscious attribution of meaning. If this is true, words and expressions would firstly function like physical stimuli, before being taken into account as symbols. Such hypothesis, once confirmed, could help explaining some links between the cultural (human communication) and the biological (stimulus-reaction mechanisms as the basis for meanings) dimension of humankind. PMID:26528419

Rapid identification of fungal pathogens in BacT/ALERT, BACTEC, and BBL MGIT media using polymerase chain reaction and DNA sequencing of the internal transcribed spacer regions.

PubMed

Pryce, Todd M; Palladino, Silvano; Price, Diane M; Gardam, Dianne J; Campbell, Peter B; Christiansen, Keryn J; Murray, Ronan J

2006-04-01

We report a direct polymerase chain reaction/sequence (d-PCRS)-based method for the rapid identification of clinically significant fungi from 5 different types of commercial broth enrichment media inoculated with clinical specimens. Media including BacT/ALERT FA (BioMérieux, Marcy l'Etoile, France) (n = 87), BACTEC Plus Aerobic/F (Becton Dickinson, Microbiology Systems, Sparks, MD) (n = 16), BACTEC Peds Plus/F (Becton Dickinson) (n = 15), BACTEC Lytic/10 Anaerobic/F (Becton Dickinson) (n = 11) bottles, and BBL MGIT (Becton Dickinson) (n = 11) were inoculated with specimens from 138 patients. A universal DNA extraction method was used combining a novel pretreatment step to remove PCR inhibitors with a column-based DNA extraction kit. Target sequences in the noncoding internal transcribed spacer regions of the rRNA gene were amplified by PCR and sequenced using a rapid (24 h) automated capillary electrophoresis system. Using sequence alignment software, fungi were identified by sequence similarity with sequences derived from isolates identified by upper-level reference laboratories or isolates defined as ex-type strains. We identified Candida albicans (n = 14), Candida parapsilosis (n = 8), Candida glabrata (n = 7), Candida krusei (n = 2), Scedosporium prolificans (n = 4), and 1 each of Candida orthopsilosis, Candida dubliniensis, Candida kefyr, Candida tropicalis, Candida guilliermondii, Saccharomyces cerevisiae, Cryptococcus neoformans, Aspergillus fumigatus, Histoplasma capsulatum, and Malassezia pachydermatis by d-PCRS analysis. All d-PCRS identifications from positive broths were in agreement with the final species identification of the isolates grown from subculture. Earlier identification of fungi using d-PCRS may facilitate prompt and more appropriate antifungal therapy.

Study of a two-stage photobase generator for photolithography in microelectronics.

PubMed

Turro, Nicholas J; Li, Yongjun; Jockusch, Steffen; Hagiwara, Yuji; Okazaki, Masahiro; Mesch, Ryan A; Schuster, David I; Willson, C Grant

2013-03-01

The investigation of the photochemistry of a two-stage photobase generator (PBG) is described. Absorption of a photon by a latent PBG (1) (first step) produces a PBG (2). Irradiation of 2 in the presence of water produces a base (second step). This two-photon sequence (1 + hν → 2 + hν → base) is an important component in the design of photoresists for pitch division technology, a method that doubles the resolution of projection photolithography for the production of microelectronic chips. In the present system, the excitation of 1 results in a Norrish type II intramolecular hydrogen abstraction to generate a 1,4-biradiacal that undergoes cleavage to form 2 and acetophenone (Φ ∼ 0.04). In the second step, excitation of 2 causes cleavage of the oxime ester (Φ = 0.56) followed by base generation after reaction with water.

Design and synthesis of digitally encoded polymers that can be decoded and erased

NASA Astrophysics Data System (ADS)

Roy, Raj Kumar; Meszynska, Anna; Laure, Chloé; Charles, Laurence; Verchin, Claire; Lutz, Jean-François

2015-05-01

Biopolymers such as DNA store information in their chains using controlled sequences of monomers. Here we describe a non-natural information-containing macromolecule that can store and retrieve digital information. Monodisperse sequence-encoded poly(alkoxyamine amide)s were synthesized using an iterative strategy employing two chemoselective steps: the reaction of a primary amine with an acid anhydride and the radical coupling of a carbon-centred radical with a nitroxide. A binary code was implemented in the polymer chains using three monomers: one nitroxide spacer and two interchangeable anhydrides defined as 0-bit and 1-bit. This methodology allows encryption of any desired sequence in the chains. Moreover, the formed sequences are easy to decode using tandem mass spectrometry. Indeed, these polymers follow predictable fragmentation pathways that can be easily deciphered. Moreover, poly(alkoxyamine amide)s are thermolabile. Thus, the digital information encrypted in the chains can be erased by heating the polymers in the solid state or in solution.

Design and synthesis of digitally encoded polymers that can be decoded and erased.

PubMed

Roy, Raj Kumar; Meszynska, Anna; Laure, Chloé; Charles, Laurence; Verchin, Claire; Lutz, Jean-François

2015-05-26

Biopolymers such as DNA store information in their chains using controlled sequences of monomers. Here we describe a non-natural information-containing macromolecule that can store and retrieve digital information. Monodisperse sequence-encoded poly(alkoxyamine amide)s were synthesized using an iterative strategy employing two chemoselective steps: the reaction of a primary amine with an acid anhydride and the radical coupling of a carbon-centred radical with a nitroxide. A binary code was implemented in the polymer chains using three monomers: one nitroxide spacer and two interchangeable anhydrides defined as 0-bit and 1-bit. This methodology allows encryption of any desired sequence in the chains. Moreover, the formed sequences are easy to decode using tandem mass spectrometry. Indeed, these polymers follow predictable fragmentation pathways that can be easily deciphered. Moreover, poly(alkoxyamine amide)s are thermolabile. Thus, the digital information encrypted in the chains can be erased by heating the polymers in the solid state or in solution.

Novel method for high-throughput colony PCR screening in nanoliter-reactors

PubMed Central

Walser, Marcel; Pellaux, Rene; Meyer, Andreas; Bechtold, Matthias; Vanderschuren, Herve; Reinhardt, Richard; Magyar, Joseph; Panke, Sven; Held, Martin

2009-01-01

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies. PMID:19282448

Telescoping Reactions with Trifluorodiazoethane-Derived Aza-Wittig Reagents and Allenyl esters.

PubMed

Zhang, Fa-Guang; Zeng, Jun-Liang; Tian, Yi-Qiang; Zheng, Yan; Cahard, Dominique; Ma, Jun-An

2018-05-28

A telescoping process involving the consecutive addition of four reagents (trifluorodiazoethane, phosphine, allenyl ester, and acetic acid) into a single reactor was developed for the novel functionalization of allenyl esters. First, new phosphazenes derived from trifluorodiazoethane and phosphines were generated and reacted with allenyl esters to give unexpected α-iminophosphoranes through the creation of C=P, C=N, and C-H bonds at the α-, β-, and γ-carbon atoms, respectively, of the allenyl esters. The α-iminophosphoranes did not react with aldehydes in a classic Wittig reaction, but instead β-enamino esters were obtained. The overall sequence of reactions offered a formal hydrohydrazonation of allenyl esters. The method was extended to other related diazo compounds and applied to the preparation of novel 5-pyrazolone derivatives. Not only is the telescoping process described herein an effective approach for truncating the multistep synthesis, but also each step has been dissected to understand and support the reaction mechanisms. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cu-catalyzed aerobic oxidative cyclizations of 3-N-hydroxyamino-1,2-propadienes with alcohols, thiols, and amines to form α-O-, S-, and N-substituted 4-methylquinoline derivatives.

PubMed

Sharma, Pankaj; Liu, Rai-Shung

2015-03-16

A one-pot, two-step synthesis of α-O-, S-, and N-substituted 4-methylquinoline derivatives through Cu-catalyzed aerobic oxidations of N-hydroxyaminoallenes with alcohols, thiols, and amines is described. This reaction sequence involves an initial oxidation of N-hydroxyaminoallenes with NuH (Nu = OH, OR, NHR, and SR) to form 3-substituted 2-en-1-ones, followed by Brønsted acid catalyzed intramolecular cyclizations of the resulting products. Our mechanistic analysis suggests that the reactions proceed through a radical-type mechanism rather than a typical nitrone-intermediate route. The utility of this new Cu-catalyzed reaction is shown by its applicability to the synthesis of several 2-amino-4-methylquinoline derivatives, which are known to be key precursors to several bioactive molecules. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An Enzyme-Catalyzed Multistep DNA Refolding Mechanism in Hairpin Telomere Formation

PubMed Central

Shi, Ke; Huang, Wai Mun; Aihara, Hideki

2013-01-01

Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting–rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions. PMID:23382649

Systematic assignment of thermodynamic constraints in metabolic network models

PubMed Central

Kümmel, Anne; Panke, Sven; Heinemann, Matthias

2006-01-01

Background The availability of genome sequences for many organisms enabled the reconstruction of several genome-scale metabolic network models. Currently, significant efforts are put into the automated reconstruction of such models. For this, several computational tools have been developed that particularly assist in identifying and compiling the organism-specific lists of metabolic reactions. In contrast, the last step of the model reconstruction process, which is the definition of the thermodynamic constraints in terms of reaction directionalities, still needs to be done manually. No computational method exists that allows for an automated and systematic assignment of reaction directions in genome-scale models. Results We present an algorithm that – based on thermodynamics, network topology and heuristic rules – automatically assigns reaction directions in metabolic models such that the reaction network is thermodynamically feasible with respect to the production of energy equivalents. It first exploits all available experimentally derived Gibbs energies of formation to identify irreversible reactions. As these thermodynamic data are not available for all metabolites, in a next step, further reaction directions are assigned on the basis of network topology considerations and thermodynamics-based heuristic rules. Briefly, the algorithm identifies reaction subsets from the metabolic network that are able to convert low-energy co-substrates into their high-energy counterparts and thus net produce energy. Our algorithm aims at disabling such thermodynamically infeasible cyclic operation of reaction subnetworks by assigning reaction directions based on a set of thermodynamics-derived heuristic rules. We demonstrate our algorithm on a genome-scale metabolic model of E. coli. The introduced systematic direction assignment yielded 130 irreversible reactions (out of 920 total reactions), which corresponds to about 70% of all irreversible reactions that are required to disable thermodynamically infeasible energy production. Conclusion Although not being fully comprehensive, our algorithm for systematic reaction direction assignment could define a significant number of irreversible reactions automatically with low computational effort. We envision that the presented algorithm is a valuable part of a computational framework that assists the automated reconstruction of genome-scale metabolic models. PMID:17123434

Integrated on-line system for DNA sequencing by capillary electrophoresis: From template to called bases

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

Ton, H.; Yeung, E.S.

1997-02-15

An integrated on-line prototype for coupling a microreactor to capillary electrophoresis for DNA sequencing has been demonstrated. A dye-labeled terminator cycle-sequencing reaction is performed in a fused-silica capillary. Subsequently, the sequencing ladder is directly injected into a size-exclusion chromatographic column operated at nearly 95{degree}C for purification. On-line injection to a capillary for electrophoresis is accomplished at a junction set at nearly 70{degree}C. High temperature at the purification column and injection junction prevents the renaturation of DNA fragments during on-line transfer without affecting the separation. The high solubility of DNA in and the relatively low ionic strength of 1 x TEmore » buffer permit both effective purification and electrokinetic injection of the DNA sample. The system is compatible with highly efficient separations by a replaceable poly(ethylene oxide) polymer solution in uncoated capillary tubes. Future automation and adaptation to a multiple-capillary array system should allow high-speed, high-throughput DNA sequencing from templates to called bases in one step. 32 refs., 5 figs.« less

A conformational switch is responsible for the reversal of the 6S RNA-dependent RNA polymerase inhibition in Escherichia coli.

PubMed

Steuten, Benedikt; Wagner, Rolf

2012-12-01

6S RNA is a bacterial transcriptional regulator,which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ 70 -containing RNA polymerase. This inhibitory RNA polymerase ∼ 6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs.Here, we used limited hydrolysis with structure-specific RNases and in-line probing of isolated 6S RNA and 6SRNA ∼ pRNA complexes to investigate the molecular details leading to the release reaction. Our results indicate that pRNA transcription induces the refolding of the 6S RNA secondary structure by disrupting part of the closing stem(conserved sequence regions CRI and CRIV) and formation of a new hairpin (conserved sequence regions CRIII and CRIV). Comparison of the dimethylsulfate modification pattern of 6S RNA in living cells at stationary growth and during outgrowth confirmed the conformational change observed in vitro. Based on our results, a model describing the individual steps of the release reaction is presented.

Electrochemical techniques on sequence-specific PCR amplicon detection for point-of-care applications.

PubMed

Luo, Xiaoteng; Hsing, I-Ming

2009-10-01

Nucleic acid based analysis provides accurate differentiation among closely affiliated species and this species- and sequence-specific detection technique would be particularly useful for point-of-care (POC) testing for prevention and early detection of highly infectious and damaging diseases. Electrochemical (EC) detection and polymerase chain reaction (PCR) are two indispensable steps, in our view, in a nucleic acid based point-of-care testing device as the former, in comparison with the fluorescence counterpart, provides inherent advantages of detection sensitivity, device miniaturization and operation simplicity, and the latter offers an effective way to boost the amount of targets to a detectable quantity. In this mini-review, we will highlight some of the interesting investigations using the combined EC detection and PCR amplification approaches for end-point detection and real-time monitoring. The promise of current approaches and the direction for future investigations will be discussed. It would be our view that the synergistic effect of the combined EC-PCR steps in a portable device provides a promising detection technology platform that will be ready for point-of-care applications in the near future.

Oxidative desulfurization-fluorination of thioethers. Application for the synthesis of fluorinated nitrogen containing building blocks.

PubMed

Hugenberg, Verena; Fröhlich, Roland; Haufe, Günter

2010-12-21

An oxidative desulfurization-fluorination protocol has been used to synthesize (2S)-2-(difluoromethyl)-N-tosylpyrrolidine (6a) and (2S)-2-(trifluoromethyl)-N-tosylpyrrolidine (7a) from the (2S)-prolinol-derived (2S)-2-(4-chlorophenylthiomethyl)-N-tosylpyrrolidine (9) or (2S)-2-(dithian-2-yl)-N-tosylpyrrolidine (5). Efforts to prepare 3,3-difluoroalanine similarly from an N-protected S-aryl-cysteine ester 17 gave only traces of the target compound 18. Instead, an unique N-(α,α-difluorobenzyl)-N-α',α'-dibromoglycine ester 19 was formed by an unprecedented sequence of reaction steps. A plausible mechanism is suggested involving a sulfur-assisted deoxygenation-difluorination of an imino oxygen and a haloform reaction like carbon-carbon bond fission as key-steps. Efforts to prepare (2S)-2-(fluoromethyl)-N-tosylpyrrolidine (12) from (2S)-N-tosylprolinol (3) by treatment with Fluolead™ (1-tert-butyl-4-trifluorosulfanyl-3,5-dimethylbenzene) gave only 5% of the target compound, but 95% of (3R)-3-fluoro-N-tosylpiperidine (11a) by ring enlargement.

Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

PubMed Central

Zanoli, Laura Maria; Spoto, Giuseppe

2012-01-01

Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

Method and apparatus for automated assembly

DOEpatents

Jones, Rondall E.; Wilson, Randall H.; Calton, Terri L.

1999-01-01

A process and apparatus generates a sequence of steps for assembly or disassembly of a mechanical system. Each step in the sequence is geometrically feasible, i.e., the part motions required are physically possible. Each step in the sequence is also constraint feasible, i.e., the step satisfies user-definable constraints. Constraints allow process and other such limitations, not usually represented in models of the completed mechanical system, to affect the sequence.

Adenine specific DNA chemical sequencing reaction.

PubMed Central

Iverson, B L; Dervan, P B

1987-01-01

Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions. Images PMID:3671067

Direct formation of element chlorides from the corresponding element oxides through microwave-assisted carbohydrochlorination reactions.

PubMed

Nordschild, Simon; Auner, Norbert

2008-01-01

A series of technically and economically important element chlorides-such as SiCl4, BCl3, AlCl3, FeCl2, PCl3 and TiCl4-was synthesized through reactions between hydrogen chloride and the corresponding element oxides in the presence of different carbon sources with microwave assistance. This process route was optimized for demonstration purposes for tetrachlorosilane formation and successfully demonstrates the broad applicability of various silicon oxide-containing minerals and materials for carbohydrochlorination. The chlorination reaction occurs at lower temperatures than with conventional heating in a tubular oven, with substantially shorter reaction times and in better yields: quantitatively in the case of tetrachlorosilane, based on the silicon content of the starting material. The experimental procedure is very simple and provides basic information about the suitability of element compounds, especially element oxides, for carbohydrochlorination. According to the general reaction sequence element oxide-->element-->element chloride used in today's technology, this one-step carbohydrochlorination with hydrogen chloride is considerably more efficient, particularly in terms of energy input and reaction times, avoiding the isolation of the pure elements required for chlorination to give the element chlorides with use of the more corrosive and toxic chlorine gas.

Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

NASA Astrophysics Data System (ADS)

Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

Transcriptome Profiling of Khat (Catha edulis) and Ephedra sinica Reveals Gene Candidates Potentially Involved in Amphetamine-Type Alkaloid Biosynthesis

PubMed Central

Groves, Ryan A.; Hagel, Jillian M.; Zhang, Ye; Kilpatrick, Korey; Levy, Asaf; Marsolais, Frédéric; Lewinsohn, Efraim; Sensen, Christoph W.; Facchini, Peter J.

2015-01-01

Amphetamine analogues are produced by plants in the genus Ephedra and by khat (Catha edulis), and include the widely used decongestants and appetite suppressants (1S,2S)-pseudoephedrine and (1R,2S)-ephedrine. The production of these metabolites, which derive from L-phenylalanine, involves a multi-step pathway partially mapped out at the biochemical level using knowledge of benzoic acid metabolism established in other plants, and direct evidence using khat and Ephedra species as model systems. Despite the commercial importance of amphetamine-type alkaloids, only a single step in their biosynthesis has been elucidated at the molecular level. We have employed Illumina next-generation sequencing technology, paired with Trinity and Velvet-Oases assembly platforms, to establish data-mining frameworks for Ephedra sinica and khat plants. Sequence libraries representing a combined 200,000 unigenes were subjected to an annotation pipeline involving direct searches against public databases. Annotations included the assignment of Gene Ontology (GO) terms used to allocate unigenes to functional categories. As part of our functional genomics program aimed at novel gene discovery, the databases were mined for enzyme candidates putatively involved in alkaloid biosynthesis. Queries used for mining included enzymes with established roles in benzoic acid metabolism, as well as enzymes catalyzing reactions similar to those predicted for amphetamine alkaloid metabolism. Gene candidates were evaluated based on phylogenetic relationships, FPKM-based expression data, and mechanistic considerations. Establishment of expansive sequence resources is a critical step toward pathway characterization, a goal with both academic and industrial implications. PMID:25806807

Chemistry and kinetics of the pyrophoric plutonium hydride-air reaction

DOE PAGES

Haschke, John M.; Dinh, Long N.

2016-12-18

The chemistry and kinetics of the pyrophoric reaction of the plutonium hydride solid solution (PuH x, 1.9 ≤ x ≤ 3) are derived from pressure-time and gas analysis data obtained after exposure of PuH 2.7 to air in a closed system. The reaction is described in this paper by two sequential steps that result in reaction of all O 2, partial reaction of N 2, and formation of H 2. Hydrogen formed by indiscriminate reaction of N 2 and O 2 at their 3.71:1 M ratio in air during the initial step is accommodated as PuH 3 inside a productmore » layer of Pu 2O 3 and PuN. H 2 is formed by reaction of O 2 and partial reaction of N 2 with PuH 3 during the second step. Both steps of reaction are described by general equations for all values of x. The rate of the first step is proportional to the square of the O 2 pressure, but independent of temperature, x, and N 2 pressure. The second step is a factor of ten slower than step one with its rate controlled by diffusion of O 2 through a boundary layer of product H 2 and unreacted N 2. Finally, rates and enthalpies of reaction are presented and anticipated effects of reactant configuration on the heat flux are discussed.« less

Stick insect locomotion in a complex environment: climbing over large gaps.

PubMed

Blaesing, Bettina; Cruse, Holk

2004-03-01

In a complex environment, animals are challenged by various types of obstacles. This requires the controller of their walking system to be highly flexible. In this study, stick insects were presented with large gaps to cross in order to observe how locomotion can be adapted to challenging environmental situations. Different approaches were used to investigate the sequence of gap-crossing behaviour. A detailed video analysis revealed that gap-crossing behaviour resembles modified walking behaviour with additional step types. The walking sequence is interrupted by an interval of exploration, in which the insect probes the gap space with its antennae and front legs. When reaching the gap, loss of contact of an antenna with the ground does not elicit any observable reactions. In contrast, an initial front leg step into the gap that often follows antennal 'non-contact' evokes slowing down of stance velocity. An ablation experiment showed that the far edge of the gap is detected by tactile antennal stimulation rather than by vision. Initial contact of an antenna or front leg with the far edge of the gap represents a 'point of no return', after which gap crossing is always successfully completed. Finally, flow chart diagrams of the gap-crossing sequence were constructed based on an ethogram of single elements of behaviour. Comparing flow charts for two gap sizes revealed differences in the frequency and succession of these elements, especially during the first part of the sequence.

Enantioselective synthesis of pactamycin, a complex antitumor antibiotic.

PubMed

Malinowski, Justin T; Sharpe, Robert J; Johnson, Jeffrey S

2013-04-12

Medicinal application of many complex natural products is precluded by the impracticality of their chemical synthesis. Pactamycin, the most structurally intricate aminocyclopentitol antibiotic, displays potent antiproliferative properties across multiple phylogenetic domains, but it is highly cytotoxic. A limited number of analogs produced by genetic engineering technologies show reduced cytotoxicity against mammalian cells, renewing promise for therapeutic applications. For decades, an efficient synthesis of pactamycin amenable to analog derivatizations has eluded researchers. Here, we present a short asymmetric total synthesis of pactamycin. An enantioselective Mannich reaction and symmetry-breaking reduction sequence was designed to enable assembly of the entire carbon core skeleton in under five steps and control critical three-dimensional (stereochemical) functional group relationships. This modular route totals 15 steps and is immediately amenable for structural analog synthesis.

Automated Synthesis of a 184-Member Library of Thiadiazepan-1, 1-dioxide-4-ones

PubMed Central

Fenster, Erik; Long, Toby R.; Zang, Qin; Hill, David; Neuenswander, Benjamin; Lushington, Gerald H.; Zhou, Aihua; Santini, Conrad; Hanson, Paul R.

2011-01-01

The construction of a 225-member (3 × 5 × 15) library of thiadiazepan-1,1-dioxide-4-ones was performed on a Chemspeed Accelerator (SLT-100) automated parallel synthesis platform, culminating in the successful preparation of 184/225 sultams. Three sultam core scaffolds were prepared based upon the utilization of an aza-Michael reaction on a multifunctional vinyl sulfonamide linchpin. The library exploits peripheral diversity in the form of a sequential, two-step [3 + 2] Huisgen cycloaddition/Pd-catalyzed Suzuki–Miyaura coupling sequence. PMID:21309582

Automated synthesis of a 184-member library of thiadiazepan-1,1-dioxide-4-ones.

PubMed

Fenster, Erik; Long, Toby R; Zang, Qin; Hill, David; Neuenswander, Benjamin; Lushington, Gerald H; Zhou, Aihua; Santini, Conrad; Hanson, Paul R

2011-05-09

The construction of a 225-member (3 × 5 × 15) library of thiadiazepan-1,1-dioxide-4-ones was performed on a Chemspeed Accelerator (SLT-100) automated parallel synthesis platform, culminating in the successful preparation of 184/225 sultams. Three sultam core scaffolds were prepared based upon the utilization of an aza-Michael reaction on a multifunctional vinyl sulfonamide linchpin. The library exploits peripheral diversity in the form of a sequential, two-step [3 + 2] Huisgen cycloaddition/Pd-catalyzed Suzuki-Miyaura coupling sequence.

PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

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

Gardner, S. N.

2013-08-08

Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

Synthesis of isochromene-type scaffolds via single-flask Diels-Alder-[4 + 2]-annulation sequence of a silyl-substituted diene with menadione.

PubMed

Lee, Jihoon; Panek, James S

2014-06-20

A sequential Diels-Alder reaction/silicon-directed [4 + 2]-annulation was developed to assemble hydroisochromene-type ring systems from menadione 2. In the first step, a Diels-Alder of the 1-silyl-substituted butadiene 1 with 2 furnished an intermediate cyclic allylsilane. Subsequently, TMSOTf promoted a [4 + 2]-annulation through trapping of an oxonium, generated by condensation between an aldehyde and the TBS protected alcohol resulted in the formation of a cis-fused hydroisochromene 13.

On the synthesis of protopine alkaloids.

PubMed

Wada, Yasuhiro; Kaga, Harumi; Uchiito, Shiho; Kumazawa, Eri; Tomiki, Miho; Onozaki, Yu; Kurono, Nobuhito; Tokuda, Masao; Ohkuma, Takeshi; Orito, Kazuhiko

2007-09-14

For the synthesis of protopine alkaloids, we studied a reaction sequence based on a ring enlargement of indeno[2,1-a][3]benzazepines by a singlet oxygen oxygenation, followed by conversion of an amide carbonyl group of the resultant 10-membered keto-lactam to a methylene group, which is the last step for completion of the synthesis. The key substances, indeno[2,1-a][3]benzazepines, were prepared by Bischler-Napieralski cyclization of alkoxy-substituted 1-(2-bromobenzyl)-3-benzazepin-2-ones. Steric effects of the substituents in this synthesis were examined.

Low-Temperature Molecular Layer Deposition Using Monofunctional Aromatic Precursors and Ozone-Based Ring-Opening Reactions.

PubMed

Svärd, Laura; Putkonen, Matti; Kenttä, Eija; Sajavaara, Timo; Krahl, Fabian; Karppinen, Maarit; Van de Kerckhove, Kevin; Detavernier, Christophe; Simell, Pekka

2017-09-26

Molecular layer deposition (MLD) is an increasingly used deposition technique for producing thin coatings consisting of purely organic or hybrid inorganic-organic materials. When organic materials are prepared, low deposition temperatures are often required to avoid decomposition, thus causing problems with low vapor pressure precursors. Monofunctional compounds have higher vapor pressures than traditional bi- or trifunctional MLD precursors, but do not offer the required functional groups for continuing the MLD growth in subsequent deposition cycles. In this study, we have used high vapor pressure monofunctional aromatic precursors in combination with ozone-triggered ring-opening reactions to achieve sustained sequential growth. MLD depositions were carried out by using three different aromatic precursors in an ABC sequence, namely with TMA + phenol + O 3 , TMA + 3-(trifluoromethyl)phenol + O 3 , and TMA + 2-fluoro-4-(trifluoromethyl)benzaldehyde + O 3 . Furthermore, the effect of hydrogen peroxide as a fourth step was evaluated for all studied processes resulting in a four-precursor ABCD sequence. According to the characterization results by ellipsometry, infrared spectroscopy, and X-ray reflectivity, self-limiting MLD processes could be obtained between 75 and 150 °C with each of the three aromatic precursors. In all cases, the GPC (growth per cycle) decreased with increasing temperature. In situ infrared spectroscopy indicated that ring-opening reactions occurred in each ABC sequence. Compositional analysis using time-of-flight elastic recoil detection indicated that fluorine could be incorporated into the film when 3-(trifluoromethyl)phenol and 2-fluoro-4-(trifluoromethyl)benzaldehyde were used as precursors.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

PubMed

Yazdi, Soroush H; Giles, Kristen L; White, Ian M

2013-11-05

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive displacement assay is applicable to detection of a variety of biological macromolecules, including proteins and proteolytic enzymes.

Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

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

Flores, J.; Sears, J.; Schael, I.P.

1990-08-01

We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated frommore » field studies.« less

Errors in Postural Preparation Lead to Increased Choice Reaction Times for Step Initiation in Older Adults

PubMed Central

Nutt, John G.; Horak, Fay B.

2011-01-01

Background. This study asked whether older adults were more likely than younger adults to err in the initial direction of their anticipatory postural adjustment (APA) prior to a step (indicating a motor program error), whether initial motor program errors accounted for reaction time differences for step initiation, and whether initial motor program errors were linked to inhibitory failure. Methods. In a stepping task with choice reaction time and simple reaction time conditions, we measured forces under the feet to quantify APA onset and step latency and we used body kinematics to quantify forward movement of center of mass and length of first step. Results. Trials with APA errors were almost three times as common for older adults as for younger adults, and they were nine times more likely in choice reaction time trials than in simple reaction time trials. In trials with APA errors, step latency was delayed, correlation between APA onset and step latency was diminished, and forward motion of the center of mass prior to the step was increased. Participants with more APA errors tended to have worse Stroop interference scores, regardless of age. Conclusions. The results support the hypothesis that findings of slow choice reaction time step initiation in older adults are attributable to inclusion of trials with incorrect initial motor preparation and that these errors are caused by deficits in response inhibition. By extension, the results also suggest that mixing of trials with correct and incorrect initial motor preparation might explain apparent choice reaction time slowing with age in upper limb tasks. PMID:21498431

Analysis of reaction schemes using maximum rates of constituent steps

PubMed Central

Motagamwala, Ali Hussain; Dumesic, James A.

2016-01-01

We show that the steady-state kinetics of a chemical reaction can be analyzed analytically in terms of proposed reaction schemes composed of series of steps with stoichiometric numbers equal to unity by calculating the maximum rates of the constituent steps, rmax,i, assuming that all of the remaining steps are quasi-equilibrated. Analytical expressions can be derived in terms of rmax,i to calculate degrees of rate control for each step to determine the extent to which each step controls the rate of the overall stoichiometric reaction. The values of rmax,i can be used to predict the rate of the overall stoichiometric reaction, making it possible to estimate the observed reaction kinetics. This approach can be used for catalytic reactions to identify transition states and adsorbed species that are important in controlling catalyst performance, such that detailed calculations using electronic structure calculations (e.g., density functional theory) can be carried out for these species, whereas more approximate methods (e.g., scaling relations) are used for the remaining species. This approach to assess the feasibility of proposed reaction schemes is exact for reaction schemes where the stoichiometric coefficients of the constituent steps are equal to unity and the most abundant adsorbed species are in quasi-equilibrium with the gas phase and can be used in an approximate manner to probe the performance of more general reaction schemes, followed by more detailed analyses using full microkinetic models to determine the surface coverages by adsorbed species and the degrees of rate control of the elementary steps. PMID:27162366

Analysis of reaction schemes using maximum rates of constituent steps

DOE PAGES

Motagamwala, Ali Hussain; Dumesic, James A.

2016-05-09

In this paper, we show that the steady-state kinetics of a chemical reaction can be analyzed analytically in terms of proposed reaction schemes composed of series of steps with stoichiometric numbers equal to unity by calculating the maximum rates of the constituent steps, r max,i, assuming that all of the remaining steps are quasi-equilibrated. Analytical expressions can be derived in terms of r max,i to calculate degrees of rate control for each step to determine the extent to which each step controls the rate of the overall stoichiometric reaction. The values of r max,i can be used to predict themore » rate of the overall stoichiometric reaction, making it possible to estimate the observed reaction kinetics. This approach can be used for catalytic reactions to identify transition states and adsorbed species that are important in controlling catalyst performance, such that detailed calculations using electronic structure calculations (e.g., density functional theory) can be carried out for these species, whereas more approximate methods (e.g., scaling relations) are used for the remaining species. Finally, this approach to assess the feasibility of proposed reaction schemes is exact for reaction schemes where the stoichiometric coefficients of the constituent steps are equal to unity and the most abundant adsorbed species are in quasi-equilibrium with the gas phase and can be used in an approximate manner to probe the performance of more general reaction schemes, followed by more detailed analyses using full microkinetic models to determine the surface coverages by adsorbed species and the degrees of rate control of the elementary steps.« less

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

Haschke, John M.; Dinh, Long N.

The chemistry and kinetics of the pyrophoric reaction of the plutonium hydride solid solution (PuH x, 1.9 ≤ x ≤ 3) are derived from pressure-time and gas analysis data obtained after exposure of PuH 2.7 to air in a closed system. The reaction is described in this paper by two sequential steps that result in reaction of all O 2, partial reaction of N 2, and formation of H 2. Hydrogen formed by indiscriminate reaction of N 2 and O 2 at their 3.71:1 M ratio in air during the initial step is accommodated as PuH 3 inside a productmore » layer of Pu 2O 3 and PuN. H 2 is formed by reaction of O 2 and partial reaction of N 2 with PuH 3 during the second step. Both steps of reaction are described by general equations for all values of x. The rate of the first step is proportional to the square of the O 2 pressure, but independent of temperature, x, and N 2 pressure. The second step is a factor of ten slower than step one with its rate controlled by diffusion of O 2 through a boundary layer of product H 2 and unreacted N 2. Finally, rates and enthalpies of reaction are presented and anticipated effects of reactant configuration on the heat flux are discussed.« less

Synthesis of perfluoroalkylene dianilines

NASA Technical Reports Server (NTRS)

Paciorek, K. L.; Ito, T. I.; Harris, D. H.; Beechan, C. M.; Nakaham, J. H.; Kratzer, R. H.

1981-01-01

The objective of this contrast was to optimize and scale-up the synthesis of 2,2-bis(4-aminophenyl)-hexafluoropropane and 1,3-bis(4-aminophenyl)hexafluoropropane, as well as to explore avenues to other perfluoroalkyl-bridged dianilines. Routes other than Friedel-Crafts reaction leading to 2,2-bis(4-aminophenyl)hexafluoropropane were investigated. The processes utilizing bisphenol-AF were all unsuccessful; reactions aimed at the production of 4-(hexafluoro-2-halo-isopropyl)aniline from the hydroxyl intermediate failed to yield the desired products. Tailoring the conditions of the Friedel-Crafts reaction of 4-(hexafluoro-2-hydroxyisopropyl)aniline, aniline, and aluminum chloride by using hydrochloride salts and selecting optimum reagent ratios, reaction times, and temperature resulted in approx. 20% yield of pure crystallized 2,2-bis(4-aminophenyl)hexafluoropropane in 0.2 mole reaction batches. Yields up to approx. 40% were realized in small, approx. 0.01 mole, batches. The synthesis of 1,3-bis(4-aminophenyl)hexafluoropropane starting with perfluoroglutarimidine was reinvestigated. The yield of the 4-step reaction sequence giving 1,3-bis(4-acetamidophenyl)hexafluoropropane was raised to 44%. The yield of the subsequent hydrolysis process was improved by a factor of approx. 2. Approaches to prepare other perfluoroalkyl-bridged dianilines were unsuccessful. Reactions reported to proceed readily with trifluoromethyl substituents failed when longer chain perfluoroalkyl groups were employed.

Oxygen reduction on a Pt(111) catalyst in HT-PEM fuel cells by density functional theory

NASA Astrophysics Data System (ADS)

Sun, Hong; Li, Jie; Almheiri, Saif; Xiao, Jianyu

2017-08-01

The oxygen reduction reaction plays an important role in the performance of high-temperature proton exchange membrane (HT-PEM) fuel cells. In this study, a molecular dynamics model, which is based on the density functional theory and couples the system's energy, the exchange-correlation energy functional, the charge density distribution function, and the simplified Kohn-Sham equation, was developed to simulate the oxygen reduction reaction on a Pt(111) surface. Additionally, an electrochemical reaction system on the basis of a four-electron reaction mechanism was also developed for this simulation. The reaction path of the oxygen reduction reaction, the product structure of each reaction step and the system's energy were simulated. It is found that the first step reaction of the first hydrogen ion with the oxygen molecule is the controlling step of the overall reaction. Increasing the operating temperature speeds up the first step reaction rate and slightly decreases its reaction energy barrier. Our results provide insight into the working principles of HT-PEM fuel cells.

Kinetic analysis of overlapping multistep thermal decomposition comprising exothermic and endothermic processes: thermolysis of ammonium dinitramide.

PubMed

Muravyev, Nikita V; Koga, Nobuyoshi; Meerov, Dmitry B; Pivkina, Alla N

2017-01-25

This study focused on kinetic modeling of a specific type of multistep heterogeneous reaction comprising exothermic and endothermic reaction steps, as exemplified by the practical kinetic analysis of the experimental kinetic curves for the thermal decomposition of molten ammonium dinitramide (ADN). It is known that the thermal decomposition of ADN occurs as a consecutive two step mass-loss process comprising the decomposition of ADN and subsequent evaporation/decomposition of in situ generated ammonium nitrate. These reaction steps provide exothermic and endothermic contributions, respectively, to the overall thermal effect. The overall reaction process was deconvoluted into two reaction steps using simultaneously recorded thermogravimetry and differential scanning calorimetry (TG-DSC) curves by considering the different physical meanings of the kinetic data derived from TG and DSC by P value analysis. The kinetic data thus separated into exothermic and endothermic reaction steps were kinetically characterized using kinetic computation methods including isoconversional method, combined kinetic analysis, and master plot method. The overall kinetic behavior was reproduced as the sum of the kinetic equations for each reaction step considering the contributions to the rate data derived from TG and DSC. During reproduction of the kinetic behavior, the kinetic parameters and contributions of each reaction step were optimized using kinetic deconvolution analysis. As a result, the thermal decomposition of ADN was successfully modeled as partially overlapping exothermic and endothermic reaction steps. The logic of the kinetic modeling was critically examined, and the practical usefulness of phenomenological modeling for the thermal decomposition of ADN was illustrated to demonstrate the validity of the methodology and its applicability to similar complex reaction processes.

Performance monitoring and response conflict resolution associated with choice stepping reaction tasks.

PubMed

Watanabe, Tatsunori; Tsutou, Kotaro; Saito, Kotaro; Ishida, Kazuto; Tanabe, Shigeo; Nojima, Ippei

2016-11-01

Choice reaction requires response conflict resolution, and the resolution processes that occur during a choice stepping reaction task undertaken in a standing position, which requires maintenance of balance, may be different to those processes occurring during a choice reaction task performed in a seated position. The study purpose was to investigate the resolution processes during a choice stepping reaction task at the cortical level using electroencephalography and compare the results with a control task involving ankle dorsiflexion responses. Twelve young adults either stepped forward or dorsiflexed the ankle in response to a visual imperative stimulus presented on a computer screen. We used the Simon task and examined the error-related negativity (ERN) that follows an incorrect response and the correct-response negativity (CRN) that follows a correct response. Error was defined as an incorrect initial weight transfer for the stepping task and as an incorrect initial tibialis anterior activation for the control task. Results revealed that ERN and CRN amplitudes were similar in size for the stepping task, whereas the amplitude of ERN was larger than that of CRN for the control task. The ERN amplitude was also larger in the stepping task than the control task. These observations suggest that a choice stepping reaction task involves a strategy emphasizing post-response conflict and general performance monitoring of actual and required responses and also requires greater cognitive load than a choice dorsiflexion reaction. The response conflict resolution processes appear to be different for stepping tasks and reaction tasks performed in a seated position.

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration.

PubMed

Stewart, Barry J; Wardle, Simon J; Haniford, David B

2002-08-15

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes.

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration

PubMed Central

Stewart, Barry J.; Wardle, Simon J.; Haniford, David B.

2002-01-01

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes. PMID:12169640

Modelling reveals kinetic advantages of co-transcriptional splicing.

PubMed

Aitken, Stuart; Alexander, Ross D; Beggs, Jean D

2011-10-01

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3' end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3' end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

PubMed

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-05-01

Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

PubMed Central

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-01-01

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses. PMID:28403327

Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

PubMed Central

Wettig, Shawn; Slavcev, Roderick A.

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction.

PubMed

Lazinski, David W; Camilli, Andrew

2013-01-01

The amplification of DNA fragments, cloned between user-defined 5' and 3' end sequences, is a prerequisite step in the use of many current applications including massively parallel sequencing (MPS). Here we describe an improved method, called homopolymer tail-mediated ligation PCR (HTML-PCR), that requires very little starting template, minimal hands-on effort, is cost-effective, and is suited for use in high-throughput and robotic methodologies. HTML-PCR starts with the addition of homopolymer tails of controlled lengths to the 3' termini of a double-stranded genomic template. The homopolymer tails enable the annealing-assisted ligation of a hybrid oligonucleotide to the template's recessed 5' ends. The hybrid oligonucleotide has a user-defined sequence at its 5' end. This primer, together with a second primer composed of a longer region complementary to the homopolymer tail and fused to a second 5' user-defined sequence, are used in a PCR reaction to generate the final product. The user-defined sequences can be varied to enable compatibility with a wide variety of downstream applications. We demonstrate our new method by constructing MPS libraries starting from nanogram and sub-nanogram quantities of Vibrio cholerae and Streptococcus pneumoniae genomic DNA.

Molecular Taxonomy of Anopheles (Nyssorhynchus) benarrochi (Diptera: Culicidae) and Malaria Epidemiology in Southern Amazonian Peru

PubMed Central

Conn, Jan E.; Moreno, Marta; Saavedra, Marlon; Bickersmith, Sara A.; Knoll, Elisabeth; Fernandez, Roberto; Vera, Hubert; Burrus, Roxanne G.; Lescano, Andres G.; Sanchez, Juan Francisco; Rivera, Esteban; Vinetz, Joseph M.

2013-01-01

Anopheline specimens were collected in 2011 by human landing catch, Shannon and CDC traps from the malaria endemic localities of Santa Rosa and San Pedro in Madre de Dios Department, Peru. Most specimens were either Anopheles (Nyssorhynchus) benarrochi B or An. (Nys.) rangeli, confirmed by polymerase chain reaction-restriction fragment length polymorphism-internal transcribed spacer 2 (PCR-RFLP-ITS2) and, for selected individuals, ITS2 sequences. A few specimens from Lupuna, Loreto Department, northern Amazonian Peru, were also identified as An. benarrochi B. A statistical parsimony network using ITS2 sequences confirmed that all Peruvian An. benarrochi B analyzed were identical to those in GenBank from Putumayo, southern Colombia. Sequences of the mtDNA COI BOLD region of specimens from all three Peruvian localities were connected using a statistical parsimony network, although there were multiple mutation steps between northern and southern Peruvian sequences. A Bayesian inference of concatenated Peruvian sequences of ITS2+COI detected a single clade with very high support for all An. benarrochi B except one individual from Lupuna that was excluded. No samples were positive for Plasmodium by CytB-PCR. PMID:23243107

A statistical approach to detection of copy number variations in PCR-enriched targeted sequencing data.

PubMed

Demidov, German; Simakova, Tamara; Vnuchkova, Julia; Bragin, Anton

2016-10-22

Multiplex polymerase chain reaction (PCR) is a common enrichment technique for targeted massive parallel sequencing (MPS) protocols. MPS is widely used in biomedical research and clinical diagnostics as the fast and accurate tool for the detection of short genetic variations. However, identification of larger variations such as structure variants and copy number variations (CNV) is still being a challenge for targeted MPS. Some approaches and tools for structural variants detection were proposed, but they have limitations and often require datasets of certain type, size and expected number of amplicons affected by CNVs. In the paper, we describe novel algorithm for high-resolution germinal CNV detection in the PCR-enriched targeted sequencing data and present accompanying tool. We have developed a machine learning algorithm for the detection of large duplications and deletions in the targeted sequencing data generated with PCR-based enrichment step. We have performed verification studies and established the algorithm's sensitivity and specificity. We have compared developed tool with other available methods applicable for the described data and revealed its higher performance. We showed that our method has high specificity and sensitivity for high-resolution copy number detection in targeted sequencing data using large cohort of samples.

A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties

PubMed Central

Somyoonsap, Peechapack; Kitpreechavanich, Vichein

2013-01-01

A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71 kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg2+ as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease. PMID:25937959

An Interactive Classroom Activity Demonstrating Reaction Mechanisms and Rate-Determining Steps

ERIC Educational Resources Information Center

Jennings, Laura D.; Keller, Steven W.

2005-01-01

An interactive classroom activity that includes two-step reaction of unwrapping and eating chocolate candies is described which brings not only the reaction intermediate, but also the reactants and products into macroscopic view. The qualitative activation barriers of both steps can be adjusted independently.

The optimal design of stepped wedge trials with equal allocation to sequences and a comparison to other trial designs.

PubMed

Thompson, Jennifer A; Fielding, Katherine; Hargreaves, James; Copas, Andrew

2017-12-01

Background/Aims We sought to optimise the design of stepped wedge trials with an equal allocation of clusters to sequences and explored sample size comparisons with alternative trial designs. Methods We developed a new expression for the design effect for a stepped wedge trial, assuming that observations are equally correlated within clusters and an equal number of observations in each period between sequences switching to the intervention. We minimised the design effect with respect to (1) the fraction of observations before the first and after the final sequence switches (the periods with all clusters in the control or intervention condition, respectively) and (2) the number of sequences. We compared the design effect of this optimised stepped wedge trial to the design effects of a parallel cluster-randomised trial, a cluster-randomised trial with baseline observations, and a hybrid trial design (a mixture of cluster-randomised trial and stepped wedge trial) with the same total cluster size for all designs. Results We found that a stepped wedge trial with an equal allocation to sequences is optimised by obtaining all observations after the first sequence switches and before the final sequence switches to the intervention; this means that the first sequence remains in the control condition and the last sequence remains in the intervention condition for the duration of the trial. With this design, the optimal number of sequences is [Formula: see text], where [Formula: see text] is the cluster-mean correlation, [Formula: see text] is the intracluster correlation coefficient, and m is the total cluster size. The optimal number of sequences is small when the intracluster correlation coefficient and cluster size are small and large when the intracluster correlation coefficient or cluster size is large. A cluster-randomised trial remains more efficient than the optimised stepped wedge trial when the intracluster correlation coefficient or cluster size is small. A cluster-randomised trial with baseline observations always requires a larger sample size than the optimised stepped wedge trial. The hybrid design can always give an equally or more efficient design, but will be at most 5% more efficient. We provide a strategy for selecting a design if the optimal number of sequences is unfeasible. For a non-optimal number of sequences, the sample size may be reduced by allowing a proportion of observations before the first or after the final sequence has switched. Conclusion The standard stepped wedge trial is inefficient. To reduce sample sizes when a hybrid design is unfeasible, stepped wedge trial designs should have no observations before the first sequence switches or after the final sequence switches.

Photofragmentation of Gas-Phase Lanthanide Cyclopentadienyl Complexes: Experimental and Time-Dependent Excited-State Molecular Dynamics

PubMed Central

2015-01-01

Unimolecular gas-phase laser-photodissociation reaction mechanisms of open-shell lanthanide cyclopentadienyl complexes, Ln(Cp)3 and Ln(TMCp)3, are analyzed from experimental and computational perspectives. The most probable pathways for the photoreactions are inferred from photoionization time-of-flight mass spectrometry (PI-TOF-MS), which provides the sequence of reaction intermediates and the distribution of final products. Time-dependent excited-state molecular dynamics (TDESMD) calculations provide insight into the electronic mechanisms for the individual steps of the laser-driven photoreactions for Ln(Cp)3. Computational analysis correctly predicts several key reaction products as well as the observed branching between two reaction pathways: (1) ligand ejection and (2) ligand cracking. Simulations support our previous assertion that both reaction pathways are initiated via a ligand-to-metal charge-transfer (LMCT) process. For the more complex chemistry of the tetramethylcyclopentadienyl complexes Ln(TMCp)3, TMESMD is less tractable, but computational geometry optimization reveals the structures of intermediates deduced from PI-TOF-MS, including several classic “tuck-in” structures and products of Cp ring expansion. The results have important implications for metal–organic catalysis and laser-assisted metal–organic chemical vapor deposition (LCVD) of insulators with high dielectric constants. PMID:24910492

Domino syntheses of bioactive tetronic and tetramic acids.

PubMed

Schobert, Rainer

2007-01-01

Natural products containing tetronic acid or tetramic acid moieties continue to attract the interest of chemists, biologists, and physicians due to their challenging structures and to the wide range of biological activities they display. This review portrays the structural varieties of tetronic and tetramic acids and the spectrum of possible therapeutically relevant effects in man for exemplary derivatives. Their biosynthetic origin from alpha-amino and alpha-hydroxy acids is briefly discussed as is the relationship between their structures and their modes of interaction with biochemical effectors such as metal cations or enzymes. A short overview of laboratory syntheses of the heterocyclic core structures of tetramic and tetronic acids is provided with an emphasis on those emulating the biosynthesis. A synthesis from the alpha-amino or alpha-hydroxy esters and the cumulated phosphorus ylide Ph(3)PCCO based upon a domino addition-intra-Wittig alkenation sequence is presented with applications to the preparation of the antibiotics reutericyclin and tenuazonic acid, the cytotoxic melophlin B, and the enzyme inhibitor RK-682. Procedural advantages of immobilizing either starting component by attaching it to a resin and its exploitation in the parallel synthesis of libraries of potential drug candidates are described. The basic domino reaction can even be extended by further C-C bond forming steps when starting from suitable alpha-hydroxy or alpha-amino allyl esters. Depending on the chosen reaction conditions, bioactive intermediates of formally three to seven step long cascades can be obtained. Among them, herbicidal 3-alkyltetronic acids and lactone endoperoxides with antiplasmodial activity exceeding that of the natural antimalarial lead artemisinin. Hence, this domino reaction gives access to diversely functionalized derivatives of tetronic and tetramic acids. As it can also be ported to solid phase, it is ideally suited for parallel and combinatorial processing. Future developments might include running such domino sequences in continuous mode in arrays of "labs on microchips".

Domino syntheses of bioactive tetronic and tetramic acids

NASA Astrophysics Data System (ADS)

Schobert, Rainer

2007-01-01

Natural products containing tetronic acid or tetramic acid moieties continue to attract the interest of chemists, biologists, and physicians due to their challenging structures and to the wide range of biological activities they display. This review portrays the structural varieties of tetronic and tetramic acids and the spectrum of possible therapeutically relevant effects in man for exemplary derivatives. Their biosynthetic origin from α-amino and α-hydroxy acids is briefly discussed as is the relationship between their structures and their modes of interaction with biochemical effectors such as metal cations or enzymes. A short overview of laboratory syntheses of the heterocyclic core structures of tetramic and tetronic acids is provided with an emphasis on those emulating the biosynthesis. A synthesis from the α-amino or α-hydroxy esters and the cumulated phosphorus ylide Ph3PCCO based upon a domino addition-intra-Wittig alkenation sequence is presented with applications to the preparation of the antibiotics reutericyclin and tenuazonic acid, the cytotoxic melophlin B, and the enzyme inhibitor RK-682. Procedural advantages of immobilizing either starting component by attaching it to a resin and its exploitation in the parallel synthesis of libraries of potential drug candidates are described. The basic domino reaction can even be extended by further C-C bond forming steps when starting from suitable α-hydroxy or α-amino allyl esters. Depending on the chosen reaction conditions, bioactive intermediates of formally three to seven step long cascades can be obtained. Among them, herbicidal 3-alkyltetronic acids and lactone endoperoxides with antiplasmodial activity exceeding that of the natural antimalarial lead artemisinin. Hence, this domino reaction gives access to diversely functionalized derivatives of tetronic and tetramic acids. As it can also be ported to solid phase, it is ideally suited for parallel and combinatorial processing. Future developments might include running such domino sequences in continuous mode in arrays of “labs on microchips”.

Comparison of a conventional and nested PCR for diagnostic confirmation and genotyping of Orientia tsutsugamushi.

PubMed

Janardhanan, Jeshina; Prakash, John Antony Jude; Abraham, Ooriapadickal C; Varghese, George M

2014-05-01

A nested polymerase chain reaction (PCR) targeting the 56-kDa antigen gene is currently the most commonly used molecular technique for confirmation of scrub typhus and genotyping of Orientia tsutsugamushi. In this study, we have compared the commonly used nested PCR (N-PCR) with a single-step conventional PCR (C-PCR) for amplification and genotyping. Eschar samples collected from 24 patients with scrub typhus confirmed by IgM enzyme-linked immunosorbent assay were used for DNA extraction following which amplifications were carried out using nested and C-PCR methods. The amplicons were sequenced and compared to other sequences in the database using BLAST. Conventional PCR showed a high positivity rate of 95.8% compared to the 75% observed using N-PCR. On sequence analysis, the N-PCR amplified region showed more variation among strains than the C-PCR amplified region. The C-PCR, which is more economical, provided faster and better results compared to N-PCR. Copyright © 2014 Elsevier Inc. All rights reserved.

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection.

PubMed

Oakley, Brian B; Line, J Eric; Berrang, Mark E; Johnson, Jessica M; Buhr, R Jeff; Cox, Nelson A; Hiett, Kelli L; Seal, Bruce S

2012-02-01

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications. Published by Elsevier Inc.

Synthesis and conformational analysis of hybrid α/β-dipeptides incorporating S-glycosyl-β(2,2)-amino acids.

PubMed

García-González, Iván; Mata, Lara; Corzana, Francisco; Jiménez-Osés, Gonzalo; Avenoza, Alberto; Busto, Jesús H; Peregrina, Jesús M

2015-01-12

We synthesized and carried out the conformational analysis of several hybrid dipeptides consisting of an α-amino acid attached to a quaternary glyco-β-amino acid. In particular, we combined a S-glycosylated β(2,2)-amino acid and two different types of α-amino acid, namely, aliphatic (alanine) and aromatic (phenylalanine and tryptophan) in the sequence of hybrid α/β-dipeptides. The key step in the synthesis involved the ring-opening reaction of a chiral cyclic sulfamidate, inserted in the peptidic sequence, with a sulfur-containing nucleophile by using 1-thio-β-D-glucopyranose derivatives. This reaction of glycosylation occurred with inversion of configuration at the quaternary center. The conformational behavior in aqueous solution of the peptide backbone and the glycosidic linkage for all synthesized hybrid glycopeptides was analyzed by using a protocol that combined NMR experiments and molecular dynamics with time-averaged restraints (MD-tar). Interestingly, the presence of the sulfur heteroatom at the quaternary center of the β-amino acid induced θ torsional angles close to 180° (anti). Notably, this value changed to 60° (gauche) when the peptidic sequence displayed aromatic α-amino acids due to the presence of CH-π interactions between the phenyl or indole ring and the methyl groups of the β-amino acid unit. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tandem catalysis of ring-closing metathesis/atom transfer radical reactions with homobimetallic ruthenium–arene complexes

PubMed Central

Borguet, Yannick; Sauvage, Xavier; Zaragoza, Guillermo; Demonceau, Albert

2010-01-01

Summary The tandem catalysis of ring-closing metathesis/atom transfer radical reactions was investigated with the homobimetallic ruthenium–indenylidene complex [(p-cymene)Ru(μ-Cl)3RuCl(3-phenyl-1-indenylidene)(PCy3)] (1) to generate active species in situ. The two catalytic processes were first carried out independently in a case study before the whole sequence was optimized and applied to the synthesis of several polyhalogenated bicyclic γ-lactams and lactones from α,ω-diene substrates bearing trihaloacetamide or trichloroacetate functionalities. The individual steps were carefully monitored by 1H and 31P NMR spectroscopies in order to understand the intimate details of the catalytic cycles. Polyhalogenated substrates and the ethylene released upon metathesis induced the clean transformation of catalyst precursor 1 into the Ru(II)–Ru(III) mixed-valence compound [(p-cymene)Ru(μ-Cl)3RuCl2(PCy3)], which was found to be an efficient promoter for atom transfer radical reactions under the adopted experimental conditions. PMID:21160564

A theoretical thermochemical study of solute-solvent dielectric effects in the displacement of codon-anticodon base pairs

NASA Astrophysics Data System (ADS)

Monajjemi, M.; Razavian, M. H.; Mollaamin, F.; Naderi, F.; Honarparvar, B.

2008-12-01

Quantum-chemical solvent effect theories describe the electronic structure of a molecular subsystem embedded in a solvent or other molecular environment. The solvation of biomolecules is important in molecular biology, since numerous processes involve proteins interacting in changing solvent-solute systems. In this theoretical study, we focus on mRNA-tRNA base pairs as a fundamental step in protein synthesis influenced by hydrogen bonding between two antiparallel trinucleotides, namely, the mRNA codon and tRNA anticodon. We use the mean reaction field theories, which describe electrostatic and polarization interactions between solute and solvent in the AAA, UUU, AAG, and UUC triplex sequences optimized in various solvent media such as water, dimethylsulfoxide, methanol, ethanol, and cyclopean using the self-consistent reaction field model. This process depends on either the reaction potential function of the solvent or charge transfer operators that appear in solute-solvent interaction. Because of codon and anticodon biological criteria, we performed nonempirical quantum-mechanical calculations at the BLYP and B3LYP/3-21G, 6-31G, and 6-31G* levels of theory in the gas phase and five solvents at three temperatures. Finally, to obtain more information, we calculated thermochemical parameters to find that the dielectric constant of solvents plays an important role in the displacement of amino acid sequences on codon-anticodon residues in proteins, which can cause some mutations in humans.

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution.

PubMed

Katz, S; Noth, J; Horch, M; Shafaat, H S; Happe, T; Hildebrandt, P; Zebger, I

2016-11-01

[FeFe] hydrogenases are biocatalytic model systems for the exploitation and investigation of catalytic hydrogen evolution. Here, we used vibrational spectroscopic techniques to characterize, in detail, redox transformations of the [FeFe] and [4Fe4S] sub-sites of the catalytic centre (H-cluster) in a monomeric [FeFe] hydrogenase. Through the application of low-temperature resonance Raman spectroscopy, we discovered a novel metastable intermediate that is characterized by an oxidized [Fe I Fe II ] centre and a reduced [4Fe4S] 1+ cluster. Based on this unusual configuration, this species is assigned to the first, deprotonated H-cluster intermediate of the [FeFe] hydrogenase catalytic cycle. Providing insights into the sequence of initial reaction steps, the identification of this species represents a key finding towards the mechanistic understanding of biological hydrogen evolution.

Performance of a visuomotor walking task in an augmented reality training setting.

PubMed

Haarman, Juliet A M; Choi, Julia T; Buurke, Jaap H; Rietman, Johan S; Reenalda, Jasper

2017-12-01

Visual cues can be used to train walking patterns. Here, we studied the performance and learning capacities of healthy subjects executing a high-precision visuomotor walking task, in an augmented reality training set-up. A beamer was used to project visual stepping targets on the walking surface of an instrumented treadmill. Two speeds were used to manipulate task difficulty. All participants (n = 20) had to change their step length to hit visual stepping targets with a specific part of their foot, while walking on a treadmill over seven consecutive training blocks, each block composed of 100 stepping targets. Distance between stepping targets was varied between short, medium and long steps. Training blocks could either be composed of random stepping targets (no fixed sequence was present in the distance between the stepping targets) or sequenced stepping targets (repeating fixed sequence was present). Random training blocks were used to measure non-specific learning and sequenced training blocks were used to measure sequence-specific learning. Primary outcome measures were performance (% of correct hits), and learning effects (increase in performance over the training blocks: both sequence-specific and non-specific). Secondary outcome measures were the performance and stepping-error in relation to the step length (distance between stepping target). Subjects were able to score 76% and 54% at first try for lower speed (2.3 km/h) and higher speed (3.3 km/h) trials, respectively. Performance scores did not increase over the course of the trials, nor did the subjects show the ability to learn a sequenced walking task. Subjects were better able to hit targets while increasing their step length, compared to shortening it. In conclusion, augmented reality training by use of the current set-up was intuitive for the user. Suboptimal feedback presentation might have limited the learning effects of the subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

Design of multi-phase dynamic chemical networks

NASA Astrophysics Data System (ADS)

Chen, Chenrui; Tan, Junjun; Hsieh, Ming-Chien; Pan, Ting; Goodwin, Jay T.; Mehta, Anil K.; Grover, Martha A.; Lynn, David G.

2017-08-01

Template-directed polymerization reactions enable the accurate storage and processing of nature's biopolymer information. This mutualistic relationship of nucleic acids and proteins, a network known as life's central dogma, is now marvellously complex, and the progressive steps necessary for creating the initial sequence and chain-length-specific polymer templates are lost to time. Here we design and construct dynamic polymerization networks that exploit metastable prion cross-β phases. Mixed-phase environments have been used for constructing synthetic polymers, but these dynamic phases emerge naturally from the growing peptide oligomers and create environments suitable both to nucleate assembly and select for ordered templates. The resulting templates direct the amplification of a phase containing only chain-length-specific peptide-like oligomers. Such multi-phase biopolymer dynamics reveal pathways for the emergence, self-selection and amplification of chain-length- and possibly sequence-specific biopolymers.

A novel cinnamyl alcohol dehydrogenase (CAD)-like reductase contributes to the structural diversity of monoterpenoid indole alkaloids in Rauvolfia.

PubMed

Geissler, Marcus; Burghard, Marie; Volk, Jascha; Staniek, Agata; Warzecha, Heribert

2016-03-01

Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites.

Reaction schemes visualized in network form: the syntheses of strychnine as an example.

PubMed

Proudfoot, John R

2013-05-24

Representation of synthesis sequences in a network form provides an effective method for the comparison of multiple reaction schemes and an opportunity to emphasize features such as reaction scale that are often relegated to experimental sections. An example of data formatting that allows construction of network maps in Cytoscape is presented, along with maps that illustrate the comparison of multiple reaction sequences, comparison of scaffold changes within sequences, and consolidation to highlight common key intermediates used across sequences. The 17 different synthetic routes reported for strychnine are used as an example basis set. The reaction maps presented required a significant data extraction and curation, and a standardized tabular format for reporting reaction information, if applied in a consistent way, could allow the automated combination of reaction information across different sources.

In situ fabrication of cleavable peptide arrays on polydimethylsiloxane and applications for kinase activity assays.

PubMed

Chen, Huang-Han; Hsiao, Yu-Chieh; Li, Jie-Ren; Chen, Shu-Hui

2015-03-20

Polydimethylsiloxane (PDMS) is widely used for microfabrication and bioanalysis; however, its surface functionalization is limited due to the lack of active functional groups and incompatibility with many solvents. We presented a novel approach for in situ fabrication of cleavable peptide arrays on polydimethylsiloxane (PDMS) viatert-butyloxycarbonyl (t-Boc)/trifluoroacetic acid (TFA) chemistry using gold nanoparticles (AuNPs) as the anchor and a disulfide/amine terminated hetero-polyethylene glycol as the cleavable linker. The method was fine tuned to use reagents compatible with the PDMS. Using 5-mer pentapeptide, Trp5, as a model, step-by-step covalent coupling during the reaction cycles was monitored by Attenuated total reflectance-Fourier transform infrared spectrometer (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), or atomic force microscopy (AFM), and further confirmed by mass spectrometry (MS) detection of the cleaved peptides. Using such a method, heptapeptides of the PKA substrate, LRRASLG (Kemptide), and its point mutated analogs were fabricated in an array format for comparative studies of cAMP-dependent protein kinase (PKA) activity. Based on on-chip detection, Kemptide sequence exhibited the highest phosphorylation activity, which was detected to a 1.5-time lesser extent for the point mutated sequence (LRRGSLG) containing the recognition motif (RRXS), and was nearly undetectable for another point mutated sequence (LRLASLG) that lacked the recognition motif. These results indicate that the reported fabrication method is able to yield highly specific peptide sequences on PDMS, leading to a highly motif-sensitive enzyme activity assay. Copyright © 2015 Elsevier B.V. All rights reserved.

A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ.

PubMed

Xu, Lijuan; Liu, Zijian; Li, Yang; Yin, Chao; Hu, Yachen; Xie, Xiaolei; Li, Qiuchun; Jiao, Xinan

2018-06-01

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90 fg/μl or 10 2 CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.

Single Molecule Spectroscopy of Amino Acids and Peptides by Recognition Tunneling

PubMed Central

Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, JongOne; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

2014-01-01

The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single molecule protein sequencing is a critical step in the search for protein biomarkers. Here we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunneling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic ‘fingerprints’ associated with each binding motif. With this recognition tunneling technique, we are able to identify D, L enantiomers, a methylated amino acid, isobaric isomers, and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. PMID:24705512

Use of Base Modifications in Primers and Amplicons to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction

PubMed Central

2011-01-01

Abstract The addition of relatively short flap sequence at the 5′-end of one of the polymerase chain reaction (PCR) primers considerably improves performance of real-time assays based on 5′-nuclease activity. This new technology, called Snake, was shown to supersede the conventional methods like TaqMan, Molecular Beacons, and Scorpions in the signal productivity and discrimination of target polymorphic variations as small as single nucleotides. The present article describes a number of reaction conditions and methods that allow further improvement of the assay performance. One of the identified approaches is the use of duplex-destabilizing modifications such as deoxyinosine and deoxyuridine in the design of the Snake primers. This approach was shown to solve the most serious problem associated with the antisense amplicon folding and cleavage. As a result, the method permits the use of relatively long—in this study—14-mer flap sequences. Investigation also revealed that only the 5′-segment of the flap requires the deoxyinosine/deoxyuridine destabilization, whereas the 3′-segment is preferably left unmodified or even stabilized using 2-amino deoxyadenosine d(2-amA) and 5-propynyl deoxyuridine d(5-PrU) modifications. The base-modification technique is especially effective when applied in combination with asymmetric three-step PCR. The most valuable discovery of the present study is the effective application of modified deoxynucleoside 5′-triphosphates d(2-amA)TP and d(5-PrU)TP in Snake PCR. This method made possible the use of very short 6-8-mer 5′-flap sequences in Snake primers. PMID:21050073

Is impaired control of reactive stepping related to falls during inpatient stroke rehabilitation?

PubMed

Mansfield, Avril; Inness, Elizabeth L; Wong, Jennifer S; Fraser, Julia E; McIlroy, William E

2013-01-01

Individuals with stroke fall more often than age-matched controls. Although many focus on the multifactorial nature of falls, the fundamental problem is likely the ability for an individual to generate reactions to recover from a loss of balance. Stepping reactions to recover balance are particularly important to balance recovery, and individuals with stroke have difficulty executing these responses to prevent a fall following a loss of balance. The purpose of this study is to determine if characteristics of balance recovery steps are related to falls during inpatient stroke rehabilitation. We conducted a retrospective review of individuals with stroke attending inpatient rehabilitation (n = 136). Details of falls experienced during inpatient rehabilitation were obtained from incident reports, nursing notes, and patient interviews. Stepping reactions were evoked using a "release-from-lean" postural perturbation. Poisson regression was used to determine characteristics of stepping reactions that were related to increased fall frequency relative to length of stay. In all, 20 individuals experienced 29 falls during inpatient rehabilitation. The characteristics of stepping reactions significantly related to increased fall rates were increased frequency of external assistance to prevent a fall to the floor, increased frequency of no-step responses, increased frequency of step responses with inadequate foot clearance, and delayed time to initiate stepping responses. Impaired control of balance recovery steps is related to increased fall rates during inpatient stroke rehabilitation. This study informs the specific features of stepping reactions that can be targeted with physiotherapy intervention during inpatient rehabilitation to improve dynamic stability control and potentially prevent falls.

Protein trans-splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display.

PubMed

Garbe, Daniel; Thiel, Ilka V; Mootz, Henning D

2010-10-01

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties. © 2010 European Peptide Society and John Wiley & Sons, Ltd.

Nucleic acid amplification using modular branched primers

DOEpatents

Ulanovsky, Levy; Raja, Mugasimangalam C.

2001-01-01

Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Beyond benzoin condensation: trimerization of aldehydes via metal-free aerobic oxidative esterification of aldehydes with benzoin products in the presence of cyanide.

PubMed

Kim, Yoo-Jin; Kim, Na Yeun; Cheon, Cheol-Hong

2014-05-02

An unusual trimerization of aldehydes in the presence of cyanide via metal-free aerobic oxidative esterification under ambient conditions is described. Various aromatic aldehydes provided the corresponding oxidative esterification products in good to excellent yields. Mechanistic studies suggested that this reaction would proceed via a two-step sequence: cyanide-catalyzed benzoin condensation of aldehydes and subsequent aerobic oxidative esterification of aldehydes with the resultant benzoin products. The usefulness of this protocol was further demonstrated by converting the resulting trimeric products into other biologically important compounds.

Training Rapid Stepping Responses in an Individual With Stroke

PubMed Central

Inness, Elizabeth L.; Komar, Janice; Biasin, Louis; Brunton, Karen; Lakhani, Bimal; McIlroy, William E.

2011-01-01

Background and Purpose Compensatory stepping reactions are important responses to prevent a fall following a postural perturbation. People with hemiparesis following a stroke show delayed initiation and execution of stepping reactions and often are found to be unable to initiate these steps with the more-affected limb. This case report describes a targeted training program involving repeated postural perturbations to improve control of compensatory stepping in an individual with stroke. Case Description Compensatory stepping reactions of a 68-year-old man were examined 52 days after left hemorrhagic stroke. He required assistance to prevent a fall in all trials administered during his initial examination because he showed weight-bearing asymmetry (with more weight borne on the more-affected right side), was unable to initiate stepping with the right leg (despite blocking of the left leg in some trials), and demonstrated delayed response times. The patient completed 6 perturbation training sessions (30–60 minutes per session) that aimed to improve preperturbation weight-bearing symmetry, to encourage stepping with the right limb, and to reduce step initiation and completion times. Outcomes Improved efficacy of compensatory stepping reactions with training and reduced reliance on assistance to prevent falling were observed. Improvements were noted in preperturbation asymmetry and step timing. Blocking the left foot was effective in encouraging stepping with the more-affected right foot. Discussion This case report demonstrates potential short-term adaptations in compensatory stepping reactions following perturbation training in an individual with stroke. Future work should investigate the links between improved compensatory step characteristics and fall risk in this vulnerable population. PMID:21511992

Generation of sequence signatures from DNA amplification fingerprints with mini-hairpin and microsatellite primers.

PubMed

Caetano-Anollés, G; Gresshoff, P M

1996-06-01

DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationships between plant tax at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure and DAF efficiency. Mini-hairpin primers with short arbitrary cores and primers complementary to simple sequence repeats present in microsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is a dual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproducibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such as primer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynodon) cultivars and detecting putatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.

Theoretical analysis of the effects of light intensity on the photocorrosion of semiconductor electrodes

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

Benito, R.M.; Nozik, A.J.

1985-07-18

A kinetic model was developed to describe the effects of light intensity on the photocorrosion of n-type semiconductor electrodes. The model is an extension of previous work by Gomes and co-workers that includes the possibility of multiple steps for the oxidation reaction of the reducing agent in the electrolyte. Six cases are considered where the semiconductor decomposition reaction is multistep (each step involves a hole); the oxidation reaction of the reducing agent is multistep (each step after the first involves a hole or a chemical intermediate), and the first steps of the competing oxidation reactions are reversible or irreversible. Itmore » was found, contrary to previous results, that the photostability of semiconductor electrodes could increase with increased light intensity if the desired oxidation reaction of the reducing agent in the electrolyte was multistep with the first step being reversible. 14 references, 5 figures, 1 table.« less

Investigation to biodiesel production by the two-step homogeneous base-catalyzed transesterification.

PubMed

Ye, Jianchu; Tu, Song; Sha, Yong

2010-10-01

For the two-step transesterification biodiesel production made from the sunflower oil, based on the kinetics model of the homogeneous base-catalyzed transesterification and the liquid-liquid phase equilibrium of the transesterification product, the total methanol/oil mole ratio, the total reaction time, and the split ratios of methanol and reaction time between the two reactors in the stage of the two-step reaction are determined quantitatively. In consideration of the transesterification intermediate product, both the traditional distillation separation process and the improved separation process of the two-step reaction product are investigated in detail by means of the rigorous process simulation. In comparison with the traditional distillation process, the improved separation process of the two-step reaction product has distinct advantage in the energy duty and equipment requirement due to replacement of the costly methanol-biodiesel distillation column. Copyright 2010 Elsevier Ltd. All rights reserved.

Nanodusty plasma chemistry: a mechanistic and variational transition state theory study of the initial steps of silyl anion-silane and silylene anion-silane polymerization reactions.

PubMed

Bao, Junwei Lucas; Seal, Prasenjit; Truhlar, Donald G

2015-06-28

The growth of nanodusty particles, which is critical in plasma chemistry, physics, and engineering. The aim of the present work is to understand the detailed reaction mechanisms of early steps in this growth. The polymerization of neutral silane with the silylene or silyl anion, which eliminates molecular hydrogen with the formation of their higher homologues, governs the silicon hydride clustering in nanodusty plasma chemistry. The detailed mechanisms of these important polymerization reactions in terms of elementary reactions have not been proposed yet. In the present work, we investigated the initial steps of these polymerization reactions, i.e., the SiH4 + Si2H4(-)/Si2H5(-) reactions, and we propose a three-step mechanism, which is also applicable to the following polymerization steps. CM5 charges of all the silicon-containing species were computed in order to analyze the character of the species in the proposed reaction mechanisms. We also calculated thermal rate constant of each step using multi-structural canonical variational transition state theory (MS-CVT) with the small-curvature tunneling (SCT) approximation, based on the minimum energy path computed using M08-HX/MG3S electronic structure method.

Protein-DNA interactions define the mechanistic aspects of circle formation and insertion reactions in IS2 transposition.

PubMed

Lewis, Leslie A; Astatke, Mekbib; Umekubo, Peter T; Alvi, Shaheen; Saby, Robert; Afrose, Jehan; Oliveira, Pedro H; Monteiro, Gabriel A; Prazeres, Duarte Mf

2012-01-26

Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments. In IS2, Step I reactions are physically and chemically asymmetric; the left imperfect, inverted repeat (IRL), the exclusive recipient end, lacks donor function. In SC I, different protection patterns of the cleavage domains (CDs) of the right imperfect inverted repeat (IRR; extensive in cis) and IRL (selective in trans) at the single active cognate IRR catalytic center (CC) are related to their donor and recipient functions. In SC II, extensive binding of the IRL CD in trans and of the abutted IRR CD in cis at this CC represents the first phase of the complex. An MCJ substrate precleaved at the 3' end of IRR revealed a temporary transition state with the IRL CD disengaged from the protein. We propose that in SC II, sequential 3' cleavages at the bound abutted CDs trigger a conformational change, allowing the IRL CD to complex to its cognate CC, producing the second phase. Corroborating data from enhanced residues and curvature propensity plots suggest that CD to CD interactions in SC I and SC II require IRL to assume a bent structure, to facilitate binding in trans. Different transpososomes are assembled in each step of the IS2 transposition pathway. Recipient versus donor end functions of the IRL CD in SC I and SC II and the conformational change in SC II that produces the phase needed for symmetrical IRL and IRR donor attacks on target DNA highlight the differences.

Protein-DNA interactions define the mechanistic aspects of circle formation and insertion reactions in IS2 transposition

PubMed Central

2012-01-01

Background Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments. Results In IS2, Step I reactions are physically and chemically asymmetric; the left imperfect, inverted repeat (IRL), the exclusive recipient end, lacks donor function. In SC I, different protection patterns of the cleavage domains (CDs) of the right imperfect inverted repeat (IRR; extensive in cis) and IRL (selective in trans) at the single active cognate IRR catalytic center (CC) are related to their donor and recipient functions. In SC II, extensive binding of the IRL CD in trans and of the abutted IRR CD in cis at this CC represents the first phase of the complex. An MCJ substrate precleaved at the 3' end of IRR revealed a temporary transition state with the IRL CD disengaged from the protein. We propose that in SC II, sequential 3' cleavages at the bound abutted CDs trigger a conformational change, allowing the IRL CD to complex to its cognate CC, producing the second phase. Corroborating data from enhanced residues and curvature propensity plots suggest that CD to CD interactions in SC I and SC II require IRL to assume a bent structure, to facilitate binding in trans. Conclusions Different transpososomes are assembled in each step of the IS2 transposition pathway. Recipient versus donor end functions of the IRL CD in SC I and SC II and the conformational change in SC II that produces the phase needed for symmetrical IRL and IRR donor attacks on target DNA highlight the differences. PMID:22277150

Serial Reaction Time Learning in Preschool- and School-Age Children.

ERIC Educational Resources Information Center

Thomas, Kathleen M.; Nelson, Charles A.

2001-01-01

Two experiments assessed visuomotor sequence learning in 4- to 10-year-olds using a serial reaction time (SRT) task with random and sequenced trials. Found that children demonstrated sequence-specific decreases in RT. Participants with explicit awareness of the sequence at the session's end showed larger sequence-specific RT decrements than…

Interaction of abiotic and microbial processes in hexachloroethane reduction in groundwater

USGS Publications Warehouse

Roberts, A. Lynn; Gschwend, Philip M.

1994-01-01

In order to gain insight into mechanisms of hexachloroethane reduction, hexa- and pentachloroethane transformation rates were measured in anaerobic groundwater samples. For samples spiked with pentachloroethane, disappearance of pentachloroethane was accompanied by tetrachloroethylene production. Transformation rates were similar in unpoisoned and in HgCl2-poisoned samples, and rates were within ±20% of predictions based on measured pH and second-order dehydrochlorination rate constants determined in clean laboratory systems, indicating that the fate of pentachloroethane in this system is dominated by abiotic reactions. No hexachloroethane transformation was observed in HgCl2-poisoned samples, whereas in unpoisoned samples, hexachloroethane disappearance was accompanied by production of tetrachloroethylene as well as traces of pentachloroethane. Although only minor amounts of pentachloroethane accumulated, as much as 30% of the hexachloroethane transformation pathway proceeds via a pentachloroethane intermediate. This suggests that the microbial reduction of hexachloroethane proceeds at least in part through a free-radical mechanism. To the extent that hexachloroethane reduction to tetrachloroethylene occurs through a pentachloroethane intermediate, the first step in the sequence, the microbially-mediated step, is the slow step; the subsequent abiotic dehydrohalogenation step occurs much more rapidly.

Dimethylsulfoniopropionate biosynthesis in a diatom Thalassiosira pseudonana: Identification of a gene encoding MTHB-methyltransferase.

PubMed

Kageyama, Hakuto; Tanaka, Yoshito; Shibata, Ayumi; Waditee-Sirisattha, Rungaroon; Takabe, Teruhiro

2018-05-01

Dimethylsulfoniopropionate (DMSP) is one of the most abundant molecules on earth and plays a pivotal role in the marine sulfur cycle. DMSP is believed to be synthesized from methionine by a four-step reaction pathway in marine algae. The genes responsible for biosynthesis of DMSP remain unidentified. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems and contributes greatly to the world's primary production. In this study, through genome search, in vivo activity and functional studies of cDNA products, a gene encoding Thalassiosira methyltransferase (TpMMT) which catalyzes the key step of DMSP synthesis formation of 4-methylthio-2-hydroxybutyrate (DMSHB) from 4-methylthio-2-oxobutyrate (MTHB), was identified. The amino acid sequence of TpMMT was homologous to the methyltransferase from Phaeodactylum tricornutum CCAP 1055/1, but not the recently identified bacterium gene. High salinity and nitrogen limitation stresses caused the increase of DMSP content and TpMMT protein in Thalassiosira. In addition to TpMMT, the enzyme activities for the first three steps could be detected and enhanced under high salinity, suggesting the importance of four-step DMSP synthetic pathway in Thalassiosira. Copyright © 2018 Elsevier Inc. All rights reserved.

A novel glucuronosyltransferase has an unprecedented ability to catalyse continuous two-step glucuronosylation of glycyrrhetinic acid to yield glycyrrhizin.

PubMed

Xu, Guojie; Cai, Wei; Gao, Wei; Liu, Chunsheng

2016-10-01

Glycyrrhizin is an important bioactive compound that is used clinically to treat chronic hepatitis and is also used as a sweetener world-wide. However, the key UDP-dependent glucuronosyltransferases (UGATs) involved in the biosynthesis of glycyrrhizin remain unknown. To discover unknown UGATs, we fully annotated potential UGATs from Glycyrrhiza uralensis using deep transcriptome sequencing. The catalytic functions of candidate UGATs were determined by an in vitro enzyme assay. Systematically screening 434 potential UGATs, we unexpectedly found one unique GuUGAT that was able to catalyse the glucuronosylation of glycyrrhetinic acid to directly yield glycyrrhizin via continuous two-step glucuronosylation. Expression analysis further confirmed the key role of GuUGAT in the biosynthesis of glycyrrhizin. Site-directed mutagenesis revealed that Gln-352 may be important for the initial step of glucuronosylation, and His-22, Trp-370, Glu-375 and Gln-392 may be important residues for the second step of glucuronosylation. Notably, the ability of GuUGAT to catalyse a continuous two-step glucuronosylation reaction was determined to be unprecedented among known glycosyltransferases of bioactive plant natural products. Our findings increase the understanding of traditional glycosyltransferases and pave the way for the complete biosynthesis of glycyrrhizin. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

From planes to bowls: Photodissociation of the bisanthenequinone cation

NASA Astrophysics Data System (ADS)

Chen, Tao; Zhen, Junfeng; Wang, Ying; Linnartz, Harold; Tielens, Alexander G. G. M.

2018-01-01

We present a combined experimental and theoretical study of the photodissociation of the bisanthenequinone (C28H12O2) cation, Bq+. The experiments show that, upon photolysis, the Bq+ cation does not dehydrogenate, but instead fragments through the sequential loss of the two neutral carbonyl groups, causing the formation of five-membered carbon cycles. Quantum chemical calculations confirm this Bq+ → [Bq - CO]+ → [Bq - 2CO]+ sequence as the energetically most favorable reaction pathway. For the first CO loss, a transition state with a barrier of ∼3.2 eV is found, substantially lower than the lowest calculated H loss dissociation pathway (∼4.9 eV). A similar situation applies for the second CO loss channel (∼3.8 eV vs. ∼4.7 eV), but where the first dissociation step does not strongly alter the planar PAH geometry, the second step transforms the molecule into a bowl-shaped one.

Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing

PubMed Central

Murphy, Danielle A.; Ely, Heather A.; Shoemaker, Robert; Boomer, Aaron; Culver, Brady P.; Hoskins, Ian; Haimes, Josh D.; Walters, Ryan D.; Fernandez, Diane; Stahl, Joshua A.; Lee, Jeeyun; Kim, Kyoung-Mee; Lamoureux, Jennifer

2017-01-01

Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens. PMID:27028240

Prebiotic replicase evolution in a surface-bound metabolic system: parasites as a source of adaptive evolution

PubMed Central

2008-01-01

Background The remarkable potential of recent forms of life for reliably passing on genetic information through many generations now depends on the coordinated action of thousands of specialized biochemical "machines" (enzymes) that were obviously absent in prebiotic times. Thus the question how a complicated system like the living cell could have assembled on Earth seems puzzling. In seeking for a scientific explanation one has to search for step-by-step evolutionary changes from prebiotic chemistry to the emergence of the first proto-cell. Results We try to sketch a plausible scenario for the first steps of prebiotic evolution by exploring the ecological feasibility of a mineral surface-bound replicator system that facilitates a primitive metabolism. Metabolism is a hypothetical network of simple chemical reactions producing monomers for the template-copying of RNA-like replicators, which in turn catalyse metabolic reactions. Using stochastic cellular automata (SCA) simulations we show that the surface-bound metabolic replicator system is viable despite internal competition among the genes and that it also maintains a set of mild "parasitic" sequences which occasionally evolve functions such as that of a replicase. Conclusion Replicase activity is shown to increase even at the expense of slowing down the replication of the evolving ribozyme itself, due to indirect mutualistic benefits in a diffuse form of group selection among neighbouring replicators. We suggest possible paths for further evolutionary changes in the metabolic replicator system leading to increased metabolic efficiency, improved replicase functionality, and membrane production. PMID:18826645

Synthesis of Bipartite Tetracysteine PNA Probes for DNA In Situ Fluorescent Labeling.

PubMed

Fang, Ge-Min; Seitz, Oliver

2017-12-24

"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A two-step database search method improves sensitivity in peptide sequence matches for metaproteomics and proteogenomics studies.

PubMed

Jagtap, Pratik; Goslinga, Jill; Kooren, Joel A; McGowan, Thomas; Wroblewski, Matthew S; Seymour, Sean L; Griffin, Timothy J

2013-04-01

Large databases (>10(6) sequences) used in metaproteomic and proteogenomic studies present challenges in matching peptide sequences to MS/MS data using database-search programs. Most notably, strict filtering to avoid false-positive matches leads to more false negatives, thus constraining the number of peptide matches. To address this challenge, we developed a two-step method wherein matches derived from a primary search against a large database were used to create a smaller subset database. The second search was performed against a target-decoy version of this subset database merged with a host database. High confidence peptide sequence matches were then used to infer protein identities. Applying our two-step method for both metaproteomic and proteogenomic analysis resulted in twice the number of high confidence peptide sequence matches in each case, as compared to the conventional one-step method. The two-step method captured almost all of the same peptides matched by the one-step method, with a majority of the additional matches being false negatives from the one-step method. Furthermore, the two-step method improved results regardless of the database search program used. Our results show that our two-step method maximizes the peptide matching sensitivity for applications requiring large databases, especially valuable for proteogenomics and metaproteomics studies. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Culture and the Sequence of Steps in Theory of Mind Development

ERIC Educational Resources Information Center

Shahaeian, Ameneh; Peterson, Candida C.; Slaughter, Virginia; Wellman, Henry M.

2011-01-01

To examine cultural contrasts in the ordered sequence of conceptual developments leading to theory of mind (ToM), we compared 135 3- to 6-year-olds (77 Australians; 58 Iranians) on an established 5-step ToM scale (Wellman & Liu, 2004). There was a cross-cultural difference in the sequencing of ToM steps but not in overall rates of ToM mastery.…

Evaluation of a rapid detection for Coxsackievirus B3 using one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP).

PubMed

Monazah, A; Zeinoddini, M; Saeeidinia, A R

2017-08-01

Coxsackievirus B3 (CVB3) is a member of the genus Enterovirus within the family Picornaviridae and is an important pathogen of viral myocarditis, which accounts for more than 50% viral myocarditis cases. VP1 is major capsid protein that this region has a low homology in both amino acid and nucleotide sequences among Enteroviruses. Therefore we have chosen this region for designed a set of RT-LAMP primers for CVB3 detection. For this the total RNA was extracted from 24-h post infected-HeLa cells with complete cytopathic effect (CPE), and applied to a one-step reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP) using CVB3-specific primers. The optimization of RT-LAMP reaction was carried out with three variables factors including MgSO 4 concentration, temperature and time of incubation. Amplification was analyzed by using 2% agarose gel electrophoresis and ethidium bromide and SYBR Green staining. Our results were shown the ladder-like pattern of the VP1 gene amplification. The LAMP reaction mix was optimized and the best result observed at 4mM MgSO 4 and 60°C for 90min incubation. RT-LAMP had high sensitivity and specificity for detection of CVB3 infection. This method can be used as a rapid and easy diagnostic test for detection of CVB3 in clinical laboratories. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrochemistry and the mechanisms of nucleation and growth of neodymium during electroreduction from LiCl-KCl eutectic salts on Mo substrate

NASA Astrophysics Data System (ADS)

Tang, Hao; Pesic, Batric

2015-03-01

The electrochemical behavior of NdCl3 was studied on a Mo electrode in molten LiCl-KCl eutectic salts. The electroreduction of Nd(III)/Nd(0) involved two reaction steps, as confirmed by three different electrochemical techniques. In the first reaction step, Nd(III) is converted into soluble Nd(II), which undergoes further reduction into metallic Nd(0) in the second reaction step. The standard reaction rate constants for each reaction step were determined by Nicholson method. The rate constant values were used in Matsuda-Ayabe's criteria for testing the electrochemical reversibility. Accordingly, both reaction steps were quasi-reversible redox reactions. The nucleation mechanisms of neodymium metal deposited on a Mo substrate were predicted by using Scharifker-Hill model, and tested for the first time by scanning electron microscopy (SEM) studies of the electrode surface. The SEM studies confirmed that for the low initial concentration of NdCl3, neodymium nucleates and grows progressively, while for higher NdCl3 concentrations, the related mechanism is instantaneous. Both are governed by the aggregative growth mechanisms based on surface mobility of formed nanoclusters.

Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction

PubMed Central

Lazinski, David W.; Camilli, Andrew

2013-01-01

The amplification of DNA fragments, cloned between user-defined 5′ and 3′ end sequences, is a prerequisite step in the use of many current applications including massively parallel sequencing (MPS). Here we describe an improved method, called homopolymer tail-mediated ligation PCR (HTML-PCR), that requires very little starting template, minimal hands-on effort, is cost-effective, and is suited for use in high-throughput and robotic methodologies. HTML-PCR starts with the addition of homopolymer tails of controlled lengths to the 3′ termini of a double-stranded genomic template. The homopolymer tails enable the annealing-assisted ligation of a hybrid oligonucleotide to the template's recessed 5′ ends. The hybrid oligonucleotide has a user-defined sequence at its 5′ end. This primer, together with a second primer composed of a longer region complementary to the homopolymer tail and fused to a second 5′ user-defined sequence, are used in a PCR reaction to generate the final product. The user-defined sequences can be varied to enable compatibility with a wide variety of downstream applications. We demonstrate our new method by constructing MPS libraries starting from nanogram and sub-nanogram quantities of Vibrio cholerae and Streptococcus pneumoniae genomic DNA. PMID:23311318

Exploring possible reaction pathways for the o-atom transfer reactions to unsaturated substrates catalyzed by a [Ni-NO2 ] ↔ [Ni-NO] redox couple using DFT methods.

PubMed

Tsipis, Athanassios C

2017-07-15

The (nitro)(N-methyldithiocarbamato)(trimethylphospane)nickel(II), [Ni(NO 2 )(S 2 CNHMe)(PMe 3 )] complex catalyses efficiently the O-atom transfer reactions to CO and acetylene. Energetically feasible sequence of elementary steps involved in the catalytic cycle of the air oxidation of CO and acetylene are proposed promoted by the Ni(NO 2 )(S 2 CNHMe)(PMe 3 )] ↔ Ni(NO 2 )(S 2 CNHMe)(PMe 3 ) redox couple using DFT methods both in vacuum and dichloromethane solutions. The catalytic air oxidation of HC≡CH involves formation of a five-member metallacycle intermediate, via a [3 + 2] cyclo-addition reaction of HC≡CH to the Ni-N = O moiety of the Ni(NO 2 )(S 2 CNHMe)(PMe 3 )] complex, followed by a β H-atom migration toward the C α carbon atom of the coordinated acetylene and release of the oxidation product (ketene). The geometric and energetic reaction profile for the reversible [Ni( κN1-NO 2 )(S 2 CNHMe)(PMe 3 )] ⇌ [Ni( κO,O2-ONO)(S 2 CNHMe)(PMe 3 )] linkage isomerization has also been modeled by DFT calculations. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A Computational Approach to Increase Time Scales in Brownian Dynamics–Based Reaction-Diffusion Modeling

PubMed Central

Frazier, Zachary

2012-01-01

Abstract Particle-based Brownian dynamics simulations offer the opportunity to not only simulate diffusion of particles but also the reactions between them. They therefore provide an opportunity to integrate varied biological data into spatially explicit models of biological processes, such as signal transduction or mitosis. However, particle based reaction-diffusion methods often are hampered by the relatively small time step needed for accurate description of the reaction-diffusion framework. Such small time steps often prevent simulation times that are relevant for biological processes. It is therefore of great importance to develop reaction-diffusion methods that tolerate larger time steps while maintaining relatively high accuracy. Here, we provide an algorithm, which detects potential particle collisions prior to a BD-based particle displacement and at the same time rigorously obeys the detailed balance rule of equilibrium reactions. We can show that for reaction-diffusion processes of particles mimicking proteins, the method can increase the typical BD time step by an order of magnitude while maintaining similar accuracy in the reaction diffusion modelling. PMID:22697237

Carbothermal Reduction of Quartz with Carbon from Natural Gas

NASA Astrophysics Data System (ADS)

Li, Fei; Tangstad, Merete

2017-04-01

Carbothermal reaction between quartz and two different carbons originating from natural gas were investigated in this paper. One of two carbons is the commercial carbon black produced from natural gas in a medium thermal production process. The other carbon is obtained from natural gas cracking at 1273 K (1000 °C) deposited directly on the quartz pellet. At the 1923 K (1650 °C) and CO atmosphere, the impact of carbon content, pellet structure, gas transfer, and heating rate are investigated in a thermo-gravimetric furnace. The reaction process can be divided into two steps: an initial SiC-producing step followed by a SiO-producing step. Higher carbon content and increased gas transfer improves the reaction rate of SiC-producing step, while the thicker carbon coating in carbon-deposited pellet hinders reaction rate. Better gas transfer of sample holder improves reaction rate but causes more SiO loss. Heating rate has almost no influence on reaction. Mass balance analysis shows that mole ratios between SiO2, free carbon, and SiC in the SiC-producing step and SiO-producing step in CO and Ar fit the reaction SiO2(s) + 3 C(s) = SiC(s) + 2 CO(g). SiC-particle and SiC-coating formation process in mixed pellet and carbon-deposited pellet are proposed. SiC whiskers formed in the voids of these two types of pellets.

Use of the challenge point framework to guide motor learning of stepping reactions for improved balance control in people with stroke: a case series.

PubMed

Pollock, Courtney L; Boyd, Lara A; Hunt, Michael A; Garland, S Jayne

2014-04-01

Stepping reactions are important for walking balance and community-level mobility. Stepping reactions of people with stroke are characterized by slow reaction times, poor coordination of motor responses, and low amplitude of movements, which may contribute to their decreased ability to recover their balance when challenged. An important aspect of rehabilitation of mobility after stroke is optimizing the motor learning associated with retraining effective stepping reactions. The Challenge Point Framework (CPF) is a model that can be used to promote motor learning through manipulation of conditions of practice to modify task difficulty, that is, the interaction of the skill of the learner and the difficulty of the task to be learned. This case series illustrates how the retraining of multidirectional stepping reactions may be informed by the CPF to improve balance function in people with stroke. Four people (53-68 years of age) with chronic stroke (>1 year) and mild to moderate motor recovery received 4 weeks of multidirectional stepping reaction retraining. Important tenets of motor learning were optimized for each person during retraining in accordance with the CPF. Participants demonstrated improved community-level walking balance, as determined with the Community Balance and Mobility Scale. These improvements were evident 1 year later. Aspects of balance-related self-efficacy and movement kinematics also showed improvements during the course of the intervention. The application of CPF motor learning principles in the retraining of stepping reactions to improve community-level walking balance in people with chronic stroke appears to be promising. The CPF provides a plausible theoretical framework for the progression of functional task training in neurorehabilitation.

Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR.

PubMed

Nomoto, Mika; Tada, Yasuomi

2018-01-01

Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Barcoded NS31/AML2 primers for sequencing of arbuscular mycorrhizal communities in environmental samples1

PubMed Central

Morgan, Benjamin S. T.; Egerton-Warburton, Louise M.

2017-01-01

Premise of the study: Arbuscular mycorrhizal fungi (AMF) are globally important root symbioses that enhance plant growth and nutrition and influence ecosystem structure and function. To better characterize levels of AMF diversity relevant to ecosystem function, deeper sequencing depth in environmental samples is needed. In this study, Illumina barcoded primers and a bioinformatics pipeline were developed and applied to study AMF diversity and community structure in environmental samples. Methods: Libraries of small subunit ribosomal RNA fragment amplicons were amplified from environmental DNA using a single-step PCR reaction with barcoded NS31/AML2 primers. Amplicons were sequenced on an Illumina MiSeq sequencer using version 2, 2 × 250-bp paired-end chemistry, and analyzed using QIIME and RDP Classifier. Results: Sequencing captured 196 to 6416 operational taxonomic units (OTUs; depending on clustering parameters) representing nine AMF genera. Regardless of clustering parameters, ∼20 OTUs dominated AMF communities (78–87% reads) with the remaining reads distributed among other OTUs. Analyses also showed significant biogeographic differences in AMF communities and that community composition could be linked to specific edaphic factors. Discussion: Barcoded NS31/AML2 primers and Illumina MiSeq sequencing provide a powerful approach to address AMF diversity and variations in fungal assemblages across host plants, ecosystems, and responses to environmental drivers including global change. PMID:28924511

A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma.

PubMed

Catteau, Aurélie; Girardi, Hélène; Monville, Florence; Poggionovo, Cécile; Carpentier, Sabrina; Frayssinet, Véronique; Voss, Jesse; Jenkins, Robert; Boisselier, Blandine; Mokhtari, Karima; Sanson, Marc; Peyro-Saint-Paul, Hélène; Giannini, Caterina

2014-06-02

Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing. The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results. The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

The Rhodium(II) Carbenoid Cyclization-Cycloaddition Cascade of α-Diazo Dihydroindolinones for the Synthesis of Novel Azapolycyclic Ring Systems‡

PubMed Central

England, Dylan B.; Eagan, James M.; Merey, Gokce; Anac, Olcay; Padwa, Albert

2008-01-01

Tandem carbonyl ylide formation-1,3-dipolar cycloaddition of α-diazo N-acetyl-tetrahydro-β-carbolin-1-one derivatives occur efficiently in the presence of a dirhodium catalyst to afford bimolecular cycloadducts in high yield. The Rh(II)-catalyzed reaction also takes place intramolecularly to give products derived from trapping of the carbonyl ylide dipole with a tethered alkene. The power of the intramolecular cascade sequence is that it rapidly assembles a pentacyclic ring system containing three new stereocenters and two adjacent quaternary centers stereospecifically in a single step and in high yield. PMID:18437248

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

French, Jarrod B.; Ealick, Steven E., E-mail: see3@cornell.edu

The crystal structure of 5-hydroxyisourate hydrolase from K. pneumoniae and the steady-state kinetic parameters of the native enzyme as well as several mutants provide insights into the catalytic mechanism of this enzyme and the possible roles of the active-site residues. The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined.more » KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.« less

Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry.

PubMed

Finnigan, William; Thomas, Adam; Cromar, Holly; Gough, Ben; Snajdrova, Radka; Adams, Joseph P; Littlechild, Jennifer A; Harmer, Nicholas J

2017-03-20

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP + , adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

Pro-inflammatory effects of a litchi protein extract in murine RAW264.7 macrophages

PubMed Central

Wang, Xiaoli; Hu, Xiaorong; Yan, Huiqing; Ma, Zhaocheng; Deng, Xiuxin

2016-01-01

It has been observed that the consumption of litchi often causes symptoms characterized by itching or sore throat, gum swelling, oral cavity ulcers and even fever and inflammation, which significantly impair the quality of life of a large population. Using the RAW264.7 cell line, a step-by-step strategy was used to screen for the components in litchi fruits that elicited adverse reactions. The adverse reaction fractions were identified by mass spectrometry and analyzed using the SMART program, and a sequence alignment of the homologous proteins was performed. MTT tests were used to determine the cytotoxicity of a litchi protein extract in RAW264.7 macrophages, and real-time PCR was applied to analyze the expression of inflammatory genes in the RAW264.7 cells treated with lipopolysaccharide or the litchi protein extract. The results showed that the litchi water-soluble protein extract could increase the production of the pro-inflammatory mediators IL-1β, iNOS and COX-2, and the anti-inflammatory mediator HO-1 in the RAW264.7 cell line. The 14-3-3-like proteins GF14 lambda, GF14 omega and GF14 upsilon were likely the candidate proteins that caused the adverse effects. PMID:27195125

One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy

PubMed Central

Casini, Arturo; MacDonald, James T.; Jonghe, Joachim De; Christodoulou, Georgia; Freemont, Paul S.; Baldwin, Geoff S.; Ellis, Tom

2014-01-01

Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications. PMID:24153110

TagDust2: a generic method to extract reads from sequencing data.

PubMed

Lassmann, Timo

2015-01-28

Arguably the most basic step in the analysis of next generation sequencing data (NGS) involves the extraction of mappable reads from the raw reads produced by sequencing instruments. The presence of barcodes, adaptors and artifacts subject to sequencing errors makes this step non-trivial. Here I present TagDust2, a generic approach utilizing a library of hidden Markov models (HMM) to accurately extract reads from a wide array of possible read architectures. TagDust2 extracts more reads of higher quality compared to other approaches. Processing of multiplexed single, paired end and libraries containing unique molecular identifiers is fully supported. Two additional post processing steps are included to exclude known contaminants and filter out low complexity sequences. Finally, TagDust2 can automatically detect the library type of sequenced data from a predefined selection. Taken together TagDust2 is a feature rich, flexible and adaptive solution to go from raw to mappable NGS reads in a single step. The ability to recognize and record the contents of raw reads will help to automate and demystify the initial, and often poorly documented, steps in NGS data analysis pipelines. TagDust2 is freely available at: http://tagdust.sourceforge.net .

Oxidation of L-phenylalanine by diperiodatoargentate(III) in aqueous alkaline medium. A Mechanistic approach

NASA Astrophysics Data System (ADS)

Lamani, S. D.; Veeresh, T. M.; Nandibewoor, S. T.

2009-12-01

The kinetics of oxidation of L-phenylalanine (L-Phe) by diperiodatoargentate(III) (DPA) in alkaline medium at a constant ionic strength of 0.25 mol/dm-3 has been studied spectrophotometrically. The reaction between DPA and L-phenylalanine in alkaline medium exhibits 1: 1 stoichiometry (L-phenylalanine: DPA). The reaction shows first order in [DPA] and has less than unit order dependence each in both [L-Phe] and [Alkali] and retarding effect of [IO{4/-}] under the reaction conditions. The active species of DPA is understood to be as monoperiodatoargentate(III) (MPA). The reaction is shown to proceed via a MPA-L-Phe complex, which decomposes in a rate-determining step to give intermediates followed by a fast step to give the products. The products were identified by spot and spectroscopic studies. The reaction constants involved in the different steps of the mechanisms were calculated. The activation parameters with respect to slow step of the mechanism were computed and discussed. The thermodynamic quantities were also determined for the reaction.

Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

PubMed

Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

2017-09-01

In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

NASA Astrophysics Data System (ADS)

Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

2017-09-01

In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

Starting with Worldviews: A Five-Step Preparatory Approach to Integrative Interdisciplinary Learning

ERIC Educational Resources Information Center

Augsburg, Tanya; Chitewere, Tendai

2013-01-01

In this article we propose a five-step sequenced approach to integrative interdisciplinary learning in undergraduate gateway courses. Drawing from the literature of interdisciplinarity, transformative learning theory, and theories of reflective learning, we utilize a sequence of five steps early in our respective undergraduate gateway courses to…

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects.

PubMed

Kwiecień, Renata A; Molinié, Roland; Paneth, Piotr; Silvestre, Virginie; Lebreton, Jacques; Robins, Richard J

2011-06-01

(15)N heavy isotope effects are especially useful when detail is sought pertaining to the reaction mechanism for the cleavage of a C-N bond. Their potential in assisting to describe the mechanism of N-demethylation of tertiary amines by the action of cytochrome P450 monooxygenase has been investigated. As a working model for the first step, oxidation of the N-methyl group to N-methoxyl, tropine and a cytochrome P450 monooxygenase reaction centre composed of a truncated heme with sulfhydryl as the axial ligand were used. It is apparent that this first step of the reaction proceeds via a hydrogen atom transfer mechanism. Transition states for this step are described for both the high spin ((4)TS(H)) and low spin ((2)TS(H)) pathways in both gas and solvation states. Hence, overall normal secondary (15)N KIE could be calculated for the reaction path modeled in the low spin state, and inverse for the reaction modeled in the high spin state. This partial reaction has been identified as the probable rate limiting step. The model for the second step, fission of the C-N bond, consisted of N-methoxylnortropine and two molecules of water. A transition state described for this step, TS(CN), gives a strongly inverse overall theoretical (15)N KIE. Copyright © 2011 Elsevier Inc. All rights reserved.

STEPS: efficient simulation of stochastic reaction-diffusion models in realistic morphologies.

PubMed

Hepburn, Iain; Chen, Weiliang; Wils, Stefan; De Schutter, Erik

2012-05-10

Models of cellular molecular systems are built from components such as biochemical reactions (including interactions between ligands and membrane-bound proteins), conformational changes and active and passive transport. A discrete, stochastic description of the kinetics is often essential to capture the behavior of the system accurately. Where spatial effects play a prominent role the complex morphology of cells may have to be represented, along with aspects such as chemical localization and diffusion. This high level of detail makes efficiency a particularly important consideration for software that is designed to simulate such systems. We describe STEPS, a stochastic reaction-diffusion simulator developed with an emphasis on simulating biochemical signaling pathways accurately and efficiently. STEPS supports all the above-mentioned features, and well-validated support for SBML allows many existing biochemical models to be imported reliably. Complex boundaries can be represented accurately in externally generated 3D tetrahedral meshes imported by STEPS. The powerful Python interface facilitates model construction and simulation control. STEPS implements the composition and rejection method, a variation of the Gillespie SSA, supporting diffusion between tetrahedral elements within an efficient search and update engine. Additional support for well-mixed conditions and for deterministic model solution is implemented. Solver accuracy is confirmed with an original and extensive validation set consisting of isolated reaction, diffusion and reaction-diffusion systems. Accuracy imposes upper and lower limits on tetrahedron sizes, which are described in detail. By comparing to Smoldyn, we show how the voxel-based approach in STEPS is often faster than particle-based methods, with increasing advantage in larger systems, and by comparing to MesoRD we show the efficiency of the STEPS implementation. STEPS simulates models of cellular reaction-diffusion systems with complex boundaries with high accuracy and high performance in C/C++, controlled by a powerful and user-friendly Python interface. STEPS is free for use and is available at http://steps.sourceforge.net/

Improved annotation through genome-scale metabolic modeling of Aspergillus oryzae

PubMed Central

Vongsangnak, Wanwipa; Olsen, Peter; Hansen, Kim; Krogsgaard, Steen; Nielsen, Jens

2008-01-01

Background Since ancient times the filamentous fungus Aspergillus oryzae has been used in the fermentation industry for the production of fermented sauces and the production of industrial enzymes. Recently, the genome sequence of A. oryzae with 12,074 annotated genes was released but the number of hypothetical proteins accounted for more than 50% of the annotated genes. Considering the industrial importance of this fungus, it is therefore valuable to improve the annotation and further integrate genomic information with biochemical and physiological information available for this microorganism and other related fungi. Here we proposed the gene prediction by construction of an A. oryzae Expressed Sequence Tag (EST) library, sequencing and assembly. We enhanced the function assignment by our developed annotation strategy. The resulting better annotation was used to reconstruct the metabolic network leading to a genome scale metabolic model of A. oryzae. Results Our assembled EST sequences we identified 1,046 newly predicted genes in the A. oryzae genome. Furthermore, it was possible to assign putative protein functions to 398 of the newly predicted genes. Noteworthy, our annotation strategy resulted in assignment of new putative functions to 1,469 hypothetical proteins already present in the A. oryzae genome database. Using the substantially improved annotated genome we reconstructed the metabolic network of A. oryzae. This network contains 729 enzymes, 1,314 enzyme-encoding genes, 1,073 metabolites and 1,846 (1,053 unique) biochemical reactions. The metabolic reactions are compartmentalized into the cytosol, the mitochondria, the peroxisome and the extracellular space. Transport steps between the compartments and the extracellular space represent 281 reactions, of which 161 are unique. The metabolic model was validated and shown to correctly describe the phenotypic behavior of A. oryzae grown on different carbon sources. Conclusion A much enhanced annotation of the A. oryzae genome was performed and a genome-scale metabolic model of A. oryzae was reconstructed. The model accurately predicted the growth and biomass yield on different carbon sources. The model serves as an important resource for gaining further insight into our understanding of A. oryzae physiology. PMID:18500999

A facile synthesis of the basic steroidal skeleton using a Pauson-Khand reaction as a key step.

PubMed

Kim, Do Han; Kim, Kwang; Chung, Young Keun

2006-10-13

A high-yield synthesis of steroid-type molecules under mild reaction conditions is achieved in two steps involving nucleophilic addition of alkynyl cerium reagent to an easily enolizable carbonyl compound (beta-tetralone) followed by an intramolecular Pauson-Khand reaction.

Step-to-step spatiotemporal variables and ground reaction forces of intra-individual fastest sprinting in a single session.

PubMed

Nagahara, Ryu; Mizutani, Mirai; Matsuo, Akifumi; Kanehisa, Hiroaki; Fukunaga, Tetsuo

2018-06-01

We aimed to investigate the step-to-step spatiotemporal variables and ground reaction forces during the acceleration phase for characterising intra-individual fastest sprinting within a single session. Step-to-step spatiotemporal variables and ground reaction forces produced by 15 male athletes were measured over a 50-m distance during repeated (three to five) 60-m sprints using a long force platform system. Differences in measured variables between the fastest and slowest trials were examined at each step until the 22nd step using a magnitude-based inferences approach. There were possibly-most likely higher running speed and step frequency (2nd to 22nd steps) and shorter support time (all steps) in the fastest trial than in the slowest trial. Moreover, for the fastest trial there were likely-very likely greater mean propulsive force during the initial four steps and possibly-very likely larger mean net anterior-posterior force until the 17th step. The current results demonstrate that better sprinting performance within a single session is probably achieved by 1) a high step frequency (except the initial step) with short support time at all steps, 2) exerting a greater mean propulsive force during initial acceleration, and 3) producing a greater mean net anterior-posterior force during initial and middle acceleration.

Development of a rapid and sensitive one-step reverse transcription-nested polymerase chain reaction in a single tube using the droplet-polymerase chain reaction machine.

PubMed

Yamaguchi, Akemi; Matsuda, Kazuyuki; Sueki, Akane; Taira, Chiaki; Uehara, Masayuki; Saito, Yasunori; Honda, Takayuki

2015-08-25

Reverse transcription (RT)-nested polymerase chain reaction (PCR) is a time-consuming procedure because it has several handling steps and is associated with the risk of cross-contamination during each step. Therefore, a rapid and sensitive one-step RT-nested PCR was developed that could be performed in a single tube using a droplet-PCR machine. The K562 BCR-ABL mRNA-positive cell line as well as bone marrow aspirates from 5 patients with chronic myelogenous leukemia (CML) and 5 controls without CML were used. We evaluated one-step RT-nested PCR using the droplet-PCR machine. One-step RT-nested PCR performed in a single tube using the droplet-PCR machine enabled the detection of BCR-ABL mRNA within 40min, which was 10(3)-fold superior to conventional RT nested PCR using three steps in separate tubes. The sensitivity of the one-step RT-nested PCR was 0.001%, with sample reactivity comparable to that of the conventional assay. One-step RT-nested PCR was developed using the droplet-PCR machine, which enabled all reactions to be performed in a single tube accurately and rapidly and with high sensitivity. This one-step RT-nested PCR may be applicable to a wide spectrum of genetic tests in clinical laboratories. Copyright © 2015 Elsevier B.V. All rights reserved.

Introduction to Polymer Chemistry.

ERIC Educational Resources Information Center

Harris, Frank W.

1981-01-01

Reviews the physical and chemical properties of polymers and the two major methods of polymer synthesis: addition (chain, chain-growth, or chain-reaction), and condensation (step-growth or step-reaction) polymerization. (JN)

Kinetics of cisplatin binding to short r(GG) containing miRNA mimics - influence of Na(+)versus K(+), temperature and hydrophobicity on reactivity.

PubMed

Alshiekh, Alak; Clausén, Maria; Elmroth, Sofi K C

2015-07-28

Nucleic acids are well recognized targets for platinum-based anticancer drugs, with RNA and DNA being kinetically comparable. In the case of RNA, previous studies have shown that the reaction between small duplex RNAs (dsRNAs) and monoaquated cisplatin (cis-Pt(NH3)2Cl(OH2)(+), ) can be followed by the metal induced hyperchromicity occurring directly after addition of to e.g. microRNA mimics. In the present study, we have used this approach to compare thermal stability and reactivity between intracellularly- and extracellularly relevant salt concentration (CNa(+) and CK(+)ca. 0.1 M), and also as a function of increased hydrophobicity (10% v/v EtOH). In addition, reactivity was studied as a function of temperature in the interval ca. 5-20 °C below the respective dsRNA melting temperatures (Tms). Four different 13- to 20-mer dsRNAs with two different central sequence motifs were used as targets containing either a central r(GG)·r(CC)- or r(GG)·r(UAU)-sequence. The reactions exhibited half-lives in the minute- to hour range at 38 °C in the presence of excess in the μM range. Further, a linear dependence was found between C and the observed pseudo-first-order rate constants. The resulting apparent second-order rate constants were significantly larger for the lower melting r(GG)·r(UAU)-containing sequences compared with that of the fully complementary ones; the higher and lower reactivities represented by RNA-1-3 and RNA-1-1 with k2,appca. 30 and 8 M(-1) s(-1) respectively at CNa(+) = 122 mM. For all RNAs a common small, but significant, trend was observed with increased reactivity in the presence of K(+) compared with Na(+), and decreased reactivity in the presence of EtOH. Finally, the temperature dependence of k2,app was evaluated using the Eyring equation. The retrieved activation parameters reveal positive values for both ΔH(≠) and ΔS(≠) for all dsRNAs, in the range ca. 23-34 kcal mol(-1) and 22-57 cal K(-1) mol(-1) respectively. These values indicate solvational effects to be important for the rate determining step of the reaction, and thus in support of a structural change of the dsRNA to take place in parallel with the adduct formation step.

The Missing Link in Leguminous Pterocarpan Biosynthesis is a Dirigent Domain-Containing Protein with Isoflavanol Dehydratase Activity

PubMed Central

Uchida, Kai; Akashi, Tomoyoshi

2017-01-01

Pterocarpan forms the basic structure of leguminous phytoalexins, and most of the isoflavonoid pathway genes encoding the enzymes responsible for its biosynthesis have been identified. However, the last step of pterocarpan biosynthesis is a ring closure reaction, and the enzyme that catalyzes this step, 2′-hydroxyisoflavanol 4,2′-dehydratase or pterocarpan synthase (PTS), remains as an unidentified ‘missing link’. This last ring formation is assumed to be the key step in determining the stereochemistry of pterocarpans, which plays a role in their antimicrobial activity. In this study, a cDNA clone encoding PTS from Glycyrrhiza echinata (GePTS1) was identified through functional expression fractionation screening of a cDNA library, which requires no sequence information, and orthologs from soybean (GmPTS1) and Lotus japonicus (LjPTS1) were also identified. These proteins were heterologously expressed in Escherichia coli and biochemically characterized. Surprisingly, the proteins were found to include amino acid motifs characteristic of dirigent proteins, some of which control stereospecific phenoxy radical coupling in lignan biosynthesis. The stereospecificity of substrates and products was examined using four substrate stereoisomers with hydroxy and methoxy derivatives at C-4′. The results showed that the 4R configuration was essential for the PTS reaction, and (−)- and (+)-pterocarpans were produced depending on the stereochemistry at C-3. In suspension-cultured soybean cells, levels of the GmPTS1 transcript increased temporarily prior to the peak in phytoalexin accumulation, strongly supporting the possible involvement of PTS in pterocarpan biosynthesis. PMID:28394400

Design and optimization of a novel reverse transcription linear-after-the-exponential PCR for the detection of foot-and-mouth disease virus.

PubMed

Pierce, K E; Mistry, R; Reid, S M; Bharya, S; Dukes, J P; Hartshorn, C; King, D P; Wangh, L J

2010-07-01

A novel molecular assay for the detection of foot-and-mouth disease virus (FMDV) was developed using linear-after-the-exponential polymerase chain reaction (LATE-PCR). Pilot experiments using synthetic DNA targets demonstrated the ability of LATE-PCR to quantify initial target concentration through endpoint detection. A two-step protocol involving reverse transcription (RT) followed by LATE-PCR was then used to confirm the ability of the assay to detect FMDV RNA. Finally, RT and LATE-PCR were combined in a one-step duplex assay for co-amplification of an FMDV RNA segment and an internal control comprised of an Armored RNA. In that form, each of the excess primers in the reaction mixture hybridize to their respective RNA targets during a short pre-incubation, then generate cDNA strands during a 3-min RT step at 60°C, and the resulting cDNA is amplified by LATE-PCR without intervening sample processing. The RT-LATE-PCR assay generates fluorescent signals at endpoint that are proportional to the starting number of RNA targets and can detect a range of sequence variants using a single mismatch-tolerant probe. In addition to offering improvements over current laboratory-based molecular diagnostic assays for FMDV, this new assay is compatible with a novel portable ('point-of-care') device, the BioSeeq II, designed for the rapid diagnosis of FMD in the field. © 2009 The Authors. Journal compilation © 2009 The Society for Applied Microbiology.

Inverted stereocontrol of iridoid synthase in snapdragon.

PubMed

Kries, Hajo; Kellner, Franziska; Kamileen, Mohamed Omar; O'Connor, Sarah E

2017-09-01

The natural product class of iridoids, found in various species of flowering plants, harbors astonishing chemical complexity. The discovery of iridoid biosynthetic genes in the medicinal plant Catharanthus roseus has provided insight into the biosynthetic origins of this class of natural product. However, not all iridoids share the exact five- to six-bicyclic ring scaffold of the Catharanthus iridoids. For instance, iridoids in the ornamental flower snapdragon ( Antirrhinum majus , Plantaginaceae family) are derived from the C7 epimer of this scaffold. Here we have cloned and characterized the iridoid synthase enzyme from A. majus (AmISY), the enzyme that is responsible for converting 8-oxogeranial into the bicyclic iridoid scaffold in a two-step reduction-cyclization sequence. Chiral analysis of the reaction products reveals that AmISY reduces C7 to generate the opposite stereoconfiguration in comparison with the Catharanthus homologue CrISY. The catalytic activity of AmISY thus explains the biosynthesis of 7-epi-iridoids in Antirrhinum and related genera. However, although the stereoselectivity of the reduction step catalyzed by AmISY is clear, in both AmISY and CrISY, the cyclization step produces a diastereomeric mixture. Although the reduction of 8-oxogeranial is clearly enzymatically catalyzed, the cyclization step appears to be subject to less stringent enzyme control. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inverted stereocontrol of iridoid synthase in snapdragon

PubMed Central

Kries, Hajo; Kellner, Franziska; Kamileen, Mohamed Omar; O'Connor, Sarah E.

2017-01-01

The natural product class of iridoids, found in various species of flowering plants, harbors astonishing chemical complexity. The discovery of iridoid biosynthetic genes in the medicinal plant Catharanthus roseus has provided insight into the biosynthetic origins of this class of natural product. However, not all iridoids share the exact five- to six-bicyclic ring scaffold of the Catharanthus iridoids. For instance, iridoids in the ornamental flower snapdragon (Antirrhinum majus, Plantaginaceae family) are derived from the C7 epimer of this scaffold. Here we have cloned and characterized the iridoid synthase enzyme from A. majus (AmISY), the enzyme that is responsible for converting 8-oxogeranial into the bicyclic iridoid scaffold in a two-step reduction–cyclization sequence. Chiral analysis of the reaction products reveals that AmISY reduces C7 to generate the opposite stereoconfiguration in comparison with the Catharanthus homologue CrISY. The catalytic activity of AmISY thus explains the biosynthesis of 7-epi-iridoids in Antirrhinum and related genera. However, although the stereoselectivity of the reduction step catalyzed by AmISY is clear, in both AmISY and CrISY, the cyclization step produces a diastereomeric mixture. Although the reduction of 8-oxogeranial is clearly enzymatically catalyzed, the cyclization step appears to be subject to less stringent enzyme control. PMID:28701463

Plant tropane alkaloid biosynthesis evolved independently in the Solanaceae and Erythroxylaceae

PubMed Central

Jirschitzka, Jan; Schmidt, Gregor W.; Reichelt, Michael; Schneider, Bernd; Gershenzon, Jonathan; D’Auria, John Charles

2012-01-01

The pharmacologically important tropane alkaloids have a scattered distribution among angiosperm families, like many other groups of secondary metabolites. To determine whether tropane alkaloids have evolved repeatedly in different lineages or arise from an ancestral pathway that has been lost in most lines, we investigated the tropinone-reduction step of their biosynthesis. In species of the Solanaceae, which produce compounds such as atropine and scopolamine, this reaction is known to be catalyzed by enzymes of the short-chain dehydrogenase/reductase family. However, in Erythroxylum coca (Erythroxylaceae), which accumulates cocaine and other tropane alkaloids, no proteins of the short-chain dehydrogenase/reductase family were found that could catalyze this reaction. Instead, purification of E. coca tropinone-reduction activity and cloning of the corresponding gene revealed that a protein of the aldo-keto reductase family carries out this reaction in E. coca. This protein, designated methylecgonone reductase, converts methylecgonone to methylecgonine, the penultimate step in cocaine biosynthesis. The protein has highest sequence similarity to other aldo-keto reductases, such as chalcone reductase, an enzyme of flavonoid biosynthesis, and codeinone reductase, an enzyme of morphine alkaloid biosynthesis. Methylecgonone reductase reduces methylecgonone (2-carbomethoxy-3-tropinone) stereospecifically to 2-carbomethoxy-3β-tropine (methylecgonine), and has its highest activity, protein level, and gene transcript level in young, expanding leaves of E. coca. This enzyme is not found at all in root tissues, which are the site of tropane alkaloid biosynthesis in the Solanaceae. This evidence supports the theory that the ability to produce tropane alkaloids has arisen more than once during the evolution of the angiosperms. PMID:22665766

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

PubMed

Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

2000-05-01

Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

The influence of tightening sequence and method on screw preload in implant superstructures.

PubMed

Al-Sahan, Maha M; Al Maflehi, Nassr S; Akeel, Riyadh F

2014-01-01

This study evaluated the effect of six screw-tightening sequences and two tightening methods on the screw preload in implant-supported superstructures. The preload was measured using strain gauges following the screw tightening of a metal framework connected to four implants. The experiment included six sequences ([1] 1-2-3-4, [2] 4-2-3-1, [3] 4-3-1-2, [4] 1-4-2-3, [5] 2-3-4-1, and [6] 3-2-4-1), two methods (onestep, three-step), and five replications. Significant differences were found between tightening sequences and methods. In the three-step method, a higher total preload was found in sequences 2 (312 ± 85 N), 3 (246 ± 54 N), and 4 (310 ± 96 N). In the one-step method, a higher total preload was found in sequences 1 (286 ± 94 N), 5 (764 ± 142 N), and 6 (350 ± 69 N). It is concluded that the highest total screw preload was achieved when anterior implants of the superstructure were first tightened in one step, followed by posterior implants.

Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

PubMed Central

Jankowsky, E; Schwenzer, B

1996-01-01

Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

Capillary electrophoresis of Big-Dye terminator sequencing reactions for human mtDNA Control Region haplotyping in the identification of human remains.

PubMed

Montesino, Marta; Prieto, Lourdes

2012-01-01

Cycle sequencing reaction with Big-Dye terminators provides the methodology to analyze mtDNA Control Region amplicons by means of capillary electrophoresis. DNA sequencing with ddNTPs or terminators was developed by (1). The progressive automation of the method by combining the use of fluorescent-dye terminators with cycle sequencing has made it possible to increase the sensibility and efficiency of the method and hence has allowed its introduction into the forensic field. PCR-generated mitochondrial DNA products are the templates for sequencing reactions. Different set of primers can be used to generate amplicons with different sizes according to the quality and quantity of the DNA extract providing sequence data for different ranges inside the Control Region.

Suitability and setup of next-generation sequencing-based method for taxonomic characterization of aquatic microbial biofilm.

PubMed

Bakal, Tomas; Janata, Jiri; Sabova, Lenka; Grabic, Roman; Zlabek, Vladimir; Najmanova, Lucie

2018-06-16

A robust and widely applicable method for sampling of aquatic microbial biofilm and further sample processing is presented. The method is based on next-generation sequencing of V4-V5 variable regions of 16S rRNA gene and further statistical analysis of sequencing data, which could be useful not only to investigate taxonomic composition of biofilm bacterial consortia but also to assess aquatic ecosystem health. Five artificial materials commonly used for biofilm growth (glass, stainless steel, aluminum, polypropylene, polyethylene) were tested to determine the one giving most robust and reproducible results. The effect of used sampler material on total microbial composition was not statistically significant; however, the non-plastic materials (glass, metal) gave more stable outputs without irregularities among sample parallels. The bias of the method is assessed with respect to the employment of a non-quantitative step (PCR amplification) to obtain quantitative results (relative abundance of identified taxa). This aspect is often overlooked in ecological and medical studies. We document that sequencing of a mixture of three merged primary PCR reactions for each sample and further evaluation of median values from three technical replicates for each sample enables to overcome this bias and gives robust and repeatable results well distinguishing among sampling localities and seasons.

A fluorescence-based centrifugal microfluidic system for parallel detection of multiple allergens

NASA Astrophysics Data System (ADS)

Chen, Q. L.; Ho, H. P.; Cheung, K. L.; Kong, S. K.; Suen, Y. K.; Kwan, Y. W.; Li, W. J.; Wong, C. K.

2010-02-01

This paper reports a robust polymer based centrifugal microfluidic analysis system that can provide parallel detection of multiple allergens in vitro. Many commercial food products (milk, bean, pollen, etc.) may introduce allergy to people. A low-cost device for rapid detection of allergens is highly desirable. With this as the objective, we have studied the feasibility of using a rotating disk device incorporating centrifugal microfluidics for performing actuationfree and multi-analyte detection of different allergen species with minimum sample usage and fast response time. Degranulation in basophils or mast cells is an indicator to demonstrate allergic reaction. In this connection, we used acridine orange (AO) to demonstrate degranulation in KU812 human basophils. It was found that the AO was released from granules when cells were stimulated by ionomycin, thus signifying the release of histamine which accounts for allergy symptoms [1-2]. Within this rotating optical platform, major microfluidic components including sample reservoirs, reaction chambers, microchannel and flow-control compartments are integrated into a single bio-compatible polydimethylsiloxane (PDMS) substrate. The flow sequence and reaction time can be controlled precisely. Sequentially through varying the spinning speed, the disk may perform a variety of steps on sample loading, reaction and detection. Our work demonstrates the feasibility of using centrifugation as a possible immunoassay system in the future.

Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach.

PubMed

Akberdin, Ilya R; Thompson, Merlin; Hamilton, Richard; Desai, Nalini; Alexander, Danny; Henard, Calvin A; Guarnieri, Michael T; Kalyuzhnaya, Marina G

2018-02-06

Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13 C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20Z R an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization in M. alcaliphilum 20Z R . A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. The computational framework of C 1 -metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.

Polymerase Chain Reaction (PCR)-based methods for detection and identification of mycotoxigenic Penicillium species using conserved genes

USDA-ARS?s Scientific Manuscript database

Polymerase chain reaction amplification of conserved genes and sequence analysis provides a very powerful tool for the identification of toxigenic as well as non-toxigenic Penicillium species. Sequences are obtained by amplification of the gene fragment, sequencing via capillary electrophoresis of d...

Classification of Foreign Body Reactions due to Industrial Silicone Injection.

PubMed

Harlim, Ago; Kanoko, Mpu; Aisah, Siti

2018-05-02

A foreign body reaction (FBR) is a typical tissue response to a biomaterial that has been injected or implanted in human body tissue. There has been a lack of data on the classification of foreign body reaction to silicone injection, which can describe the pattern of body tissue responses to silicone. Determine the foreign body reaction to silicone injection. We modified the classification proposed by Duranti and colleagues, which has categorized a FBR to hyaluronic acid injection into a new classification of an FBR to silicone injection. A cohort study of 31 women suffering from silicone-induced granulomas on their chin was conducted. Granulomatous tissue and submental skin were stained with hematoxylin-eosin and evaluated. Our data revealed that there were at least 7 categories of FBRs to silicone injection that could be developed. Categories 1 to 4 showed inflammatory activity, and categories 5 to 8 showed tissue repair by fibrosis. Using histopathological staining, we are able to sequence the steps of body reactions to silicone injection. Initial inflammatory reaction is then replaced by fibrosis process repairing the damaged tissues. The process depends on the host immune tolerance.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Green Rust Reduction of Chromium Part 2: Comparison of Heterogeneous and Homogeneous Chromate Reduction

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

Wander, Matthew C.; Schoonen, Martin A.

White and green rusts are the active chemical reagents of buried scrap iron pollutant remediation. In this work, a comparison of the initial electron-transfer step for the reduction of CrO{sub 4}{sup -2} by Fe{sub (aq)}{sup 2+} and Fe(OH){sub 2}(s) is presented. Using hybrid density functional theory and Hartree-Fock cluster calculations for the aqueous reaction, the rate constant for the homogeneous reduction of chromium by ferrous iron was determined to be 5 x 10{sup -2} M{sup -1} s{sup -1} for the initial electron transfer. Using a combination of Hartree-Fock slab and cluster calculations for the heterogeneous reaction, the initial electron transfermore » for the heterogeneous reduction of chromium by ferrous iron was determined to be 1 x 10{sup 2} s{sup -1}. The difference in rates is driven by the respective free energies of reaction: 33.4 vs -653.2 kJ/mol. This computational result is apparently the opposite of what has been observed experimentally, but further analysis suggests that these results are fully convergent with experiment. The experimental heterogeneous rate is limited by surface passivation from slow intersheet electron transfer, while the aqueous reaction may be an autocatalytic heterogeneous reaction involving the iron oxyhydroxide product. As a result, it is possible to produce a clear model of the pollutant reduction reaction sequence for these two reactants.« less

Kinect-based choice reaching and stepping reaction time tests for clinical and in-home assessment of fall risk in older people: a prospective study.

PubMed

Ejupi, Andreas; Gschwind, Yves J; Brodie, Matthew; Zagler, Wolfgang L; Lord, Stephen R; Delbaere, Kim

2016-01-01

Quick protective reactions such as reaching or stepping are important to avoid a fall or minimize injuries. We developed Kinect-based choice reaching and stepping reaction time tests (Kinect-based CRTs) and evaluated their ability to differentiate between older fallers and non-fallers and the feasibility of administering them at home. A total of 94 community-dwelling older people were assessed on the Kinect-based CRTs in the laboratory and were followed-up for falls for 6 months. Additionally, a subgroup (n = 20) conducted the Kinect-based CRTs at home. Signal processing algorithms were developed to extract features for reaction, movement and the total time from the Kinect skeleton data. Nineteen participants (20.2 %) reported a fall in the 6 months following the assessment. The reaction time (fallers: 797 ± 136 ms, non-fallers: 714 ± 89 ms), movement time (fallers: 392 ± 50 ms, non-fallers: 358 ± 51 ms) and total time (fallers: 1189 ± 170 ms, non-fallers: 1072 ± 109 ms) of the reaching reaction time test differentiated well between the fallers and non-fallers. The stepping reaction time test did not significantly discriminate between the two groups in the prospective study. The correlations between the laboratory and in-home assessments were 0.689 for the reaching reaction time and 0.860 for stepping reaction time. The study findings indicate that the Kinect-based CRT tests are feasible to administer in clinical and in-home settings, and thus represents an important step towards the development of sensor-based fall risk self-assessments. With further validation, the assessments may prove useful as a fall risk screen and home-based assessment measures for monitoring changes over time and effects of fall prevention interventions.

Effect of a perturbation-based balance training program on compensatory stepping and grasping reactions in older adults: a randomized controlled trial.

PubMed

Mansfield, Avril; Peters, Amy L; Liu, Barbara A; Maki, Brian E

2010-04-01

Compensatory stepping and grasping reactions are prevalent responses to sudden loss of balance and play a critical role in preventing falls. The ability to execute these reactions effectively is impaired in older adults. The purpose of this study was to evaluate a perturbation-based balance training program designed to target specific age-related impairments in compensatory stepping and grasping balance recovery reactions. This was a double-blind randomized controlled trial. The study was conducted at research laboratories in a large urban hospital. Thirty community-dwelling older adults (aged 64-80 years) with a recent history of falls or self-reported instability participated in the study. Participants were randomly assigned to receive either a 6-week perturbation-based (motion platform) balance training program or a 6-week control program involving flexibility and relaxation training. Features of balance reactions targeted by the perturbation-based program were: (1) multi-step reactions, (2) extra lateral steps following anteroposterior perturbations, (3) foot collisions following lateral perturbations, and (4) time to complete grasping reactions. The reactions were evoked during testing by highly unpredictable surface translation and cable pull perturbations, both of which differed from the perturbations used during training. /b> Compared with the control program, the perturbation-based training led to greater reductions in frequency of multi-step reactions and foot collisions that were statistically significant for surface translations but not cable pulls. The perturbation group also showed significantly greater reduction in handrail contact time compared with the control group for cable pulls and a possible trend in this direction for surface translations. Further work is needed to determine whether a maintenance program is needed to retain the training benefits and to assess whether these benefits reduce fall risk in daily life. Perturbation-based training shows promise as an effective intervention to improve the ability of older adults to prevent themselves from falling when they lose their balance.

Asymmetric catalytic cascade reactions for constructing diverse scaffolds and complex molecules.

PubMed

Wang, Yao; Lu, Hong; Xu, Peng-Fei

2015-07-21

With the increasing concerns about chemical pollution and sustainability of resources, among the significant challenges facing synthetic chemists are the development and application of elegant and efficient methods that enable the concise synthesis of natural products, drugs, and related compounds in a step-, atom- and redox-economic manner. One of the most effective ways to reach this goal is to implement reaction cascades that allow multiple bond-forming events to occur in a single vessel. This Account documents our progress on the rational design and strategic application of asymmetric catalytic cascade reactions in constructing diverse scaffolds and synthesizing complex chiral molecules. Our research is aimed at developing robust cascade reactions for the systematic synthesis of a range of interesting molecules that contain structural motifs prevalent in natural products, pharmaceuticals, and biological probes. The strategies employed to achieve this goal can be classified into three categories: bifunctional base/Brønsted acid catalysis, covalent aminocatalysis/N-heterocyclic carbene catalysis, and asymmetric organocatalytic relay cascades. By the use of rationally designed substrates with properly reactive sites, chiral oxindole, chroman, tetrahydroquinoline, tetrahydrothiophene, and cyclohexane scaffolds were successfully assembled under bifunctional base/Brønsted acid catalysis from simple and readily available substances such as imines and nitroolefins. We found that some of these reactions are highly efficient since catalyst loadings as low as 1 mol % can promote the multistep sequences affording complex architectures with high stereoselectivities and yields. Furthermore, one of the bifunctional base/Brønsted acid-catalyzed cascade reactions for the synthesis of chiral cyclohexanes has been used as a key step in the construction of the tetracyclic core of lycorine-type alkaloids and the formal synthesis of α-lycorane. Guided by the principles of covalent aminocatalysis and N-heterocyclic carbene catalysis, we synthesized chiral piperidine, indole, and cyclobutane derivatives. The synthesis of chiral cyclobutanes and pyrroloindolones showed unprecedented reactivity of substrates and catalysts. The development of the strategy of asymmetric organocatalytic relay cascades has provided a useful tool for the controlled synthesis of specific diastereomers in complex molecules. This Account gives a panoramic view and the logic of our research on the design, development, and applications of asymmetric catalytic cascade reactions that will potentially provide useful insights into exploring new reactions.

TG study of the Li0.4Fe2.4Zn0.2O4 ferrite synthesis

NASA Astrophysics Data System (ADS)

Lysenko, E. N.; Nikolaev, E. V.; Surzhikov, A. P.

2016-02-01

In this paper, the kinetic analysis of Li-Zn ferrite synthesis was studied using thermogravimetry (TG) method through the simultaneous application of non-linear regression to several measurements run at different heating rates (multivariate non-linear regression). Using TG-curves obtained for the four heating rates and Netzsch Thermokinetics software package, the kinetic models with minimal adjustable parameters were selected to quantitatively describe the reaction of Li-Zn ferrite synthesis. It was shown that the experimental TG-curves clearly suggest a two-step process for the ferrite synthesis and therefore a model-fitting kinetic analysis based on multivariate non-linear regressions was conducted. The complex reaction was described by a two-step reaction scheme consisting of sequential reaction steps. It is established that the best results were obtained using the Yander three-dimensional diffusion model at the first stage and Ginstling-Bronstein model at the second step. The kinetic parameters for lithium-zinc ferrite synthesis reaction were found and discussed.

Programmed release triggered by osmotic gradients in multicomponent vesicles

NASA Astrophysics Data System (ADS)

Dong, Ruo-Yu; Jang, Hyun-Sook; Granick, Steve

Polymersomes, a good candidate for encapsulation and delivery of active ingredients, can be constructed with inter-connected multiple compartments. These so-called multisomes on the one hand enable the spatial separation of various incompatible contents or processes, and on the other hand provide an efficient route for inter-compartment communication via the interface semipermeable membrane. Here we show that by establishing osmotic imbalances between different compartments, interesting synergetic morphology changes of the multisomes can be observed. And by further carefully adjusting the osmotic gradients and the arrangement of compartments, we can realize a cascade rupture of these individual units, which may be a new step towards controlled mixing and timed sequences of chemical reactions.

Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

Rapid Total Synthesis of DARPin pE59 and RNase B. a

PubMed Central

Mong, Surin K.; Vinogradov, Alexander A.; Simon, Mark D.

2014-01-01

Here we report the convergent total synthesis of two proteins: DARPin pE59 and RNase B. a. (Barnase). Leveraging our recently developed fast flow peptide synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides and were able to mitigate common side reactions including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues. PMID:24616257

In vitro V(D)J recombination: signal joint formation.

PubMed

Cortes, P; Weis-Garcia, F; Misulovin, Z; Nussenzweig, A; Lai, J S; Li, G; Nussenzweig, M C; Baltimore, D

1996-11-26

The first step of V(D)J recombination, specific cleavage at the recombination signal sequence (RSS), can be carried out by the recombination activating proteins RAG1 and RAG2. In vivo, the cleaved coding and signal ends must be rejoined to generate functional antigen receptors and maintain chromosomal integrity. We have investigated signal joint formation using deletion and inversion substrates in a cell free system. RAG1 and RAG2 alone or in combination were unable to generate signal joints. However, RAG1 and RAG2 complemented with nuclear extracts were able to recombine an extrachromosomal substrate and form precise signal joints. The in vitro reaction resembled authentic V(D)J recombination in being Ku-antigen-dependent.

More Genetic Engineering With Cloned Hemoglobin Genes

NASA Technical Reports Server (NTRS)

Bailey, James E.

1992-01-01

Cells modified to enhance growth and production of proteins. Method for enhancing both growth of micro-organisms in vitro and production of various proteins or metalbolites in these micro-organisms provides for incorporation of selected chromosomal or extrachormosomal deoxyribonucleic acid (DNA) sequences into micro-organisms from other cells or from artificial sources. Incorporated DNA includes parts encoding desired product(s) or characteristic(s) of cells and parts that control expression of productor characteristic-encoding parts in response to variations in environment. Extended method enables increased research into growth of organisms in oxygen-poor environments. Industrial applications found in enhancement of processing steps requiring oxygen in fermentation, enzymatic degradation, treatment of wastes containing toxic chemicals, brewing, and some oxidative chemical reactions.

Tin-containing zeolites are highly active catalysts for the isomerization of glucose in water.

PubMed

Moliner, Manuel; Román-Leshkov, Yuriy; Davis, Mark E

2010-04-06

The isomerization of glucose into fructose is a large-scale reaction for the production of high-fructose corn syrup (HFCS; reaction performed by enzyme catalysts) and recently is being considered as an intermediate step in the possible route of biomass to fuels and chemicals. Here, it is shown that a large-pore zeolite that contains tin (Sn-Beta) is able to isomerize glucose to fructose in aqueous media with high activity and selectivity. Specifically, a 10% (wt/wt) glucose solution containing a catalytic amount of Sn-Beta (150 Sn:glucose molar ratio) gives product yields of approximately 46% (wt/wt) glucose, 31% (wt/wt) fructose, and 9% (wt/wt) mannose after 30 min and 12 min of reaction at 383 K and 413 K, respectively. This reactivity is achieved also when a 45 wt% glucose solution is used. The properties of the large-pore zeolite greatly influence the reaction behavior because the reaction does not proceed with a medium-pore zeolite, and the isomerization activity is considerably lower when the metal centers are incorporated in ordered mesoporous silica (MCM-41). The Sn-Beta catalyst can be used for multiple cycles, and the reaction stops when the solid is removed, clearly indicating that the catalysis is occurring heterogeneously. Most importantly, the Sn-Beta catalyst is able to perform the isomerization reaction in highly acidic, aqueous environments with equivalent activity and product distribution as in media without added acid. This enables Sn-Beta to couple isomerizations with other acid-catalyzed reactions, including hydrolysis/isomerization or isomerization/dehydration reaction sequences [starch to fructose and glucose to 5-hydroxymethylfurfural (HMF) demonstrated here].

Tin-containing zeolites are highly active catalysts for the isomerization of glucose in water

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

Moliner, Manuel; Roman-Leshkov, Yuriy; Davis, Mark E.

The isomerization of glucose into fructose is a large-scale reaction for the production of high-fructose corn syrup (HFCS; reaction performed by enzyme catalysts) and recently is being considered as an intermediate step in the possible route of biomass to fuels and chemicals. Here, it is shown that a large-pore zeolite that contains tin (Sn-Beta) is able to isomerize glucose to fructose in aqueous media with high activity and selectivity. Specifically, a 10% (wt/wt) glucose solution containing a catalytic amount of Sn-Beta (1:50 Sn:glucose molar ratio) gives product yields of approximately 46% (wt/wt) glucose, 31% (wt/wt) fructose, and 9% (wt/wt) mannosemore » after 30 min and 12 min of reaction at 383 K and 413 K, respectively. This reactivity is achieved also when a 45 wt% glucose solution is used. The properties of the large-pore zeolite greatly influence the reaction behavior because the reaction does not proceed with a medium-pore zeolite, and the isomerization activity is considerably lower when the metal centers are incorporated in ordered mesoporous silica (MCM-41). The Sn-Beta catalyst can be used for multiple cycles, and the reaction stops when the solid is removed, clearly indicating that the catalysis is occurring heterogeneously. Most importantly, the Sn-Beta catalyst is able to perform the isomerization reaction in highly acidic, aqueous environments with equivalent activity and product distribution as in media without added acid. This enables Sn-Beta to couple isomerizations with other acid-catalyzed reactions, including hydrolysis/isomerization or isomerization/dehydration reaction sequences [starch to fructose and glucose to 5-hydroxymethylfurfural (HMF) demonstrated here].« less

Calculation of biochemical net reactions and pathways by using matrix operations.

PubMed Central

Alberty, R A

1996-01-01

Pathways for net biochemical reactions can be calculated by using a computer program that solves systems of linear equations. The coefficients in the linear equations are the stoichiometric numbers in the biochemical equations for the system. The solution of the system of linear equations is a vector of the stoichiometric numbers of the reactions in the pathway for the net reaction; this is referred to as the pathway vector. The pathway vector gives the number of times the various reactions have to occur to produce the desired net reaction. Net reactions may involve unknown numbers of ATP, ADP, and Pi molecules. The numbers of ATP, ADP, and Pi in a desired net reaction can be calculated in a two-step process. In the first step, the pathway is calculated by solving the system of linear equations for an abbreviated stoichiometric number matrix without ATP, ADP, Pi, NADred, and NADox. In the second step, the stoichiometric numbers in the desired net reaction, which includes ATP, ADP, Pi, NADred, and NADox, are obtained by multiplying the full stoichiometric number matrix by the calculated pathway vector. PMID:8804633

Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution† †Electronic supplementary information (ESI) available: Complementary resonance Raman and infrared spectroscopic data. See DOI: 10.1039/c6sc01098a Click here for additional data file.

PubMed Central

Katz, S.; Noth, J.; Shafaat, H. S.; Happe, T.; Hildebrandt, P.

2016-01-01

[FeFe] hydrogenases are biocatalytic model systems for the exploitation and investigation of catalytic hydrogen evolution. Here, we used vibrational spectroscopic techniques to characterize, in detail, redox transformations of the [FeFe] and [4Fe4S] sub-sites of the catalytic centre (H-cluster) in a monomeric [FeFe] hydrogenase. Through the application of low-temperature resonance Raman spectroscopy, we discovered a novel metastable intermediate that is characterized by an oxidized [FeIFeII] centre and a reduced [4Fe4S]1+ cluster. Based on this unusual configuration, this species is assigned to the first, deprotonated H-cluster intermediate of the [FeFe] hydrogenase catalytic cycle. Providing insights into the sequence of initial reaction steps, the identification of this species represents a key finding towards the mechanistic understanding of biological hydrogen evolution. PMID:28451119

Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus.

PubMed

Sun, Zhihao; Qin, Tao; Meng, Feifei; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

2017-10-18

Nine influenza virus neuraminidase (NA) subtypes have been identified in poultry and wild birds. Few methods are available for rapid and simple NA subtyping. Here we developed a multiplex probe combination-based one-step real-time reverse transcriptase PCR (rRT-PCR) to detect nine avian influenza virus NA subtypes. Nine primer-probe pairs were assigned to three groups based on the different fluorescent dyes of the probes (FAM, HEX, or Texas Red). Each probe detected only one NA subtype, without cross reactivity. The detection limit was less than 100 EID 50 or 100 copies of cDNA per reaction. Data obtained using this method with allantoic fluid samples isolated from live bird markets and H9N2-infected chickens correlated well with data obtained using virus isolation and sequencing, but was more sensitive. This new method provides a specific and sensitive alternative to conventional NA-subtyping methods.

Automatic procedures for the synthesis of difficult peptides using oxyma as activating reagent: A comparative study on the use of bases and on different deprotection and agitation conditions.

PubMed

Caporale, A; Doti, N; Monti, A; Sandomenico, A; Ruvo, M

2018-04-01

Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as "difficult" poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well-known "difficult peptides" prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N-methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of "difficult peptides". Copyright © 2018 Elsevier Inc. All rights reserved.

Kinetics and mechanisms of iron sulfide reductions in hydrogen and in carbon monoxide

USGS Publications Warehouse

Wiltowski, T.; Hinckley, C.C.; Smith, Gerard V.; Nishizawa, T.; Saporoschenko, Mykola; Shiley, R.H.; Webster, J.R.

1987-01-01

The reduction of iron sulfides by hydrogen and by carbon monoxide has been studied using plug flow and thermogravimetric methods. The reactions were studied in the 523-723??K temperature range and were found to be first-order processes. Plug flow studies were used to correlate reaction rates between pyrite and the gases as a function of the surface area of the pyrite. The rate of H2S formation increases with the surface area of the pyrite sample. The results of thermogravimetric experiments indicate that the reactions consist of several steps. Rate constants for the pyrite reduction by H2 and by CO were obtained. The activation energies increased with degree of reduction. Values of Ea were 113.2 (step I) and 122.5 kJ/mole (step II) for pyrite reduction with CO and 99.4 (step I), 122.4 (step II), 125.2 (step III), and 142.6 kJ/mole (step IV) for pyrite reduction with hydrogen. ?? 1987.

Kinetically governed polymorphism of d(G₄T₄G₃) quadruplexes in K+ solutions.

PubMed

Prislan, Iztok; Lah, Jurij; Milanic, Matija; Vesnaver, Gorazd

2011-03-01

It has been generally recognized that understanding the molecular basis of some important cellular processes is hampered by the lack of knowledge of forces that drive spontaneous formation/disruption of G-quadruplex structures in guanine-rich DNA sequences. According to numerous biophysical and structural studies G-quadruplexes may occur in the presence of K(+) and Na(+) ions as polymorphic structures formed in kinetically governed processes. The reported kinetic models suggested to describe this polymorphism should be considered inappropriate since, as a rule, they include bimolecular single-step associations characterized by negative activation energies. In contrast, our approach in studying polymorphic behavior of G-quadruplexes is based on model mechanisms that involve only elementary folding/unfolding transitions and structural conversion steps that are characterized by positive activation energies. Here, we are investigating a complex polymorphism of d(G(4)T(4)G(3)) quadruplexes in K(+) solutions. On the basis of DSC, circular dichroism and UV spectroscopy and polyacrylamide gel electrophoresis experiments we propose a kinetic model that successfully describes the observed thermally induced conformational transitions of d(G(4)T(4)G(3)) quadruplexes in terms of single-step reactions that involve besides single strands also one tetramolecular and three bimolecular quadruplex structures.

Use of ion conductors in the pyrochemical reduction of oxides

DOEpatents

Miller, William E.; Tomczuk, Zygmunt

1994-01-01

An electrochemical process and electrochemical cell for reducing a metal oxide are provided. First the oxide is separated as oxygen gas using, for example, a ZrO.sub.2 oxygen ion conductor anode and the metal ions from the reduction salt are reduced and deposited on an ion conductor cathode, for example, sodium ion reduced on a .beta.-alumina sodium ion conductor cathode. The generation of and separation of oxygen gas avoids the problem with chemical back reaction of oxygen with active metals in the cell. The method also is characterized by a sequence of two steps where an inert cathode electrode is inserted into the electrochemical cell in the second step and the metallic component in the ion conductor is then used as the anode to cause electrochemical reduction of the metal ions formed in the first step from the metal oxide where oxygen gas formed at the anode. The use of ion conductors serves to isolate the active components from chemically reacting with certain chemicals in the cell. While applicable to a variety of metal oxides, the invention has special importance for reducing CaO to Ca.degree. used for reducing UO.sub.2 and PuO.sub.2 to U and Pu.

Analyzing the dependence of oxygen incorporation current density on overpotential and oxygen partial pressure in mixed conducting oxide electrodes.

PubMed

Guan, Zixuan; Chen, Di; Chueh, William C

2017-08-30

The oxygen incorporation reaction, which involves the transformation of an oxygen gas molecule to two lattice oxygen ions in a mixed ionic and electronic conducting solid, is a ubiquitous and fundamental reaction in solid-state electrochemistry. To understand the reaction pathway and to identify the rate-determining step, near-equilibrium measurements have been employed to quantify the exchange coefficients as a function of oxygen partial pressure and temperature. However, because the exchange coefficient contains contributions from both forward and reverse reaction rate constants and depends on both oxygen partial pressure and oxygen fugacity in the solid, unique and definitive mechanistic assessment has been challenging. In this work, we derive a current density equation as a function of both oxygen partial pressure and overpotential, and consider both near and far from equilibrium limits. Rather than considering specific reaction pathways, we generalize the multi-step oxygen incorporation reaction into the rate-determining step, preceding and following quasi-equilibrium steps, and consider the number of oxygen ions and electrons involved in each. By evaluating the dependence of current density on oxygen partial pressure and overpotential separately, one obtains the reaction orders for oxygen gas molecules and for solid-state species in the electrode. We simulated the oxygen incorporation current density-overpotential curves for praseodymium-doped ceria for various candidate rate-determining steps. This work highlights a promising method for studying the exchange kinetics far away from equilibrium.

The Photosynthetic Cycle

DOE R&D Accomplishments Database

Calvin, Melvin

1955-03-21

A cyclic sequence of transformations, including the carboxylation of RuDP (ribulose diphosphate) and its re-formation, has been deduced as the route for the creation of reduced carbon compounds in photosynthetic organisms. With the demonstration of RuDP as substrate for the carboxylation in a cell-free system, each of the reactions has now been carried out independently in vitro. Further purification of this last enzyme system has confirmed the deduction that the carboxylation of RuDP leads directly to the two molecules of PGA (phosphoglyceric acid) involving an internal dismutation and suggesting the name "carboxydismutase" for the enzyme. As a consequence of this knowledge of each of the steps in the photosynthetic CO{sub 2} reduction cycle, it is possible to define the reagent requirements to maintain it. The net requirement for the reduction of one molecule of CO{sub 2} is four equivalents of [H]and three molecules of ATP (adenine triphosphate). These must ultimately be supplied by the photochemical reaction. Some possible ways in which this may be accomplished are discussed.

Trypanosoma cruzi: sequence of phagocytosis and cytotoxicity by human polymorphonuclear leucocytes.

PubMed Central

Rimoldi, M T; Cardoni, R L; Olabuenaga, S E; de Bracco, M M

1981-01-01

We have studied the relationship between phagocytosis and cytotoxicity of human polymorphonuclear leucocytes (PMN) to sensitized Trypanosoma cruzi. Assays were done simultaneously using [3H]-uridine labelled epimastigotes as target cells. Phagocytosis was evaluated by the uptake and cytotoxicity by the release of parasite associated [3H]-uridine. Both reactions reached maximum levels at the same effector- to target-cell ratio and antibody concentration. Uptake of epimastigotes by PMN was highest at 30 min and intracellular disruption and release of parasite debris took place later. In conditions that precluded repeated uptake of sensitized radiolabelled T. cruzi, the release profile of [3H]-uridine from PMN that contained intracellular parasites was similar to that of the standard cytotoxic assay. However, as the ingestion phase was separated from the release step, no lag in the onset of the reaction was observed. Although we cannot rule out extracellular killing, the results of this study demonstrate that the bulk of damaged T. cruzi epimastigotes had been previously internalized by the PMN. PMID:7016743

Re-examination of the Neisser—Wechsberg (antibody prozone) phenomenon

PubMed Central

Muschel, L. H.; Gustafson, Linda; Larsen, Linda J.

1969-01-01

Although the Neisser—Wechsberg phenomenon resembles the inhibition of agglutination systems by excess antibody, the experimental results have indicated that with heat-inactivated antiserum the phenomenon results both from an excess of specific antibody and of non-specific anti-complementary activity. Complement (C) has been shown to be fixed in the presence of the excess antiserum which inhibits the bactericidal reaction. The inhibition was overcome by an excess of the third complement component factors indicating that the excess of antiserum interfered with the activation or function of the components acting at one of the late steps in the reaction sequence. The prozone phenomenon was relatively slight when unheated antiserum was used or when sensitized organisms were washed to remove serum substances unrelated to antibody. Non-specific anti-complementary activity, therefore, is a major contributor to the prozone phenomenon. Both IgM and IgG fractions of rabbit antisera elicited a prozone although the former had relatively greater bactericidal than inhibitory activity. PMID:5352361

Simplified jet fuel reaction mechanism for lean burn combustion application

NASA Technical Reports Server (NTRS)

Lee, Chi-Ming; Kundu, Krishna; Ghorashi, Bahman

1993-01-01

Successful modeling of combustion and emissions in gas turbine engine combustors requires an adequate description of the reaction mechanism. Detailed mechanisms contain a large number of chemical species participating simultaneously in many elementary kinetic steps. Current computational fluid dynamic models must include fuel vaporization, fuel-air mixing, chemical reactions, and complicated boundary geometries. A five-step Jet-A fuel mechanism which involves pyrolysis and subsequent oxidation of paraffin and aromatic compounds is presented. This mechanism is verified by comparing with Jet-A fuel ignition delay time experimental data, and species concentrations obtained from flametube experiments. This five-step mechanism appears to be better than the current one- and two-step mechanisms.

Demonstration of Nondeclarative Sequence Learning in Mice: Development of an Animal Analog of the Human Serial Reaction Time Task

ERIC Educational Resources Information Center

Christie, Michael A.; Hersch, Steven M.

2004-01-01

In this paper, we demonstrate nondeclarative sequence learning in mice using an animal analog of the human serial reaction time task (SRT) that uses a within-group comparison of behavior in response to a repeating sequence versus a random sequence. Ten female B6CBA mice performed eleven 96-trial sessions containing 24 repetitions of a 4-trial…

Efficient reactive Brownian dynamics

DOE PAGES

Donev, Aleksandar; Yang, Chiao-Yu; Kim, Changho

2018-01-21

We develop a Split Reactive Brownian Dynamics (SRBD) algorithm for particle simulations of reaction-diffusion systems based on the Doi or volume reactivity model, in which pairs of particles react with a specified Poisson rate if they are closer than a chosen reactive distance. In our Doi model, we ensure that the microscopic reaction rules for various association and dissociation reactions are consistent with detailed balance (time reversibility) at thermodynamic equilibrium. The SRBD algorithm uses Strang splitting in time to separate reaction and diffusion and solves both the diffusion-only and reaction-only subproblems exactly, even at high packing densities. To efficiently processmore » reactions without uncontrolled approximations, SRBD employs an event-driven algorithm that processes reactions in a time-ordered sequence over the duration of the time step. A grid of cells with size larger than all of the reactive distances is used to schedule and process the reactions, but unlike traditional grid-based methods such as reaction-diffusion master equation algorithms, the results of SRBD are statistically independent of the size of the grid used to accelerate the processing of reactions. We use the SRBD algorithm to compute the effective macroscopic reaction rate for both reaction-limited and diffusion-limited irreversible association in three dimensions and compare to existing theoretical predictions at low and moderate densities. We also study long-time tails in the time correlation functions for reversible association at thermodynamic equilibrium and compare to recent theoretical predictions. Finally, we compare different particle and continuum methods on a model exhibiting a Turing-like instability and pattern formation. Our studies reinforce the common finding that microscopic mechanisms and correlations matter for diffusion-limited systems, making continuum and even mesoscopic modeling of such systems difficult or impossible. We also find that for models in which particles diffuse off lattice, such as the Doi model, reactions lead to a spurious enhancement of the effective diffusion coefficients.« less

Efficient reactive Brownian dynamics

NASA Astrophysics Data System (ADS)

Donev, Aleksandar; Yang, Chiao-Yu; Kim, Changho

2018-01-01

We develop a Split Reactive Brownian Dynamics (SRBD) algorithm for particle simulations of reaction-diffusion systems based on the Doi or volume reactivity model, in which pairs of particles react with a specified Poisson rate if they are closer than a chosen reactive distance. In our Doi model, we ensure that the microscopic reaction rules for various association and dissociation reactions are consistent with detailed balance (time reversibility) at thermodynamic equilibrium. The SRBD algorithm uses Strang splitting in time to separate reaction and diffusion and solves both the diffusion-only and reaction-only subproblems exactly, even at high packing densities. To efficiently process reactions without uncontrolled approximations, SRBD employs an event-driven algorithm that processes reactions in a time-ordered sequence over the duration of the time step. A grid of cells with size larger than all of the reactive distances is used to schedule and process the reactions, but unlike traditional grid-based methods such as reaction-diffusion master equation algorithms, the results of SRBD are statistically independent of the size of the grid used to accelerate the processing of reactions. We use the SRBD algorithm to compute the effective macroscopic reaction rate for both reaction-limited and diffusion-limited irreversible association in three dimensions and compare to existing theoretical predictions at low and moderate densities. We also study long-time tails in the time correlation functions for reversible association at thermodynamic equilibrium and compare to recent theoretical predictions. Finally, we compare different particle and continuum methods on a model exhibiting a Turing-like instability and pattern formation. Our studies reinforce the common finding that microscopic mechanisms and correlations matter for diffusion-limited systems, making continuum and even mesoscopic modeling of such systems difficult or impossible. We also find that for models in which particles diffuse off lattice, such as the Doi model, reactions lead to a spurious enhancement of the effective diffusion coefficients.

Efficient reactive Brownian dynamics

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

Donev, Aleksandar; Yang, Chiao-Yu; Kim, Changho

We develop a Split Reactive Brownian Dynamics (SRBD) algorithm for particle simulations of reaction-diffusion systems based on the Doi or volume reactivity model, in which pairs of particles react with a specified Poisson rate if they are closer than a chosen reactive distance. In our Doi model, we ensure that the microscopic reaction rules for various association and dissociation reactions are consistent with detailed balance (time reversibility) at thermodynamic equilibrium. The SRBD algorithm uses Strang splitting in time to separate reaction and diffusion and solves both the diffusion-only and reaction-only subproblems exactly, even at high packing densities. To efficiently processmore » reactions without uncontrolled approximations, SRBD employs an event-driven algorithm that processes reactions in a time-ordered sequence over the duration of the time step. A grid of cells with size larger than all of the reactive distances is used to schedule and process the reactions, but unlike traditional grid-based methods such as reaction-diffusion master equation algorithms, the results of SRBD are statistically independent of the size of the grid used to accelerate the processing of reactions. We use the SRBD algorithm to compute the effective macroscopic reaction rate for both reaction-limited and diffusion-limited irreversible association in three dimensions and compare to existing theoretical predictions at low and moderate densities. We also study long-time tails in the time correlation functions for reversible association at thermodynamic equilibrium and compare to recent theoretical predictions. Finally, we compare different particle and continuum methods on a model exhibiting a Turing-like instability and pattern formation. Our studies reinforce the common finding that microscopic mechanisms and correlations matter for diffusion-limited systems, making continuum and even mesoscopic modeling of such systems difficult or impossible. We also find that for models in which particles diffuse off lattice, such as the Doi model, reactions lead to a spurious enhancement of the effective diffusion coefficients.« less

Sequence tagging reveals unexpected modifications in toxicoproteomics

PubMed Central

Dasari, Surendra; Chambers, Matthew C.; Codreanu, Simona G.; Liebler, Daniel C.; Collins, Ben C.; Pennington, Stephen R.; Gallagher, William M.; Tabb, David L.

2010-01-01

Toxicoproteomic samples are rich in posttranslational modifications (PTMs) of proteins. Identifying these modifications via standard database searching can incur significant performance penalties. Here we describe the latest developments in TagRecon, an algorithm that leverages inferred sequence tags to identify modified peptides in toxicoproteomic data sets. TagRecon identifies known modifications more effectively than the MyriMatch database search engine. TagRecon outperformed state of the art software in recognizing unanticipated modifications from LTQ, Orbitrap, and QTOF data sets. We developed user-friendly software for detecting persistent mass shifts from samples. We follow a three-step strategy for detecting unanticipated PTMs in samples. First, we identify the proteins present in the sample with a standard database search. Next, identified proteins are interrogated for unexpected PTMs with a sequence tag-based search. Finally, additional evidence is gathered for the detected mass shifts with a refinement search. Application of this technology on toxicoproteomic data sets revealed unintended cross-reactions between proteins and sample processing reagents. Twenty five proteins in rat liver showed signs of oxidative stress when exposed to potentially toxic drugs. These results demonstrate the value of mining toxicoproteomic data sets for modifications. PMID:21214251

Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method.

PubMed

Honda, Shozo; Morichika, Keisuke; Kirino, Yohei

2016-03-01

RNA digestions catalyzed by many ribonucleases generate RNA fragments that contain a 2',3'-cyclic phosphate (cP) at their 3' termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named cP-RNA-seq that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3' termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment; hence, subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ∼6 d, excluding the time required for sequencing and bioinformatics analyses, which are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ∼3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5'-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes.

Decoding DNA labels by melting curve analysis using real-time PCR.

PubMed

Balog, József A; Fehér, Liliána Z; Puskás, László G

2017-12-01

Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Code amplification and decoding can be done in two steps using quantitative PCR (qPCR). To obtain a DNA barcode with high complexity, we defined 8 template groups, each having 4 different DNA templates, yielding 158 (>2.5 billion) combinations of different individual melting temperature (Tm) values and corresponding ID codes. The reproducibility and specificity of the decoding was confirmed by using the most complex template mixture, which had 32 different products in 8 groups with different Tm values. The industrial applicability of our protocol was also demonstrated by labeling a drone with an oil-based paint containing a predefined DNA code, which was then successfully decoded. The method presented here consists of a simple code system based on a small number of synthetic DNA sequences and a cost-effective, rapid decoding protocol using a few qPCR reactions, enabling a wide range of authentication applications.

Interaction of tetraethoxysilane with OH-terminated SiO2 (0 0 1) surface: A first principles study

NASA Astrophysics Data System (ADS)

Deng, Xiaodi; Song, Yixu; Li, Jinchun; Pu, Yikang

2014-06-01

First principles calculates have been performed to investigate the surface reaction mechanism of tetraethoxysilane (TEOS) with fully hydroxylated SiO2(0 0 1) substrate. In semiconductor industry, this is the key step to understand and control the SiO2 film growth in chemical vapor deposition (CVD) and atomic layer deposition (ALD) processes. During the calculation, we proposed a model which breaks the surface dissociative chemisorption into two steps and we calculated the activation barriers and thermochemical energies for each step. Our calculation result for step one shows that the first half reaction is thermodynamically favorable. For the second half reaction, we systematically studied the two potential reaction pathways. The comparing result indicates that the pathway which is more energetically favorable will lead to formation of crystalline SiO2 films while the other will lead to formation of disordered SiO2 films.

One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses.

PubMed

Park, Sun-Whan; Lee, Ye-Ji; Lee, Won-Ja; Jee, Youngmee; Choi, WooYoung

2016-06-01

Ebola and Marburg viruses (EBOVs and MARVs, respectively) are causative agents of severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. In 2014, there was a major Ebola outbreak in various countries in West Africa, including Guinea, Liberia, Republic of Sierra Leone, and Nigeria. EBOV and MARV are clinically difficult to diagnose and distinguish from other African epidemic diseases. Therefore, in this study, we aimed to develop a method for rapid identification of the virus to prevent the spread of infection. We established a conventional one-step reverse transcription-polymerase chain reaction (RT-PCR) assay for these pathogens based on the Superscript Reverse Transcriptase-Platinum Taq polymerase enzyme mixture. All assays were thoroughly optimized using in vitro-transcribed RNA. We designed seven primer sets of nucleocapsid protein (NP) genes based on sequences from seven filoviruses, including five EBOVs and two MARVs. To evaluate the sensitivity of the RT-PCR assay for each filovirus, 10-fold serial dilutions of synthetic viral RNA transcripts of EBOV or MARV NP genes were used to assess detection limits of viral RNA copies. The potential for these primers to cross react with other filoviruses was also examined. The results showed that the primers were specific for individual genotype detection in the examined filoviruses. The assay established in this study may facilitate rapid, reliable laboratory diagnosis in suspected cases of Ebola and Marburg hemorrhagic fevers.

Evolution of atomic structure during nanoparticle formation

DOE PAGES

Tyrsted, Christoffer; Lock, Nina; Jensen, Kirsten M. Ø.; ...

2014-04-14

Understanding the mechanism of nanoparticle formation during synthesis is a key prerequisite for the rational design and engineering of desirable materials properties, yet remains elusive due to the difficulty of studying structures at the nanoscale under real conditions. Here, the first comprehensive structural description of the formation of a nanoparticle, yttria-stabilized zirconia (YSZ), all the way from its ionic constituents in solution to the final crystal, is presented. The transformation is a complicated multi-step sequence of atomic reorganizations as the material follows the reaction pathway towards the equilibrium product. Prior to nanoparticle nucleation, reagents reorganize into polymeric species whose structuremore » is incompatible with the final product. Instead of direct nucleation of clusters into the final product lattice, a highly disordered intermediate precipitate forms with a local bonding environment similar to the product yet lacking the correct topology. During maturation, bond reforming occurs by nucleation and growth of distinct domains within the amorphous intermediary. The present study moves beyond kinetic modeling by providing detailed real-time structural insight, and it is demonstrated that YSZ nanoparticle formation and growth is a more complex chemical process than accounted for in conventional models. This level of mechanistic understanding of the nanoparticle formation is the first step towards more rational control over nanoparticle synthesis through control of both solution precursors and reaction intermediaries.« less

Optimization of calcium phosphate fine ceramic powders preparation

NASA Astrophysics Data System (ADS)

Sezanova, K.; Tepavitcharova, S.; Rabadjieva, D.; Gergulova, R.; Ilieva, R.

2013-12-01

The effect of biomimetic synthesis method, reaction medium and further precursor treatments on the chemical and phase composition, crystal size and morphology of calcium phosphates was examined. Nanosized calcium phosphate precursors were biomimetically precipitated by the method of continuous precipitation in three types of reaction media at pH 8: (i) SBF as an inorganic electrolyte system; (ii) organic (glycerine) modified SBF (volume ratio of 1:1); (iii) polymer (10 g/l xanthan gum or 10 g/l guar gum) modified SBF (volume ratio of 1:1). After maturation (24 h) the samples were lyophilized, calcinated at 300°C for 3 hours, and washed with water, followed by new gelation, lyophilization and step-wise (200, 400, 600, 800, and 1000°C, each for 3 hours) sintering. The reaction medium influenced the chemical composition and particle size but not the morphology of the calcium phosphate powders. In all studied cases bi-phase calcium phosphate fine powders with well-shaped spherical grains, consisting of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) with a Ca/P ratio of 1.3 - 1.6 were obtained. The SBF modifiers decreased the particle size of the product in the sequence guar gum ˜ xanthan gum < glycerin < SBF medium.

Stability and Unimolecular Reactivity of Palladate(II) Complexes [Ln PdR3 ]- (L=Phosphine, R=Organyl, n=0 and 1).

PubMed

Kolter, Marlene; Koszinowski, Konrad

2016-10-24

The reduction of Pd II precatalysts to catalytically active Pd 0 species is a key step in many palladium-mediated cross-coupling reactions. Besides phosphines, the stoichiometrically used organometallic reagents can afford this reduction, but do so in a poorly understood way. To elucidate the mechanism of this reaction, we have treated solutions of Pd(OAc) 2 and a phosphine ligand L in tetrahydrofuran with RMgCl (R=Ph, Bn, Bu) as well as other organometallic reagents. Analysis of these model systems by electrospray- ionization mass spectrometry found palladate(II) complexes [L n PdR 3 ] - (n=0 and 1), thus pointing to the occurrence of transmetallation reactions. Upon gas-phase fragmentation, the [L n PdR 3 ] - anions preferentially underwent a reductive elimination to yield Pd 0 species. The sequence of the transmetallation and reductive elimination, thus, constitutes a feasible mechanism for the reduction of the Pd(OAc) 2 precatalyst. Other species of interest observed include the Pd IV complex [PdBn 5 ] - , which did not fragment via a reductive elimination but lost BnH instead. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduced Order Models for Reactions of Energetic Materials

NASA Astrophysics Data System (ADS)

Kober, Edward

The formulation of reduced order models for the reaction chemistry of energetic materials under high pressures is needed for the development of mesoscale models in the areas of initiation, deflagration and detonation. Phenomenologically, 4-8 step models have been formulated from the analysis of cook-off data by analyzing the temperature rise of heated samples. Reactive molecular dynamics simulations have been used to simulate many of these processes, but reducing the results of those simulations to simple models has not been achieved. Typically, these efforts have focused on identifying molecular species and detailing specific chemical reactions. An alternative approach is presented here that is based on identifying the coordination geometries of each atom in the simulation and tracking classes of reactions by correlated changes in these geometries. Here, every atom and type of reaction is documented for every time step; no information is lost from unsuccessful molecular identification. Principal Component Analysis methods can then be used to map out the effective chemical reaction steps. For HMX and TATB decompositions simulated with ReaxFF, 90% of the data can be explained by 4-6 steps, generating models similar to those from the cook-off analysis. By performing these simulations at a variety of temperatures and pressures, both the activation and reaction energies and volumes can then be extracted.

On the mechanism of the palladium catalyzed intramolecular Pauson-Khand-type reaction.

PubMed

Lan, Yu; Deng, Lujiang; Liu, Jing; Wang, Can; Wiest, Olaf; Yang, Zhen; Wu, Yun-Dong

2009-07-17

Density functional theory calculations and experimental studies have been carried out on the intramolecular Pauson-Khand-Type reaction mediated by a PdCl(2)-thiourea catalyst, which proceeds under mild reaction conditions and provides a useful alternative to traditional Pauson-Khand reactions. The classical mechanism of the Pauson-Khand reaction involving the alkyne/alkene C-C bond formation as the key step has been found to be energetically unfavorable and is not in line with the experimental observations. A novel reaction mechanism has been proposed for the reaction. The first step involves the cis-halometalation of the alkyne, followed by sequential alkene and carbonyl insertion. The rate-determining fourth step is an intramolecular C-Cl oxidative addition, leading to a Pd(IV) intermediate. A C-C bond formation by reductive elimination completes the reaction. The mechanism is in agreement with the key experimental observations including (1) the need of a chloride for catalytic activity and the absence of catalysis with Pd(OAc)(2) alone; (2) the rate acceleration by the addition of LiCl; both with PdCl(2) and Pd(OAc)(2) catalysts; and (3) the preferred formation of the trans diastereomer in substituted cases. The cis halometalation and the formation and stability of the Pd(IV) intermediate is studied in detail and provides general insights into these novel steps.

A trifunctional mesoporous silica-based, highly active catalyst for one-pot, three-step cascade reactions.

PubMed

Biradar, Ankush V; Patil, Vijayshinha S; Chandra, Prakash; Doke, Dhananjay S; Asefa, Tewodros

2015-05-18

We report the synthesis of a trifunctional catalyst containing amine, sulphonic acid and Pd nanoparticle catalytic groups anchored on the pore walls of SBA-15. The catalyst efficiently catalyzes one-pot three-step cascade reactions comprising deacetylation, Henry reaction and hydrogenation, giving up to ∼100% conversion and 92% selectivity to the final product.

Investigation of the chemical pathway of gaseous nitrogen dioxide formation during flue gas desulfurization with dry sodium bicarbonate injection

NASA Astrophysics Data System (ADS)

Stein, Antoinette Weil

The chemical reaction pathway for the viable flue gas desulfurization process, dry sodium bicarbonate injection, was investigated to mitigate undesirable plume discoloration. Based on a foundation of past findings, a simplified three-step reaction pathway was hypothesized for the formation of the plume-discoloring constituent, NO2. As the first step, it was hypothesized that sodium sulfite formed by sodium bicarbonate reaction with flue gas SO 2. As the second step, it was hypothesized that sodium nitrate formed by sodium sulfite reaction with flue gas NO. And as the third step, it was hypothesized that NO2 and sodium sulfate formed by sodium nitrate reaction with SO2. The second and third hypothesized steps were experimentally investigated using an isothermal fixed bed reactor. As reported in the past, technical grade sodium sulfite was found to be un-reactive with NO and O2. Freshly prepared sodium sulfite, maintained unexposed to moist air, was shown to react with NO and O2 resulting in a mixture of sodium nitrite and sodium nitrate together with a significant temperature rise. This reaction was found to proceed only when oxygen was present in the flue gas. As reported in the past, technical grade sodium nitrate was shown to be un-reactive with SO2. But freshly formed sodium nitrate kept unexposed to humidity was found to be reactive with SO2 and O 2 resulting in the formation of NO2 and sodium sulfate polymorphic Form I. The NO2 formation by this reaction was shown to be temperature dependent with maximum formation at 175°C. Plume mitigation methods were studied based on the validated three-step reaction pathway. Mitigation of NO2 was exhibited by limiting oxygen concentration in the flue gas to a level below 5%. It was also shown that significant NO2 mitigation was achieved by operating below 110°C or above 250°C. An innovative NO2 mitigation method was patented as a result of the findings of this study. The patented process incorporated a process step of sodium sulfite injection to remove flue gas NO prior to sodium bicarbonate injection.

Mechanisms of formation of 8-oxoguanine due to reactions of one and two OH* radicals and the H2O2 molecule with guanine: A quantum computational study.

PubMed

Jena, N R; Mishra, P C

2005-07-28

Mechanisms of formation of the mutagenic product 8-oxoguanine (8OG) due to reactions of guanine with two separate OH* radicals and with H2O2 were investigated at the B3LYP/6-31G, B3LYP/6-311++G, and B3LYP/AUG-cc-pVDZ levels of theory. Single point energy calculations were carried out with the MP2/AUG-cc-pVDZ method employing the optimized geometries at the B3LYP/AUG-cc-pVDZ level. Solvent effect was treated using the PCM and IEF-PCM models. Reactions of two separate OH* radicals and H2O2 with the C2 position of 5-methylimidazole (5MI) were investigated taking 5MI as a model to study reactions at the C8 position of guanine. The addition reaction of an OH* radical at the C8 position of guanine is found to be nearly barrierless while the corresponding adduct is quite stable. The reaction of a second OH* radical at the C8 position of guanine leading to the formation of 8OG complexed with a water molecule can take place according to two different mechanisms, involving two steps each. According to one mechanism, at the first step, 8-hydroxyguanine (8OHG) complexed with a water molecule is formed ,while at the second step, 8OHG is tautomerized to 8OG. In the other mechanism, at the first step, an intermediate complexed (IC) with a water molecule is formed, the five-membered ring of which is open, while at the second step, the five-membered ring is closed and a hydrogen bonded complex of 8OG with a water molecule is formed. The reaction of H2O2 with guanine leading to the formation of 8OG complexed with a water molecule can also take place in accordance with two different mechanisms having two steps each. At the first step of one mechanism, H2O2 is dissociated into two OH* groups that react with guanine to form the same IC as that formed in the reaction with two separate OH* radicals, and the subsequent step of this mechanism is also the same as that of the reaction of guanine with two separate OH* radicals. At the first step of the other mechanism of the reaction of guanine with H2O2, the latter molecule is dissociated into a hydrogen atom and an OOH* group which become bonded to the N7 and C8 atoms of guanine, respectively. At the second step of this mechanism, the OOH* group is dissociated into an oxygen atom and an OH* group, the former becomes bonded to the C8 atom of guanine while the latter abstracts the H8 atom bonded to C8, thus producing 8OG complexed with a water molecule. Solvent effects of the aqueous medium on certain reaction barriers and released energies are appreciable. 5MI works as a satisfactory model for a qualitative study of the reactions of two separate OH* radicals or H2O2 occurring at the C8 position of guanine.

[Optimization and assessment of a reverse hybridization system for the detection of HBV drug-resistant mutations].

PubMed

Liu, Yan-chen; Huang, Ai-long; Hu, Yuan; Hu, Jie-li; Lai, Guo-qi; Zhang, Wen-lu

2011-12-01

To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system. 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively. Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test. Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

i-rDNA: alignment-free algorithm for rapid in silico detection of ribosomal gene fragments from metagenomic sequence data sets.

PubMed

Mohammed, Monzoorul Haque; Ghosh, Tarini Shankar; Chadaram, Sudha; Mande, Sharmila S

2011-11-30

Obtaining accurate estimates of microbial diversity using rDNA profiling is the first step in most metagenomics projects. Consequently, most metagenomic projects spend considerable amounts of time, money and manpower for experimentally cloning, amplifying and sequencing the rDNA content in a metagenomic sample. In the second step, the entire genomic content of the metagenome is extracted, sequenced and analyzed. Since DNA sequences obtained in this second step also contain rDNA fragments, rapid in silico identification of these rDNA fragments would drastically reduce the cost, time and effort of current metagenomic projects by entirely bypassing the experimental steps of primer based rDNA amplification, cloning and sequencing. In this study, we present an algorithm called i-rDNA that can facilitate the rapid detection of 16S rDNA fragments from amongst millions of sequences in metagenomic data sets with high detection sensitivity. Performance evaluation with data sets/database variants simulating typical metagenomic scenarios indicates the significantly high detection sensitivity of i-rDNA. Moreover, i-rDNA can process a million sequences in less than an hour on a simple desktop with modest hardware specifications. In addition to the speed of execution, high sensitivity and low false positive rate, the utility of the algorithmic approach discussed in this paper is immense given that it would help in bypassing the entire experimental step of primer-based rDNA amplification, cloning and sequencing. Application of this algorithmic approach would thus drastically reduce the cost, time and human efforts invested in all metagenomic projects. A web-server for the i-rDNA algorithm is available at http://metagenomics.atc.tcs.com/i-rDNA/

Scanning sequences after Gibbs sampling to find multiple occurrences of functional elements

PubMed Central

Tharakaraman, Kannan; Mariño-Ramírez, Leonardo; Sheetlin, Sergey L; Landsman, David; Spouge, John L

2006-01-01

Background Many DNA regulatory elements occur as multiple instances within a target promoter. Gibbs sampling programs for finding DNA regulatory elements de novo can be prohibitively slow in locating all instances of such an element in a sequence set. Results We describe an improvement to the A-GLAM computer program, which predicts regulatory elements within DNA sequences with Gibbs sampling. The improvement adds an optional "scanning step" after Gibbs sampling. Gibbs sampling produces a position specific scoring matrix (PSSM). The new scanning step resembles an iterative PSI-BLAST search based on the PSSM. First, it assigns an "individual score" to each subsequence of appropriate length within the input sequences using the initial PSSM. Second, it computes an E-value from each individual score, to assess the agreement between the corresponding subsequence and the PSSM. Third, it permits subsequences with E-values falling below a threshold to contribute to the underlying PSSM, which is then updated using the Bayesian calculus. A-GLAM iterates its scanning step to convergence, at which point no new subsequences contribute to the PSSM. After convergence, A-GLAM reports predicted regulatory elements within each sequence in order of increasing E-values, so users have a statistical evaluation of the predicted elements in a convenient presentation. Thus, although the Gibbs sampling step in A-GLAM finds at most one regulatory element per input sequence, the scanning step can now rapidly locate further instances of the element in each sequence. Conclusion Datasets from experiments determining the binding sites of transcription factors were used to evaluate the improvement to A-GLAM. Typically, the datasets included several sequences containing multiple instances of a regulatory motif. The improvements to A-GLAM permitted it to predict the multiple instances. PMID:16961919

Micellar induced regioselectivity in the two-step consecutive reaction of SO3(2-) with Br-(CH2CH2)n-Br (n=2-5).

PubMed

Currie, Fredrik; Jarvoll, Patrik; Holmberg, Krister; Romsted, Laurence S; Gunaseelan, Krishnan

2007-08-15

High field (800 MHz) (1)H NMR was used to monitor the two-step consecutive reaction of excess SO(3)(2-) with symmetrical bifunctional alpha,omega-dibromoalkanes with butane (DBB), hexane (DBH), octane (DBO), and decane (DBD) chains in CTAB micelles at 25 degrees C. The first-order rate constant for the first substitution step for DBB and DBH is about 5 times faster than for the second, but the kinetics for DBO and DBD were not cleanly first-order. After 40 min, the solution contained about 80% of the intermediate bromoalkanesulfonate from DBB and DBH and the remainder is alkanedisulfonate and unreacted starting material. The same reactions were carried out in homogeneous MeOH/D(2)O solutions at 50 degrees C. The rate constants for all four alpha,omega-dibromoalkanes were first-order throughout the time course of the reaction and the same within +/-10%. However, because micellar solutions are organized on the nanoscale and bring together lipophilic and hydrophilic reactants into a small reaction volume at the micellar interface, they speed this substitution reaction considerably compared to reaction in MeOH/D(2)O. The CTAB micelles also induce a significant regioselectivity in product formation by speeding the first step of the consecutive reaction more than the second. The results are consistent with the bromoalkanesulfonate intermediates having a radial orientation within the micelles with the -CH(2)SO(3)(-) group in the interfacial region and the -CH(2)Br group directed into the micellar core such that the concentration of -CH(2)Br groups in the reactive zone, i.e., the micellar interface, is significantly reduced. These results provide the first example of self-assembled surfactant system altering the relative rates of the reaction steps of a consecutive reaction and, in doing so, enhancing monosubstitution of a symmetrically disubstituted species.

Sequence verification as quality-control step for production of cDNA microarrays.

PubMed

Taylor, E; Cogdell, D; Coombes, K; Hu, L; Ramdas, L; Tabor, A; Hamilton, S; Zhang, W

2001-07-01

To generate cDNA arrays in our core laboratory, we amplified about 2300 PCR products from a human, sequence-verified cDNA clone library. As a quality-control step, we sequenced the PCR products immediately before printing. The sequence information was used to search the GenBank database to confirm the identities. Although these clones were previously sequence verified by the company, we found that only 79% of the clones matched the original database after handling. Our experience strongly indicates the necessity to sequence verify the clones at the final stage before printing on microarray slides and to modify the gene list accordingly.

Protein sequences and redox titrations indicate that the electron acceptors in reaction centers from heliobacteria are similar to Photosystem I

NASA Technical Reports Server (NTRS)

Trost, J. T.; Brune, D. C.; Blankenship, R. E.

1992-01-01

Photosynthetic reaction centers isolated from Heliobacillus mobilis exhibit a single major protein on SDS-PAGE of 47 000 Mr. Attempts to sequence the reaction center polypeptide indicated that the N-terminus is blocked. After enzymatic and chemical cleavage, four peptide fragments were sequenced from the Heliobacillus mobilis apoprotein. Only one of these sequences showed significant specific similarity to any of the protein and deduced protein sequences in the GenBank data base. This fragment is identical with 56% of the residues, including both cysteines, found in highly conserved region that is proposed to bind iron-sulfur center Fx in the Photosystem I reaction center peptide that is the psaB gene product. The similarity to the psaA gene product in this region is 48%. Redox titrations of laser-flash-induced photobleaching with millisecond decay kinetics on isolated reaction centers from Heliobacterium gestii indicate a midpoint potential of -414 mV with n = 2 titration behavior. In membranes, the behavior is intermediate between n = 1 and n = 2, and the apparent midpoint potential is -444 mV. This is compared to the behavior in Photosystem I, where the intermediate electron acceptor A1, thought to be a phylloquinone molecule, has been proposed to undergo a double reduction at low redox potentials in the presence of viologen redox mediators. These results strongly suggest that the acceptor side electron transfer system in reaction centers from heliobacteria is indeed analogous to that found in Photosystem I. The sequence similarities indicate that the divergence of the heliobacteria from the Photosystem I line occurred before the gene duplication and subsequent divergence that lead to the heterodimeric protein core of the Photosystem I reaction center.

Metabolism of monoterpenes: early steps in the metabolism of d-neomenthyl-. beta. -D-glucoside in peppermint (Mentha piperita) rhizomes

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

Croteau, R.; Sood, V.K.; Renstroem, B.

1984-11-01

Previous studies have shown that the monoterpene ketone l-(G-/sup 3/H) menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaves of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to methyl acetate while the bulk of the neomenthol is transformed to neomenthyl-..beta..-D-glucoside which is then transported to the rhizome. Analysis of the disposition of l-(G)/sup 3/H)menthone applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis and transport of the monoterpenyl glucoside to be determined, and gave strong indication that the glucoside was subsequently metabolized in the rhizome. Studiesmore » with d-(G-/sup 3/H)neomenthyl-..beta..-D-glucoside as substrate, using excised rhizomes or rhizome segments, confirmed the hydrolysis of the glucoside as an early step in metabolism at this site, and revealed that the terpenoid moiety was further converted to a series of ether-soluble, methanol-soluble, and water-soluble products. The conversion of menthone to the lactone, and of the lactone to more polar products, were confirmed in vivo using l-(G-/sup 3/H)menthone and l-(G-/sup 3/H)-3,4-menthone lactone as substrates. Additional oxidation products were formed in vivo via the desaturation of labeled neomenthol and/or menthone, but none of these transformations appeared to lead to ring opening of the p-menthane skeleton. Each step in the main reaction sequence, from hydrolysis of neomenthyl glucoside to lactonization of menthone, was demonstrated in cell-free extracts from the rhizomes of flowering mint plants. The lactomization step is of particular significance in providing a means of cleaving the p-methane ring to afford an acyclic carbon skeleton that can be further degraded by modifications of the well-known ..beta..-oxidation sequence. 41 references, 3 figures, 1 table.« less

Fast dye salts provide fast access to azidoarene synthons in multi-step one-pot tandem click transformations

PubMed Central

Fletcher, James T.; Reilly, Jacquelline E.

2012-01-01

This study examined whether commercially available diazonium salts could be used as efficient aromatic azide precursors in one-pot multi-step click transformations. Seven different diazonium salts, including Fast Red RC, Fast Blue B, Fast Corinth V and Variamine Blue B were surveyed under aqueous click reaction conditions of CuSO4/Na ascorbate catalyst with 1:1 t-BuOH:H2O solvent. Two-step tandem reactions with terminal alkyne and diyne co-reactants led to 1,2,3-triazole products in 66%-88% yields, while three-step tandem reactions with trimethylsilyl-protected alkyne and diyne co-reactants led to 1,2,3-triazole products in 61%-78% yields. PMID:22368306

A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA

PubMed Central

Lavery, Richard; Zakrzewska, Krystyna; Beveridge, David; Bishop, Thomas C.; Case, David A.; Cheatham, Thomas; Dixit, Surjit; Jayaram, B.; Lankas, Filip; Laughton, Charles; Maddocks, John H.; Michon, Alexis; Osman, Roman; Orozco, Modesto; Perez, Alberto; Singh, Tanya; Spackova, Nada; Sponer, Jiri

2010-01-01

It is well recognized that base sequence exerts a significant influence on the properties of DNA and plays a significant role in protein–DNA interactions vital for cellular processes. Understanding and predicting base sequence effects requires an extensive structural and dynamic dataset which is currently unavailable from experiment. A consortium of laboratories was consequently formed to obtain this information using molecular simulations. This article describes results providing information not only on all 10 unique base pair steps, but also on all possible nearest-neighbor effects on these steps. These results are derived from simulations of 50–100 ns on 39 different DNA oligomers in explicit solvent and using a physiological salt concentration. We demonstrate that the simulations are converged in terms of helical and backbone parameters. The results show that nearest-neighbor effects on base pair steps are very significant, implying that dinucleotide models are insufficient for predicting sequence-dependent behavior. Flanking base sequences can notably lead to base pair step parameters in dynamic equilibrium between two conformational sub-states. Although this study only provides limited data on next-nearest-neighbor effects, we suggest that such effects should be analyzed before attempting to predict the sequence-dependent behavior of DNA. PMID:19850719

Automated property optimization via ab initio O(N) elongation method: Application to (hyper-)polarizability in DNA

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

Orimoto, Yuuichi, E-mail: orimoto.yuuichi.888@m.kyushu-u.ac.jp; Aoki, Yuriko; Japan Science and Technology Agency, CREST, 4-1-8 Hon-chou, Kawaguchi, Saitama 332-0012

An automated property optimization method was developed based on the ab initio O(N) elongation (ELG) method and applied to the optimization of nonlinear optical (NLO) properties in DNA as a first test. The ELG method mimics a polymerization reaction on a computer, and the reaction terminal of a starting cluster is attacked by monomers sequentially to elongate the electronic structure of the system by solving in each step a limited space including the terminal (localized molecular orbitals at the terminal) and monomer. The ELG-finite field (ELG-FF) method for calculating (hyper-)polarizabilities was used as the engine program of the optimization method,more » and it was found to show linear scaling efficiency while maintaining high computational accuracy for a random sequenced DNA model. Furthermore, the self-consistent field convergence was significantly improved by using the ELG-FF method compared with a conventional method, and it can lead to more feasible NLO property values in the FF treatment. The automated optimization method successfully chose an appropriate base pair from four base pairs (A, T, G, and C) for each elongation step according to an evaluation function. From test optimizations for the first order hyper-polarizability (β) in DNA, a substantial difference was observed depending on optimization conditions between “choose-maximum” (choose a base pair giving the maximum β for each step) and “choose-minimum” (choose a base pair giving the minimum β). In contrast, there was an ambiguous difference between these conditions for optimizing the second order hyper-polarizability (γ) because of the small absolute value of γ and the limitation of numerical differential calculations in the FF method. It can be concluded that the ab initio level property optimization method introduced here can be an effective step towards an advanced computer aided material design method as long as the numerical limitation of the FF method is taken into account.« less

Automated property optimization via ab initio O(N) elongation method: Application to (hyper-)polarizability in DNA.

PubMed

Orimoto, Yuuichi; Aoki, Yuriko

2016-07-14

An automated property optimization method was developed based on the ab initio O(N) elongation (ELG) method and applied to the optimization of nonlinear optical (NLO) properties in DNA as a first test. The ELG method mimics a polymerization reaction on a computer, and the reaction terminal of a starting cluster is attacked by monomers sequentially to elongate the electronic structure of the system by solving in each step a limited space including the terminal (localized molecular orbitals at the terminal) and monomer. The ELG-finite field (ELG-FF) method for calculating (hyper-)polarizabilities was used as the engine program of the optimization method, and it was found to show linear scaling efficiency while maintaining high computational accuracy for a random sequenced DNA model. Furthermore, the self-consistent field convergence was significantly improved by using the ELG-FF method compared with a conventional method, and it can lead to more feasible NLO property values in the FF treatment. The automated optimization method successfully chose an appropriate base pair from four base pairs (A, T, G, and C) for each elongation step according to an evaluation function. From test optimizations for the first order hyper-polarizability (β) in DNA, a substantial difference was observed depending on optimization conditions between "choose-maximum" (choose a base pair giving the maximum β for each step) and "choose-minimum" (choose a base pair giving the minimum β). In contrast, there was an ambiguous difference between these conditions for optimizing the second order hyper-polarizability (γ) because of the small absolute value of γ and the limitation of numerical differential calculations in the FF method. It can be concluded that the ab initio level property optimization method introduced here can be an effective step towards an advanced computer aided material design method as long as the numerical limitation of the FF method is taken into account.

Hybrid Quantum/Classical Molecular Dynamics Simulations of the Proton Transfer Reactions Catalyzed by Ketosteroid Isomerase: Analysis of Hydrogen Bonding, Conformational Motions, and Electrostatics

PubMed Central

Chakravorty, Dhruva K.; Soudackov, Alexander V.; Hammes-Schiffer, Sharon

2009-01-01

Hybrid quantum/classical molecular dynamics simulations of the two proton transfer reactions catalyzed by ketosteroid isomerase are presented. The potential energy surfaces for the proton transfer reactions are described with the empirical valence bond method. Nuclear quantum effects of the transferring hydrogen increase the rates by a factor of ~8, and dynamical barrier recrossings decrease the rates by a factor of 3–4. For both proton transfer reactions, the donor-acceptor distance decreases substantially at the transition state. The carboxylate group of the Asp38 side chain, which serves as the proton acceptor and donor in the first and second steps, respectively, rotates significantly between the two proton transfer reactions. The hydrogen bonding interactions within the active site are consistent with the hydrogen bonding of both Asp99 and Tyr14 to the substrate. The simulations suggest that a hydrogen bond between Asp99 and the substrate is present from the beginning of the first proton transfer step, whereas the hydrogen bond between Tyr14 and the substrate is virtually absent in the first part of this step but forms nearly concurrently with the formation of the transition state. Both hydrogen bonds are present throughout the second proton transfer step until partial dissociation of the product. The hydrogen bond between Tyr14 and Tyr55 is present throughout both proton transfer steps. The active site residues are more mobile during the first step than during the second step. The van der Waals interaction energy between the substrate and the enzyme remains virtually constant along the reaction pathway, but the electrostatic interaction energy is significantly stronger for the dienolate intermediate than for the reactant and product. Mobile loop regions distal to the active site exhibit significant structural rearrangements and, in some cases, qualitative changes in the electrostatic potential during the catalytic reaction. These results suggest that relatively small conformational changes of the enzyme active site and substrate strengthen the hydrogen bonds that stabilize the intermediate, thereby facilitating the proton transfer reactions. Moreover, the conformational and electrostatic changes associated with these reactions are not limited to the active site but rather extend throughout the entire enzyme. PMID:19799395

Enantioselectivity in CPA-catalyzed Friedel-Crafts reaction of indole and N-tosylimines: a challenge for guiding models.

PubMed

Simón, Luis

2018-03-28

Qualitative reaction models or predicting guides are a very useful outcome of theoretical investigations of organocatalytic reaction mechanism that allow forecasting of the degree and sense of the enantioselectivity of reactions involving novel substrates. However, application of these models can be unexpectedly challenging in reactions affected by a large number of conformations and potential control of the enantioselectivity by different reaction steps. The QM/MM study of the Friedel-Crafts reaction between indole and the N-tosylimide of benzaldehyde catalysed by different CPA reveals that the reaction consists of two CPA-assisted steps: the addition of the two reagents to yield a Wheland intermediate, and its re-aromatization. The relevance of the second step depends on the catalyst: it changes the sense of the expected stereoselectivity for a BINOP-derived CPA but is irrelevant in the reaction catalysed by a VAPOL-derived imidodiphosphoric acid catalyst. Although the relative energies of the TSs can be rationalized considering the steric interactions with the catalyst, the possibility of additional H-bonds, or the relative stability of the conformation of the reagents, predicting the enantioselectivity is not possible using qualitative guides.

Addition reaction of methyl cinnamate with 2-amino-4- nitrophenol

NASA Astrophysics Data System (ADS)

Suryanti, Venty; Rakhman Wibowo, Fajar; Pranoto; Robingatun Isnaeni, Siti; Ratna Kumala Sari, Meiyanti; Handayani, Sekar

2016-02-01

A novel compound which have one N-H fragment and nitrophenyl group has been designed and synthesized from cinnamaldehyde. The reaction was conducted in 3 step reactions to give the final product. Firstly, cinnamaldehyde was converted into cinnamic acid, which was then esterified with methyl alcohol to obtained methyl cinnamate. The last step was the addition reaction between methyl cinnamate and 2-amino-4-nitrophenol to give a cinnamaldehyde derivative, namely methyl-3-(2-hidroksi-5-nitrophenyl amino)-3- phenylpropanoate.

The alignment of enzymatic steps reveals similar metabolic pathways and probable recruitment events in Gammaproteobacteria.

PubMed

Poot-Hernandez, Augusto Cesar; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

2015-11-17

It is generally accepted that gene duplication followed by functional divergence is one of the main sources of metabolic diversity. In this regard, there is an increasing interest in the development of methods that allow the systematic identification of these evolutionary events in metabolism. Here, we used a method not based on biomolecular sequence analysis to compare and identify common and variable routes in the metabolism of 40 Gammaproteobacteria species. The metabolic maps deposited in the KEGG database were transformed into linear Enzymatic Step Sequences (ESS) by using the breadth-first search algorithm. These ESS represent subsequent enzymes linked to each other, where their catalytic activities are encoded in the Enzyme Commission numbers. The ESS were compared in an all-against-all (pairwise comparisons) approach by using a dynamic programming algorithm, leaving only a set of significant pairs. From these comparisons, we identified a set of functionally conserved enzymatic steps in different metabolic maps, in which cell wall components and fatty acid and lysine biosynthesis were included. In addition, we found that pathways associated with biosynthesis share a higher proportion of similar ESS than degradation pathways and secondary metabolism pathways. Also, maps associated with the metabolism of similar compounds contain a high proportion of similar ESS, such as those maps from nucleotide metabolism pathways, in particular the inosine monophosphate pathway. Furthermore, diverse ESS associated with the low part of the glycolysis pathway were identified as functionally similar to multiple metabolic pathways. In summary, our comparisons may help to identify similar reactions in different metabolic pathways and could reinforce the patchwork model in the evolution of metabolism in Gammaproteobacteria.

(-)-Menthol biosynthesis and molecular genetics

NASA Astrophysics Data System (ADS)

Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

2005-12-01

(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

The Missing Link in Leguminous Pterocarpan Biosynthesis is a Dirigent Domain-Containing Protein with Isoflavanol Dehydratase Activity.

PubMed

Uchida, Kai; Akashi, Tomoyoshi; Aoki, Toshio

2017-02-01

Pterocarpan forms the basic structure of leguminous phytoalexins, and most of the isoflavonoid pathway genes encoding the enzymes responsible for its biosynthesis have been identified. However, the last step of pterocarpan biosynthesis is a ring closure reaction, and the enzyme that catalyzes this step, 2'-hydroxyisoflavanol 4,2'-dehydratase or pterocarpan synthase (PTS), remains as an unidentified 'missing link'. This last ring formation is assumed to be the key step in determining the stereochemistry of pterocarpans, which plays a role in their antimicrobial activity. In this study, a cDNA clone encoding PTS from Glycyrrhiza echinata (GePTS1) was identified through functional expression fractionation screening of a cDNA library, which requires no sequence information, and orthologs from soybean (GmPTS1) and Lotus japonicus (LjPTS1) were also identified. These proteins were heterologously expressed in Escherichia coli and biochemically characterized. Surprisingly, the proteins were found to include amino acid motifs characteristic of dirigent proteins, some of which control stereospecific phenoxy radical coupling in lignan biosynthesis. The stereospecificity of substrates and products was examined using four substrate stereoisomers with hydroxy and methoxy derivatives at C-4'. The results showed that the 4R configuration was essential for the PTS reaction, and (-)- and (+)-pterocarpans were produced depending on the stereochemistry at C-3. In suspension-cultured soybean cells, levels of the GmPTS1 transcript increased temporarily prior to the peak in phytoalexin accumulation, strongly supporting the possible involvement of PTS in pterocarpan biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.

Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype

PubMed Central

Bekaert, Michaël; Bakheit, Mohammed; Frischmann, Sieghard; Patel, Pranav; Simon-Loriere, Etienne; Lambrechts, Louis; Duong, Veasna; Dussart, Philippe; Harold, Graham; Fall, Cheikh; Faye, Oumar; Sall, Amadou Alpha; Weidmann, Manfred

2018-01-01

Background 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia Methodology/Principal findings 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. Conclusions/Significance We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters. PMID:29813062

Nonequilibrium thermodynamics and a fluctuation theorem for individual reaction steps in a chemical reaction network

NASA Astrophysics Data System (ADS)

Pal, Krishnendu; Das, Biswajit; Banerjee, Kinshuk; Gangopadhyay, Gautam

2015-09-01

We have introduced an approach to nonequilibrium thermodynamics of an open chemical reaction network in terms of the propensities of the individual elementary reactions and the corresponding reverse reactions. The method is a microscopic formulation of the dissipation function in terms of the relative entropy or Kullback-Leibler distance which is based on the analogy of phase space trajectory with the path of elementary reactions in a network of chemical process. We have introduced here a fluctuation theorem valid for each opposite pair of elementary reactions which is useful in determining the contribution of each sub-reaction on the nonequilibrium thermodynamics of overall reaction. The methodology is applied to an oligomeric enzyme kinetics at a chemiostatic condition that leads the reaction to a nonequilibrium steady state for which we have estimated how each step of the reaction is energy driven or entropy driven to contribute to the overall reaction.

High-resolution melt PCR analysis for genotyping of Ureaplasma parvum isolates directly from clinical samples.

PubMed

Payne, Matthew S; Tabone, Tania; Kemp, Matthew W; Keelan, Jeffrey A; Spiller, O Brad; Newnham, John P

2014-02-01

Ureaplasma sp. infection in neonates and adults underlies a variety of disease pathologies. Of the two human Ureaplasma spp., Ureaplasma parvum is clinically the most common. We have developed a high-resolution melt (HRM) PCR assay for the differentiation of the four serovars of U. parvum in a single step. Currently U. parvum strains are separated into four serovars by sequencing the promoter and coding region of the multiple-banded antigen (MBA) gene. We designed primers to conserved sequences within this region for PCR amplification and HRM analysis to generate reproducible and distinct melt profiles that distinguish clonal representatives of serovars 1, 3, 6, and 14. Furthermore, our HRM PCR assay could classify DNA extracted from 74 known (MBA-sequenced) test strains with 100% accuracy. Importantly, HRM PCR was also able to identify U. parvum serovars directly from 16 clinical swabs. HRM PCR performed with DNA consisting of mixtures of combined known serovars yielded profiles that were easily distinguished from those for single-serovar controls. These profiles mirrored clinical samples that contained mixed serovars. Unfortunately, melt curve analysis software is not yet robust enough to identify the composition of mixed serovar samples, only that more than one serovar is present. HRM PCR provides a single-step, rapid, cost-effective means to differentiate the four serovars of U. parvum that did not amplify any of the known 10 serovars of Ureaplasma urealyticum tested in parallel. Choice of reaction reagents was found to be crucial to allow sufficient sensitivity to differentiate U. parvum serovars directly from clinical swabs rather than requiring cell enrichment using microbial culture techniques.

Problem-Solving Test: Pyrosequencing

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2013-01-01

Terms to be familiar with before you start to solve the test: Maxam-Gilbert sequencing, Sanger sequencing, gel electrophoresis, DNA synthesis reaction, polymerase chain reaction, template, primer, DNA polymerase, deoxyribonucleoside triphosphates, orthophosphate, pyrophosphate, nucleoside monophosphates, luminescence, acid anhydride bond,…

[The detection and sequence analysis of the simian T-lymphotrophic retrovirus (STLV-1 Papio) by using the polymerase chain reaction].

PubMed

D'iachenko, A G; Dzhalagoniia, B E; Kapanadze, B I

1993-01-01

The gene amplification technique was used for detection and sequence analysis of STLV-1 Papio proviral DNA. The polymerase chain reaction was performed with a primer pair at tax region of HTLV-1, 7336-7354, sense strand, and 7516-7494, antisense strand. One microgram of DNAs isolated from LUG-4 cells and autopsies was used in a reaction volume of 50 microliters involving 30 cycles of amplifications. The reaction product was blunt-end cloned into pUC19 cut with Smal. The sequence was done with T7-polymerase using 32P-dATR as a label. Our results indicate that STLV-1 Papio provirus is actually present in the cells of a lymphoid cell line and tumor cells of lymphomatous monkeys. There are some differences between STLV-1 Papio and reported sequences of HTLV-1 and STLV-1.

Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR.

PubMed

Krych, Łukasz; Kot, Witold; Bendtsen, Katja M B; Hansen, Axel K; Vogensen, Finn K; Nielsen, Dennis S

2018-01-01

The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Learning to Predict Chemical Reactions

PubMed Central

Kayala, Matthew A.; Azencott, Chloé-Agathe; Chen, Jonathan H.

2011-01-01

Being able to predict the course of arbitrary chemical reactions is essential to the theory and applications of organic chemistry. Approaches to the reaction prediction problems can be organized around three poles corresponding to: (1) physical laws; (2) rule-based expert systems; and (3) inductive machine learning. Previous approaches at these poles respectively are not high-throughput, are not generalizable or scalable, or lack sufficient data and structure to be implemented. We propose a new approach to reaction prediction utilizing elements from each pole. Using a physically inspired conceptualization, we describe single mechanistic reactions as interactions between coarse approximations of molecular orbitals (MOs) and use topological and physicochemical attributes as descriptors. Using an existing rule-based system (Reaction Explorer), we derive a restricted chemistry dataset consisting of 1630 full multi-step reactions with 2358 distinct starting materials and intermediates, associated with 2989 productive mechanistic steps and 6.14 million unproductive mechanistic steps. And from machine learning, we pose identifying productive mechanistic steps as a statistical ranking, information retrieval, problem: given a set of reactants and a description of conditions, learn a ranking model over potential filled-to-unfilled MO interactions such that the top ranked mechanistic steps yield the major products. The machine learning implementation follows a two-stage approach, in which we first train atom level reactivity filters to prune 94.00% of non-productive reactions with a 0.01% error rate. Then, we train an ensemble of ranking models on pairs of interacting MOs to learn a relative productivity function over mechanistic steps in a given system. Without the use of explicit transformation patterns, the ensemble perfectly ranks the productive mechanism at the top 89.05% of the time, rising to 99.86% of the time when the top four are considered. Furthermore, the system is generalizable, making reasonable predictions over reactants and conditions which the rule-based expert does not handle. A web interface to the machine learning based mechanistic reaction predictor is accessible through our chemoinformatics portal (http://cdb.ics.uci.edu) under the Toolkits section. PMID:21819139

A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

PubMed

Matsunaga, Hiroko; Goto, Mari; Arikawa, Koji; Shirai, Masataka; Tsunoda, Hiroyuki; Huang, Huan; Kambara, Hideki

2015-02-15

Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes. Copyright © 2014 Elsevier Inc. All rights reserved.

Patome: a database server for biological sequence annotation and analysis in issued patents and published patent applications.

PubMed

Lee, Byungwook; Kim, Taehyung; Kim, Seon-Kyu; Lee, Kwang H; Lee, Doheon

2007-01-01

With the advent of automated and high-throughput techniques, the number of patent applications containing biological sequences has been increasing rapidly. However, they have attracted relatively little attention compared to other sequence resources. We have built a database server called Patome, which contains biological sequence data disclosed in patents and published applications, as well as their analysis information. The analysis is divided into two steps. The first is an annotation step in which the disclosed sequences were annotated with RefSeq database. The second is an association step where the sequences were linked to Entrez Gene, OMIM and GO databases, and their results were saved as a gene-patent table. From the analysis, we found that 55% of human genes were associated with patenting. The gene-patent table can be used to identify whether a particular gene or disease is related to patenting. Patome is available at http://www.patome.org/; the information is updated bimonthly.

Patome: a database server for biological sequence annotation and analysis in issued patents and published patent applications

PubMed Central

Lee, Byungwook; Kim, Taehyung; Kim, Seon-Kyu; Lee, Kwang H.; Lee, Doheon

2007-01-01

With the advent of automated and high-throughput techniques, the number of patent applications containing biological sequences has been increasing rapidly. However, they have attracted relatively little attention compared to other sequence resources. We have built a database server called Patome, which contains biological sequence data disclosed in patents and published applications, as well as their analysis information. The analysis is divided into two steps. The first is an annotation step in which the disclosed sequences were annotated with RefSeq database. The second is an association step where the sequences were linked to Entrez Gene, OMIM and GO databases, and their results were saved as a gene–patent table. From the analysis, we found that 55% of human genes were associated with patenting. The gene–patent table can be used to identify whether a particular gene or disease is related to patenting. Patome is available at ; the information is updated bimonthly. PMID:17085479

Culture and the sequence of steps in theory of mind development.

PubMed

Shahaeian, Ameneh; Peterson, Candida C; Slaughter, Virginia; Wellman, Henry M

2011-09-01

To examine cultural contrasts in the ordered sequence of conceptual developments leading to theory of mind (ToM), we compared 135 3- to 6-year-olds (77 Australians; 58 Iranians) on an established 5-step ToM scale (Wellman & Liu, 2004). There was a cross-cultural difference in the sequencing of ToM steps but not in overall rates of ToM mastery. In line with our predictions, the children from Iran conformed to a distinctive sequence previously observed only in children in China. In contrast to the case with children from Australia (and the United States), knowledge access was understood earlier than opinion diversity in children from Iran, consistent with this collectivist culture's emphasis on filial respect, dispute avoidance, and acquiring knowledge. Having a sibling was linked with faster overall ToM progress in Australia only and was not related to scale sequences in either culture.

WO3 and W Thermal Atomic Layer Etching Using "Conversion-Fluorination" and "Oxidation-Conversion-Fluorination" Mechanisms.

PubMed

Johnson, Nicholas R; George, Steven M

2017-10-04

The thermal atomic layer etching (ALE) of WO 3 and W was demonstrated with new "conversion-fluorination" and "oxidation-conversion-fluorination" etching mechanisms. Both of these mechanisms are based on sequential, self-limiting reactions. WO 3 ALE was achieved by a "conversion-fluorination" mechanism using an AB exposure sequence with boron trichloride (BCl 3 ) and hydrogen fluoride (HF). BCl 3 converts the WO 3 surface to a B 2 O 3 layer while forming volatile WO x Cl y products. Subsequently, HF spontaneously etches the B 2 O 3 layer producing volatile BF 3 and H 2 O products. In situ spectroscopic ellipsometry (SE) studies determined that the BCl 3 and HF reactions were self-limiting versus exposure. The WO 3 ALE etch rates increased with temperature from 0.55 Å/cycle at 128 °C to 4.19 Å/cycle at 207 °C. W served as an etch stop because BCl 3 and HF could not etch the underlying W film. W ALE was performed using a three-step "oxidation-conversion-fluorination" mechanism. In this ABC exposure sequence, the W surface is first oxidized to a WO 3 layer using O 2 /O 3 . Subsequently, the WO 3 layer is etched with BCl 3 and HF. SE could simultaneously monitor the W and WO 3 thicknesses and conversion of W to WO 3 . SE measurements showed that the W film thickness decreased linearly with number of ABC reaction cycles. W ALE was shown to be self-limiting with respect to each reaction in the ABC process. The etch rate for W ALE was ∼2.5 Å/cycle at 207 °C. An oxide thickness of ∼20 Å remained after W ALE, but could be removed by sequential BCl 3 and HF exposures without affecting the W layer. These new etching mechanisms will enable the thermal ALE of a variety of additional metal materials including those that have volatile metal fluorides.

Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme.

PubMed

Mukherjee, J J; Dekker, E E

1987-10-25

Starting with 100 g (wet weight) of a mutant of Escherichia coli K-12 forced to grow on L-threonine as sole carbon source, we developed a 6-step procedure that provides 30-40 mg of homogeneous 2-amino-3-ketobutyrate CoA ligase (also called aminoacetone synthetase or synthase). This ligase, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, has an apparent molecular weight approximately equal to 85,000 and consists of two identical (or nearly identical) subunits with Mr = 42,000. Computer analysis of amino acid composition data, which gives the best fit nearest integer ratio for each residue, indicates a total of 387 amino acids/subunit with a calculated Mr = 42,093. Stepwise Edman degradation provided the N-terminal sequence of the first 21 amino acids. It is a pyridoxal phosphate-dependent enzyme since (a) several carbonyl reagents caused greater than 90% loss of activity, (b) dialysis against buffer containing hydroxylamine resulted in 89% loss of activity coincident with an 86% decrease in absorptivity at 428 nm, (c) incubation of the apoenzyme with 20 microM pyridoxal phosphate showed a parallel recovery (greater than 90%) of activity and 428-nm absorptivity, and (d) reduction of the holoenzyme with NaBH4 resulted in complete inactivation, disappearance of a new absorption maximum at 333 nm. Strict specificity for glycine is shown but acetyl-CoA (100%), n-propionyl-CoA (127%), or n-butyryl-CoA (16%) is utilized in the condensation reaction. Apparent Km values for acetyl-CoA, n-propionyl-CoA, and glycine are 59 microM, 80 microM, and 12 mM, respectively; the pH optimum = 7.5. Added divalent metal ions or sulfhydryl compounds inhibited catalysis of the condensation reaction.

Multiscale Informatics for Low-Temperature Propane Oxidation: Further Complexities in Studies of Complex Reactions

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

Burke, Michael P.; Goldsmith, C. Franklin; Klippenstein, Stephen J.

2015-07-16

We have developed a multi-scale approach (Burke, M. P.; Klippenstein, S. J.; Harding, L. B. Proc. Combust. Inst. 2013, 34, 547–555.) to kinetic model formulation that directly incorporates elementary kinetic theories as a means to provide reliable, physics-based extrapolation to unexplored conditions. Here, we extend and generalize the multi-scale modeling strategy to treat systems of considerable complexity – involving multi-well reactions, potentially missing reactions, non-statistical product branching ratios, and non-Boltzmann (i.e. non-thermal) reactant distributions. The methodology is demonstrated here for a subsystem of low-temperature propane oxidation, as a representative system for low-temperature fuel oxidation. A multi-scale model is assembled andmore » informed by a wide variety of targets that include ab initio calculations of molecular properties, rate constant measurements of isolated reactions, and complex systems measurements. Active model parameters are chosen to accommodate both “parametric” and “structural” uncertainties. Theoretical parameters (e.g. barrier heights) are included as active model parameters to account for parametric uncertainties in the theoretical treatment; experimental parameters (e.g. initial temperatures) are included to account for parametric uncertainties in the physical models of the experiments. RMG software is used to assess potential structural uncertainties due to missing reactions. Additionally, branching ratios among product channels are included as active model parameters to account for structural uncertainties related to difficulties in modeling sequences of multiple chemically activated steps. The approach is demonstrated here for interpreting time-resolved measurements of OH, HO2, n-propyl, i-propyl, propene, oxetane, and methyloxirane from photolysis-initiated low-temperature oxidation of propane at pressures from 4 to 60 Torr and temperatures from 300 to 700 K. In particular, the multi-scale informed model provides a consistent quantitative explanation of both ab initio calculations and time-resolved species measurements. The present results show that interpretations of OH measurements are significantly more complicated than previously thought – in addition to barrier heights for key transition states considered previously, OH profiles also depend on additional theoretical parameters for R + O2 reactions, secondary reactions, QOOH + O2 reactions, and treatment of non-Boltzmann reaction sequences. Extraction of physically rigorous information from those measurements may require more sophisticated treatment of all of those model aspects, as well as additional experimental data under more conditions, to discriminate among possible interpretations and ensure model reliability. Keywords: Optimization, Uncertainty quantification, Chemical mechanism, Low-Temperature Oxidation, Non-Boltzmann« less

Oligopeptides and copeptides of homochiral sequence, via beta-sheets, from mixtures of racemic alpha-amino acids, in a one-pot reaction in water; relevance to biochirogenesis.

PubMed

Illos, Roni A; Bisogno, Fabricio R; Clodic, Gilles; Bolbach, Gerard; Weissbuch, Isabelle; Lahav, Meir

2008-07-09

As part of our studies on the biochirogenesis of peptides of homochiral sequence during early evolution, the formation of oligopeptides composed of 14-24 residues of the same handedness in the polymerization of dl-leucine (Leu), dl-phenylalanine (Phe), and dl-valine (Val) in aqueous solutions, by activation with N, N'-carbonyldiimidazole and then initiation with a primary amine, in a one-pot reaction, was demonstrated by MALDI-TOF MS using deuterium enantio-labeled alpha-amino acids. The formation of long isotactic peptides is rationalized by the following steps occurring in tandem: (i) creation of a library of short diasteroisomeric oligopeptides containing isotactic peptides in excess in comparison to a binomial kinetics, as a result of an asymmetric induction exerted by the N-terminal residue of a given handedness; (ii) precipitation of the less soluble racemic isotactic penta- and hexapeptides in the form of beta-sheets that are delineated by homochiral rims; (iii) regio-enantiospecific chain elongation occurring heterogeneously at the beta-sheets/solution interface. Polymerization of l-Leu with l-isoleucine (Ile) or l-Phe with l- (1) N-Me-histidine yielded mixtures of copeptides containing both residues. In contrast, in the polymerization of the corresponding mixtures of l- + d-alpha-amino acids, the long oligopeptides were composed mainly from oligo- l-Leu and oligo- d-Ile in the first system and oligo- d-Phe in the second. Furthermore, in the polymerization of mixtures of hydrophobic racemic alpha-amino acids dl-Leu, dl-Val, and dl-Phe and with added racemic dl-alanine and dl-tyrosine, copeptides of homochiral sequences are most dominantly represented. Possible routes for a spontaneous "mirror-symmetry breaking" process of the racemic mixtures of homochiral peptides are presented.

Standard Free Droplet Digital Polymerase Chain Reaction as a New Tool for the Quality Control of High-Capacity Adenoviral Vectors in Small-Scale Preparations

PubMed Central

Boehme, Philip; Stellberger, Thorsten; Solanki, Manish; Zhang, Wenli; Schulz, Eric; Bergmann, Thorsten; Liu, Jing; Doerner, Johannes; Baiker, Armin E.

2015-01-01

Abstract High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy. PMID:25640117

Characterization of a recombinant type II 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Helicobacter pylori.

PubMed

Webby, Celia J; Patchett, Mark L; Parker, Emily J

2005-08-15

DAH7P (3-Deoxy-D-arabino-heptulosonate 7-phosphate) synthase catalyses the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) as the first committed step in the biosynthesis of aromatic compounds in plants and micro-organisms. Previous work has identified two families of DAH7P synthases based on sequence similarity and molecular mass, with the majority of the mechanistic and structural studies being carried out on the type I paralogues from Escherichia coli. Whereas a number of organisms possess genes encoding both type I and type II DAH7P synthases, the pathogen Helicobacter pylori has only a single, type II, enzyme. Recombinant DAH7P synthase from H. pylori was partially solubilized by co-expression with chaperonins GroEL/GroES in E. coli, and purified to homogeneity. The enzyme reaction follows an ordered sequential mechanism with the following kinetic parameters: K(m) (PEP), 3 microM; K(m) (E4P), 6 microM; and kcat, 3.3 s(-1). The enzyme reaction involves interaction of the si face of PEP with the re face of E4P. H. pylori DAH7P synthase is not inhibited by phenylalanine, tyrosine, tryptophan or chorismate. EDTA inactivates the enzyme, and activity is restored by a range of bivalent metal ions, including (in order of decreasing effectiveness) Co2+, Mn2+, Ca2+, Mg2+, Cu2+ and Zn2+. Analysis of type II DAH7P synthase sequences reveals several highly conserved motifs, and comparison with the type I enzymes suggests that catalysis by these two enzyme types occurs on a similar active-site scaffold and that the two DAH7P synthase families may indeed be distantly related.

Urea degradation by electrochemically generated reactive chlorine species: products and reaction pathways.

PubMed

Cho, Kangwoo; Hoffmann, Michael R

2014-10-07

This study investigated the transformation of urea by electrochemically generated reactive chlorine species (RCS). Solutions of urea with chloride ions were electrolyzed using a bismuth doped TiO2 (BiOx/TiO2) anode coupled with a stainless steel cathode at applied anodic potentials (Ea) of either +2.2 V or +3.0 V versus the normal hydrogen electrode. In NaCl solution, the current efficiency of RCS generation was near 30% at both potentials. In divided cell experiments, the pseudo-first-order rate of total nitrogen decay was an order of magnitude higher at Ea of +3.0 V than at +2.2 V, presumably because dichlorine radical (Cl2(-)·) ions facilitate the urea transformation primary driven by free chlorine. Quadrupole mass spectrometer analysis of the reactor headspace revealed that N2 and CO2 are the primary gaseous products of the oxidation of urea, whose urea-N was completely transformed into N2 (91%) and NO3(-) (9%). The higher reaction selectivity with respect to N2 production can be ascribed to a low operational ratio of free available chlorine to N. The mass-balance analysis recovered urea-C as CO2 at 77%, while CO generation most likely accounts for the residual carbon. In light of these results, we propose a reaction mechanism involving chloramines and chloramides as reaction intermediates, where the initial chlorination is the rate-determining step in the overall sequence of reactions.

Theoretical aspects of methyl acetate and methanol activation on MgO(100) and (501) catalyst surfaces with application in FAME production

NASA Astrophysics Data System (ADS)

Man, Isabela-Costinela; Soriga, Stefan Gabriel; Parvulescu, Vasile

2017-01-01

Density functional theory (DFT) calculations were carried out to study the activation of methyl acetate and methanol on MgO(100) and MgO(501) surfaces and integrated in the context of transesterification, interesterification and glycerolysis reactions used in biodiesel industry. First results indicate the importance of including of dispersion forces in the calculations. On MgO(100) the reverse reactions steps of Csbnd O and Csbnd H dissociations and on MgO(501) the same reverse reaction step of Csbnd H dissociations of methyl acetate are energetically favorable, while the dissociation of Csbnd O bond into methoxide and acetate fragments on the edge of MgO(501) was found to be exothermic with a low activation energy. For methanol, the dissociation of Osbnd H bond on MgO(100) surface in the presence of the second coadsorbed methanol molecule becomes more energetically favoured compared to the isolated molecule, due to the fact that the methoxide fragment is stabilized by intermolecular hydrogen bonding. This is reflected by the decrease of the activation energy of the forward reaction step and the increase of the activation energy of the backward reaction step, increasing the probability to have dissociated molecules among the undissociated ones. These results represent a step forward for better understanding from atomistic point of view the paths of these reactions on these surfaces for the corresponding catalytic processes.

Fundamental Reaction Pathway for Peptide Metabolism by Proteasome: Insights from First-principles Quantum Mechanical/Molecular Mechanical Free Energy Calculations

PubMed Central

Wei, Donghui; Fang, Lei; Tang, Mingsheng; Zhan, Chang-Guo

2013-01-01

Proteasome is the major component of the crucial nonlysosomal protein degradation pathway in the cells, but the detailed reaction pathway is unclear. In this study, first-principles quantum mechanical/molecular mechanical free energy calculations have been performed to explore, for the first time, possible reaction pathways for proteasomal proteolysis/hydrolysis of a representative peptide, succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-AMC). The computational results reveal that the most favorable reaction pathway consists of six steps. The first is a water-assisted proton transfer within proteasome, activating Thr1-Oγ. The second is a nucleophilic attack on the carbonyl carbon of a Tyr residue of substrate by the negatively charged Thr1-Oγ, followed by the dissociation of the amine AMC (third step). The fourth step is a nucleophilic attack on the carbonyl carbon of the Tyr residue of substrate by a water molecule, accompanied by a proton transfer from the water molecule to Thr1-Nz. Then, Suc-LLVY is dissociated (fifth step), and Thr1 is regenerated via a direct proton transfer from Thr1-Nz to Thr1-Oγ. According to the calculated energetic results, the overall reaction energy barrier of the proteasomal hydrolysis is associated with the transition state (TS3b) for the third step involving a water-assisted proton transfer. The determined most favorable reaction pathway and the rate-determining step have provided a reasonable interpretation of the reported experimental observations concerning the substituent and isotopic effects on the kinetics. The calculated overall free energy barrier of 18.2 kcal/mol is close to the experimentally-derived activation free energy of ~18.3–19.4 kcal/mol, suggesting that the computational results are reasonable. PMID:24111489

Kinetic aspects of chain growth in Fischer-Tropsch synthesis.

PubMed

Filot, Ivo A W; Zijlstra, Bart; Broos, Robin J P; Chen, Wei; Pestman, Robert; Hensen, Emiel J M

2017-04-28

Microkinetics simulations are used to investigate the elementary reaction steps that control chain growth in the Fischer-Tropsch reaction. Chain growth in the FT reaction on stepped Ru surfaces proceeds via coupling of CH and CR surface intermediates. Essential to the growth mechanism are C-H dehydrogenation and C hydrogenation steps, whose kinetic consequences have been examined by formulating two novel kinetic concepts, the degree of chain-growth probability control and the thermodynamic degree of chain-growth probability control. For Ru the CO conversion rate is controlled by the removal of O atoms from the catalytic surface. The temperature of maximum CO conversion rate is higher than the temperature to obtain maximum chain-growth probability. Both maxima are determined by Sabatier behavior, but the steps that control chain-growth probability are different from those that control the overall rate. Below the optimum for obtaining long hydrocarbon chains, the reaction is limited by the high total surface coverage: in the absence of sufficient vacancies the CHCHR → CCHR + H reaction is slowed down. Beyond the optimum in chain-growth probability, CHCR + H → CHCHR and OH + H → H 2 O limit the chain-growth process. The thermodynamic degree of chain-growth probability control emphasizes the critical role of the H and free-site coverage and shows that at high temperature, chain depolymerization contributes to the decreased chain-growth probability. That is to say, during the FT reaction chain growth is much faster than chain depolymerization, which ensures high chain-growth probability. The chain-growth rate is also fast compared to chain-growth termination and the steps that control the overall CO conversion rate, which are O removal steps for Ru.

Redefinition of rubisco carboxylase reaction reveals origin of water for hydration and new roles for active-site residues.

PubMed

Kannappan, Babu; Gready, Jill E

2008-11-12

Crystallographic, mutagenesis, kinetic, and computational studies on Rubisco over three decades have revealed much about its catalytic mechanism and the role played by several active-site residues. However, key questions remain unanswered. Specific details of the carboxylase and oxygenase mechanisms, required to underpin the rational re-engineering of Rubisco, are still speculative. Here we address critical gaps in knowledge with a definitive comprehensive computational investigation of the mechanism of carboxylase activity at the Rubisco active site. Density functional theory calculations (B3LYP/6-31G(d,p)) were performed on active-site fragment models of a size up to 77 atoms, not previously possible computationally. All amino acid residues suspected to play roles in the acid-base chemistry in the multistep reaction, and interacting directly with the central Mg (2+) atom and the reactive moiety of substrate and intermediates, were included. The results provide a firm basis for us to propose a novel mechanism for the entire sequence of reactions in the carboxylase catalysis and to define precise roles for the active-site residues, singly and in concert. In this mechanism, the carbamylated LYS201 plays a more limited role than previously proposed but is crucial for initiating the reaction by acting as a base in the enolization. We suggest a wider role for HIS294, with involvement in the carboxylation, hydration, and C2-C3 bond-scission steps, consistent with the suggestion of Harpel et al. (1998) but contrary to the consensus view of Cleland et al. (1998). In contrast to the common assumption that the water molecule for the hydration step comes from within the active site, we propose that the Mg-coordinated water is not dissociated at the start of the gas-addition reaction but rather remains coordinated and is used for the hydration of the C3 carbon atom. New roles are also proposed for LYS175, GLU204, and HIS294. The mechanism suggests roles in the gas-addition step for residues in three spatially distinct regions of the active site, HIS294 and LYS334 in the C-terminal domain of the large subunit (LSU), but also hitherto unsuspected roles for a cluster of three residues (ASN123, GLU60, and TYR20) in the N-terminal domain of the partner LSU of the dimer containing the active site. Our new mechanism is supported by existing experimental data, provides new convincing interpretations of previously puzzling data, and allows new insights into mutational strategies for improving Rubisco activity.

Anticipation measures of sequence learning: manual versus oculomotor versions of the serial reaction time task.

PubMed

Vakil, Eli; Bloch, Ayala; Cohen, Haggar

2017-03-01

The serial reaction time (SRT) task has generated a very large amount of research. Nevertheless the debate continues as to the exact cognitive processes underlying implicit sequence learning. Thus, the first goal of this study is to elucidate the underlying cognitive processes enabling sequence acquisition. We therefore compared reaction time (RT) in sequence learning in a standard manual activated (MA) to that in an ocular activated (OA) version of the task, within a single experimental setting. The second goal is to use eye movement measures to compare anticipation, as an additional indication of sequence learning, between the two versions of the SRT. Performance of the group given the MA version of the task (n = 29) was compared with that of the group given the OA version (n = 30). The results showed that although overall, RT was faster for the OA group, the rate of sequence learning was similar to that of the MA group performing the standard version of the SRT. Because the stimulus-response association is automatic and exists prior to training in the OA task, the decreased reaction time in this version of the task reflects a purer measure of the sequence learning that occurs in the SRT task. The results of this study show that eye tracking anticipation can be measured directly and can serve as a direct measure of sequence learning. Finally, using the OA version of the SRT to study sequence learning presents a significant methodological contribution by making sequence learning studies possible among populations that struggle to perform manual responses.

Transition-metal-free synthesis of imidazo[2,1-b]thiazoles and thiazolo[3,2-a]benzimidazoles via an S-propargylation/5-exo-dig cyclization/isomerization sequence using propargyl tosylates as substrates.

PubMed

Omar, Mohamed A; Frey, Wolfgang; Conrad, Jürgen; Beifuss, Uwe

2014-11-07

A transition-metal-free route for the synthesis of several N-fused heterocycles, including thiazolo[3,2-a]benzimidazoles and imidazo[2,1-b]thiazoles, is reported. The reaction between propargyl tosylates and 2-mercaptobenzimidazoles under basic conditions results in 3-substituted thiazolo[3,2-a]benzimidazoles, in yields up to 92% in a single synthesis step. With 2-mercaptoimidazoles as the substrate, the corresponding imidazo[2,1-b]thiazoles were exclusively obtained. The transformation is considered to proceed as an intermolecular S-propargylation that is followed by 5-exo-dig ring closure and double-bond isomerization.

From Structure-Function Analyses to Protein Engineering for Practical Applications of DNA Ligase

PubMed Central

Tanabe, Maiko; Nishida, Hirokazu

2015-01-01

DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures. PMID:26508902

RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing.

PubMed

Chang, Lun-Ching; Das, Biswajit; Lih, Chih-Jian; Si, Han; Camalier, Corinne E; McGregor, Paul M; Polley, Eric

2016-01-01

With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96-0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.

Involvement of α-, γ- and δ-Tocopherol Isomers from
Pumpkin (Cucurbita pepo L.) Seed Oil or Oil Mixtures in
the Biphasic DPPH˙ Disappearance Kinetics

PubMed Central

Broznić, Dalibor; Milin, Čedomila

2016-01-01

Summary The antioxidant activity of three types of pumpkin seed oil or oil mixtures (cold- -pressed, produced from roasted seed paste and salad) produced in the northern part of Croatia and the kinetics of their behaviour as free radical scavengers were investigated using DPPH˙. In addition, the involvement of oil tocopherol isomers (α-, γ- and δ-) in different steps of DPPH˙ disappearance and their impact on the rate of reaction were analysed. The kinetics of DPPH˙ disappearance is a two-step process. In the first step, rapid disappearance of DPPH˙ occurs during the first 11 min of the reaction, depending on the oil type, followed by a slower decline in the second step. To describe DPPH˙ disappearance kinetics, six mathematical models (mono- and biphasic) were tested. Our findings showed that γ- and δ-tocopherols affected DPPH˙ disappearance during the first step, and α-tocopherol in the second step of the reaction. Moreover, α-tocopherol demonstrated 30 times higher antioxidant activity than γ- and δ-tocopherols. The results indicated the biphasic double-exponential behaviour of DPPH˙ disappearance in oil samples, due to the complexity of reactions that involve different tocopherol isomers and proceed through different chemical pathways. PMID:27904410

Involvement of α-, γ- and δ-Tocopherol Isomers from
Pumpkin (Cucurbita pepo L.) Seed Oil or Oil Mixtures in
the Biphasic DPPH˙ Disappearance Kinetics.

PubMed

Broznić, Dalibor; Jurešić, Gordana Čanadi; Milin, Čedomila

2016-06-01

The antioxidant activity of three types of pumpkin seed oil or oil mixtures (cold- -pressed, produced from roasted seed paste and salad) produced in the northern part of Croatia and the kinetics of their behaviour as free radical scavengers were investigated using DPPH˙. In addition, the involvement of oil tocopherol isomers (α-, γ- and δ-) in different steps of DPPH˙ disappearance and their impact on the rate of reaction were analysed. The kinetics of DPPH˙ disappearance is a two-step process. In the first step, rapid disappearance of DPPH˙ occurs during the first 11 min of the reaction, depending on the oil type, followed by a slower decline in the second step. To describe DPPH˙ disappearance kinetics, six mathematical models (mono- and biphasic) were tested. Our findings showed that γ- and δ-tocopherols affected DPPH˙ disappearance during the first step, and α-tocopherol in the second step of the reaction. Moreover, α-tocopherol demonstrated 30 times higher antioxidant activity than γ- and δ-tocopherols. The results indicated the biphasic double-exponential behaviour of DPPH˙ disappearance in oil samples, due to the complexity of reactions that involve different tocopherol isomers and proceed through different chemical pathways.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

PubMed

Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

2018-05-15

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase - The importance of accommodating the active site water.

PubMed

Heyes, Logan C; Reichau, Sebastian; Cross, Penelope J; Jameson, Geoffrey B; Parker, Emily J

2014-12-01

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9±0.4μM. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated. Copyright © 2014 Elsevier Inc. All rights reserved.

Rapid detection of duck hepatitis A virus genotype C using reverse transcription loop-mediated isothermal amplification.

PubMed

Li, Chuanfeng; Chen, Zongyan; Meng, Chunchun; Liu, Guangqing

2014-02-01

A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was used and optimized to develop a rapid and sensitive detection system for duck hepatitis A virus genotype C (DHAV-C) RNA. A set of four specific primers was designed against highly conserved sequences located within the 3D gene from DHAV (strain GX1201). Under optimal reaction conditions, the sensitivity of DHAV-C-specific RT-LAMP was 100-fold higher than that of reverse transcriptase-polymerase chain reaction (RT-PCR), with a detection limit of 0.3pg (6.59×10(4) copies) per reaction. No cross-reactivity was observed from the samples of other duck viruses, which is in good accordance with RT-PCR. Furthermore, a positive reaction can be visually inspected by observing turbidity or color change after the addition of SYBR green I dye. The DHAV-C-specific RT-LAMP assay was applied to the samples and compared with RT-PCR. The positive-sample ratios were 26.7% (12 of 45) by RT-LAMP and 20% (9 of 45) by RT-PCR. Therefore, the newly developed RT-LAMP assay is a rapid, specific, sensitive, and cost-effective method of DHAV-C detection. This assay has potential applications in both clinical diagnosis and field surveillance of DHAV-C infection. Copyright © 2013. Published by Elsevier B.V.

Methane utilization in Methylomicrobium alcaliphilum 20Z R: a systems approach

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

Akberdin, Ilya R.; Thompson, Merlin; Hamilton, Richard

Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20Z R an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization inmore » M. alcaliphilum 20Z R. A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. As a result, the computational framework of C 1-metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.« less

Methane utilization in Methylomicrobium alcaliphilum 20Z R: a systems approach

DOE PAGES

Akberdin, Ilya R.; Thompson, Merlin; Hamilton, Richard; ...

2018-02-06

Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20Z R an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization inmore » M. alcaliphilum 20Z R. A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. As a result, the computational framework of C 1-metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.« less

Multiplex polymerase chain reaction-capillary gel electrophoresis: a promising tool for GMO screening--assay for simultaneous detection of five genetically modified cotton events and species.

PubMed

Nadal, Anna; Esteve, Teresa; Pla, Maria

2009-01-01

A multiplex polymerase chain reaction assay coupled to capillary gel electrophoresis for amplicon identification by size and color (multiplex PCR-CGE-SC) was developed for simultaneous detection of cotton species and 5 events of genetically modified (GM) cotton. Validated real-time-PCR reactions targeting Bollgard, Bollgard II, Roundup Ready, 3006-210-23, and 281-24-236 junction sequences, and the cotton reference gene acp1 were adapted to detect more than half of the European Union-approved individual or stacked GM cotton events in one reaction. The assay was fully specific (<1.7% of false classification rate), with limit of detection values of 0.1% for each event, which were also achieved with simulated mixtures at different relative percentages of targets. The assay was further combined with a second multiplex PCR-CGE-SC assay to allow simultaneous detection of 6 cotton and 5 maize targets (two endogenous genes and 9 GM events) in two multiplex PCRs and a single CGE, making the approach more economic. Besides allowing simultaneous detection of many targets with adequate specificity and sensitivity, the multiplex PCR-CGE-SC approach has high throughput and automation capabilities, while keeping a very simple protocol, e.g., amplification and labeling in one step. Thus, it is an easy and inexpensive tool for initial screening, to be complemented with quantitative assays if necessary.

Heat damaged forages: effects on forage energy content

USDA-ARS?s Scientific Manuscript database

Traditionally, educational materials describing the effects of heat damage within baled hays have focused on reduced bioavailability of crude protein as a result of Maillard reactions. These reactions are not simple, but actually occur in complex, multi-step pathways. Typically, the initial step inv...

The effect of different screw-tightening techniques on the stress generated on an internal-connection implant superstructure.

PubMed

Choi, Jung-Han; Lim, Young-Jun; Kim, Chang-Whe; Kim, Myung-Joo

2009-01-01

This study evaluated the effect of different screw-tightening sequences, forces, and methods on the stresses generated on a well-fitting internal-connection implant (Astra Tech) superstructure. A metal framework directly connected to four parallel implants was fabricated on a fully edentulous mandibular resin model. Six stone casts with four implant replicas were made from a pickup impression of the superstructure to represent a "well-fitting" situation. Stresses generated by four screw-tightening sequences (1-2-3-4, 4-3-2-1, 2-4-3-1, and 2-3-1-4), two forces (10 and 20 Ncm), and two methods (one-step and two-step) were evaluated. In the two-step method, screws were tightened to the initial torque (10 Ncm) in a predetermined screw-tightening sequence and then to the final torque (20 Ncm) in the same sequence. Stresses were recorded twice by three strain gauges attached to the framework (superior face midway between abutments). Deformation data were analyzed using multiple analysis of variance at a .05 level of statistical significance. In all stone casts, stresses were produced by the superstructure connection, regardless of screw-tightening sequence, force, and method. No statistically significant differences for superstructure preload stresses were found based on screw-tightening sequences (-180.0 to -181.6 microm/m) or forces (-163.4 and -169.2 microm/m) (P > .05). However, different screw-tightening methods induced different stresses on the superstructure. The two-step screw-tightening method (-180.1 microm/m) produced significantly higher stress than the one-step method (-169.2 microm/m) (P = .0457). Within the limitations of this in vitro study, screw-tightening sequence and force were not critical factors in the stress generated on a well-fitting internal-connection implant superstructure. The stress caused by the two-step method was greater than that produced using the one-step method. Further studies are needed to evaluate the effect of screw-tightening techniques on preload stress in various different clinical situations.

Molecular variation and horizontal gene transfer of the homocysteine methyltransferase gene mmuM and its distribution in clinical pathogens.

PubMed

Ying, Jianchao; Wang, Huifeng; Bao, Bokan; Zhang, Ying; Zhang, Jinfang; Zhang, Cheng; Li, Aifang; Lu, Junwan; Li, Peizhen; Ying, Jun; Liu, Qi; Xu, Teng; Yi, Huiguang; Li, Jinsong; Zhou, Li; Zhou, Tieli; Xu, Zuyuan; Ni, Liyan; Bao, Qiyu

2015-01-01

The homocysteine methyltransferase encoded by mmuM is widely distributed among microbial organisms. It is the key enzyme that catalyzes the last step in methionine biosynthesis and plays an important role in the metabolism process. It also enables the microbial organisms to tolerate high concentrations of selenium in the environment. In this research, 533 mmuM gene sequences covering 70 genera of the bacteria were selected from GenBank database. The distribution frequency of mmuM is different in the investigated genera of bacteria. The mapping results of 160 mmuM reference sequences showed that the mmuM genes were found in 7 species of pathogen genomes sequenced in this work. The polymerase chain reaction products of one mmuM genotype (NC_013951 as the reference) were sequenced and the sequencing results confirmed the mapping results. Furthermore, 144 representative sequences were chosen for phylogenetic analysis and some mmuM genes from totally different genera (such as the genes between Escherichia and Klebsiella and between Enterobacter and Kosakonia) shared closer phylogenetic relationship than those from the same genus. Comparative genomic analysis of the mmuM encoding regions on plasmids and bacterial chromosomes showed that pKF3-140 and pIP1206 plasmids shared a 21 kb homology region and a 4.9 kb fragment in this region was in fact originated from the Escherichia coli chromosome. These results further suggested that mmuM gene did go through the gene horizontal transfer among different species or genera of bacteria. High-throughput sequencing combined with comparative genomics analysis would explore distribution and dissemination of the mmuM gene among bacteria and its evolution at a molecular level.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

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

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Generation of RNA in abiotic conditions.

NASA Astrophysics Data System (ADS)

di Mauro, Ernesto

Generation of RNA in abiotic conditions. Ernesto Di Mauro Dipartimento di Genetica Bi-ologia Molecolare, Universit` "Sapienza" Roma, Italy. a At least four conditions must be satisfied for the spontaneous generation of (pre)-genetic poly-mers: 1) availability of precursors that are activated enough to spontaneously polymerize. Preliminary studies showed that (a) nucleic bases and acyclonucleosides can be synthesized from formamide H2NCOH by simply heating with prebiotically available mineral catalysts [last reviewed in (1)], and that b) nucleic bases can be phosphorylated in every possible posi-tion [2'; 3'; 5'; cyclic 2',3'; cyclic 3',5' (2)]. The higher stability of the cyclic forms allows their accumulation. 2) A polymerization mechanism. A reaction showing the formation of RNA polymers starting from prebiotically plausible precursors (3',5' cyclic GMP and 3', 5'cyclic AMP) was recently reported (3). Polymerization in these conditions is thermodynamically up-hill and an equilibrium is attained that limits the maximum length of the polymer produced to about 40 nucleotides for polyG and 100 nucleotides for polyA. 3) Ligation of the synthesized oligomers. If this type of reaction could occur according to a terminal-joining mechanism and could generate canonical 3',5' phosphodiester bonds, exponential growth would be obtained of the generated oligomers. This type of reaction has been reported (4) , limited to homogeneous polyA sequences and leading to the production of polyA dimers and tetramers. What is still missing are: 4) mechanisms that provide the proof of principle for the generation of sequence complexity. We will show evidence for two mechanisms providing this proof of principle for simple complementary sequences. Namely: abiotic sequence complementary-driven terminal ligation and sequence-complementary terminal growth. In conclusion: all the steps leading to the generation of RNA in abiotic conditions are satisfied. (1) R Saladino, C Crestini, F. Ciciriello, S. Pino, G. Costanzo, E. Di Mauro. From formamide to RNA: the roles of formamide and water in the evolution of chemical information. Research In Microbiology, Special Issue on The Origin of life and Microbiology (2009) 160:441-448. (2) Costanzo, G., Saladino, R., Crestini, C., Ciciriello, F., and Di Mauro, E. Nucleoside phos-phorylation by phosphate minerals. J. Biol. Chem. (2007) 282: 16729-16735. (3) Samanta Pino, Fabiana Ciciriello, Giovanna Costanzo and E. Di Mauro, Nonenzymatic RNA Ligation in Water J. Biol. Chem. (2008), 283: 36494-36503 (4) Costanzo, G., Pino, S., Ciciriello, F., and Di Mauro, E. Generation of long RNA chains in water. J Biol Chem. (2009) 284:33206-33216.

Kinetic concepts of thermally stimulated reactions in solids

NASA Astrophysics Data System (ADS)

Vyazovkin, Sergey

Historical analysis suggests that the basic kinetic concepts of reactions in solids were inherited from homogeneous kinetics. These concepts rest upon the assumption of a single-step reaction that disagrees with the multiple-step nature of solid-state processes. The inadequate concepts inspire such unjustified anticipations of kinetic analysis as evaluating constant activation energy and/or deriving a single-step reaction mechanism for the overall process. A more adequate concept is that of the effective activation energy, which may vary with temperature and extent of conversion. The adequacy of this concept is illustrated by literature data as well as by experimental data on the thermal dehydration of calcium oxalate monohydrate and thermal decomposition of calcium carbonate, ammonium nitrate and 1,3,5,7- tetranitro-1,3,5,7-tetrazocine.

Array automated assembly task low cost silicon solar array project, phase 2

NASA Technical Reports Server (NTRS)

Olson, C.

1980-01-01

Analyses of solar cell and module process steps for throughput rate, cost effectiveness, and reproductibility are reported. In addition to the concentration on cell and module processing sequences, an investigation was made into the capability of using microwave energy in the diffusion, sintering, and thick film firing steps of cell processing. Although the entire process sequence was integrated, the steps are treated individually with test and experimental data, conclusions, and recommendations.

Comparing the efficacy of metronome beeps and stepping stones to adjust gait: steps to follow!

PubMed

Bank, Paulina J M; Roerdink, Melvyn; Peper, C E

2011-03-01

Acoustic metronomes and visual targets have been used in rehabilitation practice to improve pathological gait. In addition, they may be instrumental in evaluating and training instantaneous gait adjustments. The aim of this study was to compare the efficacy of two cue types in inducing gait adjustments, viz. acoustic temporal cues in the form of metronome beeps and visual spatial cues in the form of projected stepping stones. Twenty healthy elderly (aged 63.2 ± 3.6 years) were recruited to walk on an instrumented treadmill at preferred speed and cadence, paced by either metronome beeps or projected stepping stones. Gait adaptations were induced using two manipulations: by perturbing the sequence of cues and by imposing switches from one cueing type to the other. Responses to these manipulations were quantified in terms of step-length and step-time adjustments, the percentage correction achieved over subsequent steps, and the number of steps required to restore the relation between gait and the beeps or stepping stones. The results showed that perturbations in a sequence of stepping stones were overcome faster than those in a sequence of metronome beeps. In switching trials, switching from metronome beeps to stepping stones was achieved faster than vice versa, indicating that gait was influenced more strongly by the stepping stones than the metronome beeps. Together these results revealed that, in healthy elderly, the stepping stones induced gait adjustments more effectively than did the metronome beeps. Potential implications for the use of metronome beeps and stepping stones in gait rehabilitation practice are discussed.

Phylogenetic tree of 16s rRNA sequences from sulfate-reducing bacteria in a sandy marine sediment

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

Devereux, R.; Mundfrom, G.W.

1994-01-01

Phylogenetic divergence among sulfate-reducing bateria in an estuarine sediment sample was investigated by PCR amplification and comparison of partial 16S rDNA sequences. Twenty unique 16S rDNA sequences were found, 12 from delta subclass bacteria based on overall sequence similarity (82-91%). Two successive PCR amplifications were used to obtain and clone the 16S rDNA. The first reaction used templates derived from phosphate-buffered saline washed sediment with primers designed to amplify nearly full-length bacterial domain 16S rDNA. A produce from a first reaction was used as template in a second reaction with primers designed to selectivity amplify a region of 16S rDNAmore » genes of sulfate-reducing bacteria. A phylogenetic tree incorporating the cloned sequences suggests the presence of yet to be cultivated lines of sulfate-reducing bacteria within the sediment sample.« less

A characterization of the two-step reaction mechanism of phenol decomposition by a Fenton reaction

NASA Astrophysics Data System (ADS)

Valdés, Cristian; Alzate-Morales, Jans; Osorio, Edison; Villaseñor, Jorge; Navarro-Retamal, Carlos

2015-11-01

Phenol is one of the worst contaminants at date, and its degradation has been a crucial task over years. Here, the decomposition process of phenol, in a Fenton reaction, is described. Using scavengers, it was observed that decomposition of phenol was mainly influenced by production of hydroxyl radicals. Experimental and theoretical activation energies (Ea) for phenol oxidation intermediates were calculated. According to these Ea, phenol decomposition is a two-step reaction mechanism mediated predominantly by hydroxyl radicals, producing a decomposition yield order given as hydroquinone > catechol > resorcinol. Furthermore, traces of reaction derived acids were detected by HPLC and GS-MS.

Unraveling the role of protein dynamics in dihydrofolate reductase catalysis

PubMed Central

Luk, Louis Y. P.; Javier Ruiz-Pernía, J.; Dawson, William M.; Roca, Maite; Loveridge, E. Joel; Glowacki, David R.; Harvey, Jeremy N.; Mulholland, Adrian J.; Tuñón, Iñaki; Moliner, Vicent; Allemann, Rudolf K.

2013-01-01

Protein dynamics have controversially been proposed to be at the heart of enzyme catalysis, but identification and analysis of dynamical effects in enzyme-catalyzed reactions have proved very challenging. Here, we tackle this question by comparing an enzyme with its heavy (15N, 13C, 2H substituted) counterpart, providing a subtle probe of dynamics. The crucial hydride transfer step of the reaction (the chemical step) occurs more slowly in the heavy enzyme. A combination of experimental results, quantum mechanics/molecular mechanics simulations, and theoretical analyses identify the origins of the observed differences in reactivity. The generally slightly slower reaction in the heavy enzyme reflects differences in environmental coupling to the hydride transfer step. Importantly, the barrier and contribution of quantum tunneling are not affected, indicating no significant role for “promoting motions” in driving tunneling or modulating the barrier. The chemical step is slower in the heavy enzyme because protein motions coupled to the reaction coordinate are slower. The fact that the heavy enzyme is only slightly less active than its light counterpart shows that protein dynamics have a small, but measurable, effect on the chemical reaction rate. PMID:24065822

One-pot, two-step synthesis of imidazo[1,2-a]benzimidazoles via a multicomponent [4 + 1] cycloaddition reaction.

PubMed

Hsiao, Ya-Shan; Narhe, Bharat D; Chang, Ying-Sheng; Sun, Chung-Ming

2013-10-14

A one-pot, two-step synthesis of imidazo[1,2-a]benzimidazoles has been achieved by a three-component reaction of 2-aminobenzimidazoles with an aromatic aldehyde and an isocyanide. The reaction involving condensation of 2-aminobenzimidazole with an aldehyde is run under microwave activation to generate an imine intermediate under basic conditions which then undergoes [4 + 1] cycloaddition with an isocyanide.

Can use of walkers or canes impede lateral compensatory stepping movements?

PubMed

Bateni, Hamid; Heung, Evelyn; Zettel, John; McLlroy, William E; Maki, Brian E

2004-08-01

Although assistive devices, such as walkers and canes are often prescribed to aid in balance control, recent studies have suggested that such devices may actually increase risk of falling. In this study, we investigated one possible mechanism: the potential for walkers or canes to interfere with, or constrain, lateral movement of the feet and thereby impede execution of compensatory stepping reactions during lateral loss of balance. Lateral stepping reactions were evoked, in 10 healthy young adults (ages 22-27 years), by means of sudden unpredictable medio-lateral support surface translation. Subjects were tested while holding and loading a standard pickup walker or single-tip cane or while using no assistive device (hands free or holding an object). Results supported the hypothesis that using a walker or cane can interfere with compensatory stepping. Collisions between the swing-foot and mobility aid were remarkably frequent when using the walker (60% of stepping reactions) and also occurred in cane trials (11% of stepping reactions). Furthermore, such collisions were associated with a significant reduction (26-37%) in lateral step length. It appeared that subjects were sometimes able to avoid collision by increasing the forward or backward displacement of the swing-foot or by moving the cane; however, attempts to lift the walker out of the way occurred rarely and were usually impeded due to collision between the contralateral walker post and stance foot. The fact that compensatory stepping behavior was altered significantly in such a healthy cohort clearly demonstrates some of the safety limitations inherent to these assistive devices, as currently designed. Copyright 2003 Elsevier B.V.

Explore the reaction mechanism of the Maillard reaction: a density functional theory study.

PubMed

Ren, Ge-Rui; Zhao, Li-Jiang; Sun, Qiang; Xie, Hu-Jun; Lei, Qun-Fang; Fang, Wen-Jun

2015-05-01

The mechanism of Maillard reaction has been investigated by means of density functional theory calculations in the gaseous phase and aqueous solution. The Maillard reaction is a cascade of consecutive and parallel reaction. In the present model system study, glucose and glycine were taken as the initial reactants. On the basis of previous experimental results, the mechanisms of Maillard reaction have been proposed, and the possibility for the formation of different compounds have been evaluated through calculating the relative energy changes for different steps of reaction under different pH conditions. Our calculations reveal that the TS3 in Amadori rearrangement reaction is the rate-determining step of Maillard reaction with the activation barriers of about 66.7 and 68.8 kcal mol(-1) in the gaseous phase and aqueous solution, respectively. The calculation results are in good agreement with previous studies and could provide insights into the reaction mechanism of Maillard reaction, since experimental evaluation of the role of intermediates in the Maillard reaction is quite complicated.

Kinetic Modelling and Experimental Studies for the Effects of Fe 2+ Ions on Xylan Hydrolysis with Dilute-Acid Pretreatment and Subsequent Enzymatic Hydrolysis

DOE PAGES

Wei, Hui; Chen, Xiaowen; Shekiro, Joseph; ...

2018-01-20

High-temperature (150-170 degrees C) pretreatment of lignocellulosic biomass with mineral acids is well established for xylan breakdown. Fe 2+ is known to be a cocatalyst of this process although kinetics of its action remains unknown. The present work addresses the effect of ferrous ion concentration on sugar yield and degradation product formation from corn stover for the entire two-step treatment, including the subsequent enzymatic cellulose hydrolysis. The feedstock was impregnated with 0.5% acid and 0.75 mM iron cocatalyst, which was found to be optimal in preliminary experiments. The detailed kinetic data of acid pretreatment, with and without iron, was satisfactorilymore » modelled with a four-step linear sequence of first-order irreversible reactions accounting for the formation of xylooligomers, xylose and furfural as intermediates to provide the values of Arrhenius activation energy. Based on this kinetic modelling, Fe 2+ turned out to accelerate all four reactions, with a significant alteration of the last two steps, that is, xylose degradation. Consistent with this model, the greatest xylan conversion occurred at the highest severity tested under 170 ⁰C/30 min with 0.75 mM Fe 2+, with a total of 8% xylan remaining in the pretreated solids, whereas the operational conditions leading to the highest xylose monomer yield, 63%, were milder, 150 degrees C with 0.75 mM Fe 2+ for 20 min. Furthermore, the subsequent enzymatic hydrolysis with the prior addition of 0.75 mM of iron(II) increased the glucose production to 56.3% from 46.3% in the control (iron-free acid). The detailed analysis indicated that conducting the process at lower temperatures yet long residence times benefits the yield of sugars. The above kinetic modelling results of Fe 2+ accelerating all four reactions are in line with our previous mechanistic research showing that the pretreatment likely targets multiple chemistries in plant cell wall polymer networks, including those represented by the C-O-C and C-H bonds in cellulose, resulting in enhanced sugar solubilization and digestibility.« less

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations.

PubMed

Hashimoto, Masahiko; Barany, Francis; Xu, Feng; Soper, Steven A

2007-09-01

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.

Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes.

PubMed

Cooper, A; Converse, C A

1976-07-13

A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.

Kinetic Modelling and Experimental Studies for the Effects of Fe 2+ Ions on Xylan Hydrolysis with Dilute-Acid Pretreatment and Subsequent Enzymatic Hydrolysis

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

Wei, Hui; Chen, Xiaowen; Shekiro, Joseph

High-temperature (150-170 degrees C) pretreatment of lignocellulosic biomass with mineral acids is well established for xylan breakdown. Fe 2+ is known to be a cocatalyst of this process although kinetics of its action remains unknown. The present work addresses the effect of ferrous ion concentration on sugar yield and degradation product formation from corn stover for the entire two-step treatment, including the subsequent enzymatic cellulose hydrolysis. The feedstock was impregnated with 0.5% acid and 0.75 mM iron cocatalyst, which was found to be optimal in preliminary experiments. The detailed kinetic data of acid pretreatment, with and without iron, was satisfactorilymore » modelled with a four-step linear sequence of first-order irreversible reactions accounting for the formation of xylooligomers, xylose and furfural as intermediates to provide the values of Arrhenius activation energy. Based on this kinetic modelling, Fe 2+ turned out to accelerate all four reactions, with a significant alteration of the last two steps, that is, xylose degradation. Consistent with this model, the greatest xylan conversion occurred at the highest severity tested under 170 ⁰C/30 min with 0.75 mM Fe 2+, with a total of 8% xylan remaining in the pretreated solids, whereas the operational conditions leading to the highest xylose monomer yield, 63%, were milder, 150 degrees C with 0.75 mM Fe 2+ for 20 min. Furthermore, the subsequent enzymatic hydrolysis with the prior addition of 0.75 mM of iron(II) increased the glucose production to 56.3% from 46.3% in the control (iron-free acid). The detailed analysis indicated that conducting the process at lower temperatures yet long residence times benefits the yield of sugars. The above kinetic modelling results of Fe 2+ accelerating all four reactions are in line with our previous mechanistic research showing that the pretreatment likely targets multiple chemistries in plant cell wall polymer networks, including those represented by the C-O-C and C-H bonds in cellulose, resulting in enhanced sugar solubilization and digestibility.« less

Immobilised enzyme microreactor for screening of multi-step bioconversions: characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols.

PubMed

Matosevic, S; Lye, G J; Baganz, F

2011-09-20

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His₆-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to L-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of L-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation. Copyright © 2011 Elsevier B.V. All rights reserved.

From Sequences to Insights in Microbial Ecology

PubMed Central

Knight, R.

2010-01-01

s4-3 Rapid declines in the cost of sequencing have made large volumes of DNA sequence data available to individual investigators. Now, data analysis is the rate-limiting step: providing a user with sequences alone typically leads to bewilderment, frustration, and skepticism about the technology. In this talk, I focus on how to extract insights from 16S rRNA data, including key lab steps (barcoding and normalization) and on which tools are available to perform routine but essential processing steps such as denoising, chimera detection, taxonomy assignment, and diversity analyses (including detection of biological clusters and gradients in the samples). Providing users with advice on these points and with a standard pipeline they can exploit (but modify if circumstances require) can greatly accelerate the rate of understanding, publication, and acquisition of funding for further studies.

Surfactant-controlled polymerization of semiconductor clusters to quantum dots through competing step-growth and living chain-growth mechanisms.

PubMed

Evans, Christopher M; Love, Alyssa M; Weiss, Emily A

2012-10-17

This article reports control of the competition between step-growth and living chain-growth polymerization mechanisms in the formation of cadmium chalcogenide colloidal quantum dots (QDs) from CdSe(S) clusters by varying the concentration of anionic surfactant in the synthetic reaction mixture. The growth of the particles proceeds by step-addition from initially nucleated clusters in the absence of excess phosphinic or carboxylic acids, which adsorb as their anionic conjugate bases, and proceeds indirectly by dissolution of clusters, and subsequent chain-addition of monomers to stable clusters (Ostwald ripening) in the presence of excess phosphinic or carboxylic acid. Fusion of clusters by step-growth polymerization is an explanation for the consistent observation of so-called "magic-sized" clusters in QD growth reactions. Living chain-addition (chain addition with no explicit termination step) produces QDs over a larger range of sizes with better size dispersity than step-addition. Tuning the molar ratio of surfactant to Se(2-)(S(2-)), the limiting ionic reagent, within the living chain-addition polymerization allows for stoichiometric control of QD radius without relying on reaction time.

Two-step carbon coating of lithium vanadium phosphate as high-rate cathode for lithium-ion batteries

NASA Astrophysics Data System (ADS)

Kuang, Quan; Zhao, Yanming

2012-10-01

Carbon-coated Li3V2(PO4)3 was firstly prepared at 850 °C via two-step reaction method combined sol-gel and conventional solid-state synthesis by using VPO4/carbon as an intermediate. Two different carbon sources, citric acid and glucose as carbon additives in sequence, ultimately deduced double carbon-coated Li3V2(PO4)3 as a high-rate cathode material. The Li3V2(PO4)3/carbon with 4.39% residual carbon has a splendid electronic conductivity of 4.76×10-2 S cm-1. Even in the voltage window of 2.5-4.8 V, the Li3V2(PO4)3/carbon cathode can retain outstanding rate ability (170.4 mAh g-1 at 1.2 C, 101.9 mAh g-1 at 17 C), and no degradation is found after 120 C current rate. These phenomena show that the two-step carbon-coated Li3V2(PO4)3 can act as a fast charge-discharge cathode material for high-power Li-ion batteries. Furthermore, it's believed that this synthesize method can be easily transplanted to prepare other lithiated vanadium-based phosphates.

Use of ion conductors in the pyrochemical reduction of oxides

DOEpatents

Miller, W.E.; Tomczuk, Z.

1994-02-01

An electrochemical process and electrochemical cell for reducing a metal oxide are provided. First the oxide is separated as oxygen gas using, for example, a ZrO[sub 2] oxygen ion conductor anode and the metal ions from the reduction salt are reduced and deposited on an ion conductor cathode, for example, sodium ion reduced on a [beta]-alumina sodium ion conductor cathode. The generation of and separation of oxygen gas avoids the problem with chemical back reaction of oxygen with active metals in the cell. The method also is characterized by a sequence of two steps where an inert cathode electrode is inserted into the electrochemical cell in the second step and the metallic component in the ion conductor is then used as the anode to cause electrochemical reduction of the metal ions formed in the first step from the metal oxide where oxygen gas formed at the anode. The use of ion conductors serves to isolate the active components from chemically reacting with certain chemicals in the cell. While applicable to a variety of metal oxides, the invention has special importance for reducing CaO to Ca[sup o] used for reducing UO[sub 2] and PuO[sub 2] to U and Pu. 2 figures.

Mobile Genome Express (MGE): A comprehensive automatic genetic analyses pipeline with a mobile device.

PubMed

Yoon, Jun-Hee; Kim, Thomas W; Mendez, Pedro; Jablons, David M; Kim, Il-Jin

2017-01-01

The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or genome. However, data analysis is still the biggest bottleneck for its wide implementation. Most laboratories still depend on manual procedures for data handling and analyses, which translates into a delay and decreased efficiency in the delivery of NGS results to doctors and patients. Thus, there is high demand for developing an automatic and an easy-to-use NGS data analyses system. We developed comprehensive, automatic genetic analyses controller named Mobile Genome Express (MGE) that works in smartphones or other mobile devices. MGE can handle all the steps for genetic analyses, such as: sample information submission, sequencing run quality check from the sequencer, secured data transfer and results review. We sequenced an Actrometrix control DNA containing multiple proven human mutations using a targeted sequencing panel, and the whole analysis was managed by MGE, and its data reviewing program called ELECTRO. All steps were processed automatically except for the final sequencing review procedure with ELECTRO to confirm mutations. The data analysis process was completed within several hours. We confirmed the mutations that we have identified were consistent with our previous results obtained by using multi-step, manual pipelines.

Save the last dance for me: unwanted serial position effects in jury evaluations.

PubMed

Bruine de Bruin, Wändi

2005-03-01

Whenever competing options are considered in sequence, their evaluations may be affected by order of appearance. Such serial position effects would threaten the fairness of competitions using jury evaluations. Randomization cannot reduce potential order effects, but it does give candidates an equal chance of being assigned to preferred serial positions. Whether, or what, serial position effects emerge may depend on the cognitive demands of the judgment task. In end-of-sequence procedures, final scores are not given until all candidates have performed, possibly burdening judges' memory. If judges' evaluations are based on how well they remember performances, serial position effects may resemble those found with free recall. Candidates may also be evaluated step-by-step, immediately after each performance. This procedure should not burden memory, though it may produce different serial position effects. Yet, this paper reports similar serial position effects with end-of-sequence and step-by-step procedures used for the Eurovision Song Contest: Ratings increased with serial position. The linear order effect was replicated in the step-by-step judgments of World and European Figure Skating Contests. It is proposed that, independent of the evaluation procedure, judges' initial impressions of sequentially appearing candidates may be formed step-by-step, yielding serial position effects.

Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection.

PubMed

Dash, Paban Kumar; Parida, Manmohan; Santhosh, S R; Saxena, Parag; Srivastava, Ambuj; Neeraja, Mamidi; Lakshmi, V; Rao, P V Lakshmana

2008-09-01

Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.

Detection of Papaya leaf distortion mosaic virus by reverse-transcription loop-mediated isothermal amplification.

PubMed

Shen, Wentao; Tuo, Decai; Yan, Pu; Li, Xiaoying; Zhou, Peng

2014-01-01

Papaya leaf distortion mosaic virus (PLDMV) can infect transgenic papaya resistant to a related pathogen, Papaya ringspot virus (PRSV), posing a substantial threat to papaya production in China. Current detection methods, however, are unable to be used for rapid detection in the field. Here, a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of PLDMV, using a set of four RT-LAMP primers designed based on the conserved sequence of PLDMV CP. The RT-LAMP method detected specifically PLDMV and was highly sensitive, with a detection limit of 1.32×10(-6) μg of total RNA per reaction. Indeed, the reaction was 10 times more sensitive than one-step RT-PCR, while also requiring significantly less time and equipment. The effectiveness of RT-LAMP and one-step RT-PCR in detecting the virus were compared using 90 field samples of non-transgenic papaya and 90 field samples of commercialized PRSV-resistant transgenic papaya from Hainan Island. None of the non-transgenic papaya tested positive for PLDMV using either method. In contrast, 19 of the commercialized PRSV-resistant transgenic papaya samples tested positive by RT-LAMP assay, and 6 of those tested negative by RT-PCR. Therefore, the PLDMV-specific RT-LAMP is a simple, rapid, sensitive, and cost-effective tool in the field diagnosis and control of PLDMV. Copyright © 2013 Elsevier B.V. All rights reserved.

Efficient Green's Function Reaction Dynamics (GFRD) simulations for diffusion-limited, reversible reactions

NASA Astrophysics Data System (ADS)

Bashardanesh, Zahedeh; Lötstedt, Per

2018-03-01

In diffusion controlled reversible bimolecular reactions in three dimensions, a dissociation step is typically followed by multiple, rapid re-association steps slowing down the simulations of such systems. In order to improve the efficiency, we first derive an exact Green's function describing the rate at which an isolated pair of particles undergoing reversible bimolecular reactions and unimolecular decay separates beyond an arbitrarily chosen distance. Then the Green's function is used in an algorithm for particle-based stochastic reaction-diffusion simulations for prediction of the dynamics of biochemical networks. The accuracy and efficiency of the algorithm are evaluated using a reversible reaction and a push-pull chemical network. The computational work is independent of the rates of the re-associations.

Domino reactions initiated by intramolecular hydride transfers from tri(di)arylmethane fragments to ketenimine and carbodiimide functions.

PubMed

Alajarin, Mateo; Bonillo, Baltasar; Ortin, Maria-Mar; Sanchez-Andrada, Pilar; Vidal, Angel; Orenes, Raul-Angel

2010-10-21

The ability of triarylmethane and diarylmethane fragments to behave as hydride donors participating in thermal [1,5]-H shift/6π-ERC tandem processes involving ketenimine and carbodiimide functions is disclosed. C-Alkyl-C-phenyl ketenimines N-substituted by a triarylmethane substructure convert into a variety of 3,3,4,4-tetrasubstituted-3,4-dihydroquinolines, as structurally related carbodiimides transform into 3,4,4-trisubstituted-3,4-dihydroquinazolines via transient ortho-azaxylylenes. The first step of these one-pot conversions, the [1,5]-H shift, is considered to be a hydride migration on the basis of the known hydricity of the tri(di)arylmethane fragment and the electrophilicity of the central heterocumulenic carbon atom, whereas the final electrocyclization involves the formation of a sterically congested C-C or C-N bond. In the cases of C,C-diphenyl substituted triarylmethane-ketenimines the usual 6π-ERC becomes prohibited by the presence of two phenyl rings at each end of the azatrienic system. This situation opens new reaction channels: (a) following the initial hydride shift, the tandem sequence continues with an alternative electrocyclization mode to give 9,10-dihydroacridines, (b) the full sequence is initiated by a rare 1,5 migration of an electron-rich aryl group, followed by a 6π-ERC which leads to 2-aryl-3,4-dihydroquinolines, or (c) a different [1,5]-H shift/6π-ERC sequence involving the initial migration of a hydrogen atom from a methyl group at the ortho position to the nitrogen atom of the ketenimine function. Diarylmethane-ketenimines bearing a methyl group at the benzylic carbon atom experience a tandem double [1,5]-H shift, the first one being the usual benzylic hydride transfer whereas the second one involves the methyl group at the initial benzylic carbon atom, the reaction products being 2-aminostyrenes. Diarylmethane-ketenimines lacking such a methyl group convert into 3,4-dihydroquinolines by the habitual tandem [1,5]-H shift/6π-ERC processes.

Quantification of anhydride groups in anhydride-based epoxy hardeners by reaction headspace gas chromatography.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2017-06-01

We demonstrate a reaction headspace gas chromatographic method for quantifying anhydride groups in anhydride-based epoxy hardeners. In this method, the conversion process of anhydride groups can be realized by two steps. In the first step, anhydride groups in anhydride-based epoxy hardeners completely reacted with water to form carboxyl groups. In the second step, the carboxyl groups reacted with sodium bicarbonate solution in a closed sample vial. After the complete reaction between the carboxyl groups and sodium bicarbonate, the CO 2 formed from this reaction was then measured by headspace gas chromatography. The data showed that the reaction in the closed headspace vial can be completed in 15 min at 55°C, the relative standard deviation of the reaction headspace gas chromatography method in the precision test was less than 3.94%, the relative differences between the new method and a reference method were no more than 9.38%. The present reaction method is automated, efficient and can be a reliable tool for quantifying the anhydride groups in anhydride-based epoxy hardeners and related research. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two steps hydrothermal growth and characterisations of BaTiO3 films composed of nanowires

NASA Astrophysics Data System (ADS)

Zawawi, Che Zaheerah Najeehah Che Mohd; Salleh, Shahril; Oon Jew, Lee; Tufail Chaudhary, Kashif; Helmi, Mohamad; Safwan Aziz, Muhammad; Haider, Zuhaib; Ali, Jalil

2018-05-01

Barium titanate (BaTiO3) films composed of nanowires have gained considerable research interest due to their lead-free composition and strong energy conversion efficiency. BaTiO3 films can be developed with a simple two steps hydrothermal reactions, which are low cost effective. In this research, BaTiO3 films were fabricated on titanium foil through two steps hydrothermal method namely, the growth of TiO2 and followed by BaTiO3 films. The structural evolutions and the dielectric properties of the films were investigated as well. The structural evolutions of titanium dioxide (TiO2) and BaTiO3 nanowires were characterized using X-ray diffraction and scanning electron microscopy. First step of hydrothermal reaction, TiO2 nanowires were prepared in varied temperatures of 160 °C, 200 °C and 250 °C respectively. Second step of hydrothermal reaction was performed to produce a layer of BaTiO3 films.

Catalysis-Enhancement via Rotary Fluctuation of F1-ATPase

PubMed Central

Watanabe, Rikiya; Hayashi, Kumiko; Ueno, Hiroshi; Noji, Hiroyuki

2013-01-01

Protein conformational fluctuations modulate the catalytic powers of enzymes. The frequency of conformational fluctuations may modulate the catalytic rate at individual reaction steps. In this study, we modulated the rotary fluctuation frequency of F1-ATPase (F1) by attaching probes with different viscous drag coefficients at the rotary shaft of F1. Individual rotation pauses of F1 between rotary steps correspond to the waiting state of a certain elementary reaction step of ATP hydrolysis. This allows us to investigate the impact of the frequency modulation of the rotary fluctuation on the rate of the individual reaction steps by measuring the duration of rotation pauses. Although phosphate release was significantly decelerated, the ATP-binding and hydrolysis steps were less sensitive or insensitive to the viscous drag coefficient of the probe. Brownian dynamics simulation based on a model similar to the Sumi-Marcus theory reproduced the experimental results, providing a theoretical framework for the role of rotational fluctuation in F1 rate enhancement. PMID:24268150

Effect of sustained elevated temperature prior to amplification on template copy number estimation using digital polymerase chain reaction.

PubMed

Bhat, Somanath; McLaughlin, Jacob L H; Emslie, Kerry R

2011-02-21

Digital polymerase chain reaction (dPCR) has the potential to enable accurate quantification of target DNA copy number provided that all target DNA molecules are successfully amplified. Following duplex dPCR analysis from a linear DNA target sequence that contains single copies of two independent template sequences, we have observed that amplification of both templates in a single partition does not always occur. To investigate this finding, we heated the target DNA solution to 95 °C for increasing time intervals and then immediately chilled on ice prior to preparing the dPCR mix. We observed an exponential decline in estimated copy number (R(2)≥ 0.98) of the two template sequences when amplified from either a linearized plasmid or a 388 base pair (bp) amplicon containing the same two template sequences. The distribution of amplifiable templates and the final concentration (copies per µL) were both affected by heat treatment of the samples at 95 °C from 0 s to 30 min. The proportion of target sequences from which only one of the two templates was amplified in a single partition (either 1507 or hmg only) increased over time, while the proportion of target sequences where both templates were amplified (1507 and hmg) in each individual partition declined rapidly from 94% to 52% (plasmid) and 88% to 31% (388 bp amplicon) suggesting an increase in number of targets from which both templates no longer amplify. A 10 min incubation at 95 °C reduced the initial amplifiable template concentration of the plasmid and the 388 bp amplicon by 59% and 91%, respectively. To determine if a similar decrease in amplifiable target occurs during the default pre-activation step of typical PCR amplification protocol, we used mastermixes with a 20 s or 10 min hot-start. The choice of mastermix and consequent pre-activation time did not affect the estimated plasmid concentration. Therefore, we conclude that prolonged exposure of this DNA template to elevated temperatures could lead to significant bias in dPCR measurements. However, care must be taken when designing PCR and non-PCR based experiments by reducing exposure of the DNA template to sustained elevated temperatures in order to improve accuracy in copy number estimation and concentration determination.

Simplified Microarray Technique for Identifying mRNA in Rare Samples

NASA Technical Reports Server (NTRS)

Almeida, Eduardo; Kadambi, Geeta

2007-01-01

Two simplified methods of identifying messenger ribonucleic acid (mRNA), and compact, low-power apparatuses to implement the methods, are at the proof-of-concept stage of development. These methods are related to traditional methods based on hybridization of nucleic acid, but whereas the traditional methods must be practiced in laboratory settings, these methods could be practiced in field settings. Hybridization of nucleic acid is a powerful technique for detection of specific complementary nucleic acid sequences, and is increasingly being used for detection of changes in gene expression in microarrays containing thousands of gene probes. A traditional microarray study entails at least the following six steps: 1. Purification of cellular RNA, 2. Amplification of complementary deoxyribonucleic acid [cDNA] by polymerase chain reaction (PCR), 3. Labeling of cDNA with fluorophores of Cy3 (a green cyanine dye) and Cy5 (a red cyanine dye), 4. Hybridization to a microarray chip, 5. Fluorescence scanning the array(s) with dual excitation wavelengths, and 6. Analysis of the resulting images. This six-step procedure must be performed in a laboratory because it requires bulky equipment.

Deducing the temporal order of cofactor function in ligand-regulated gene transcription: theory and experimental verification.

PubMed

Dougherty, Edward J; Guo, Chunhua; Simons, S Stoney; Chow, Carson C

2012-01-01

Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available.

Deducing the Temporal Order of Cofactor Function in Ligand-Regulated Gene Transcription: Theory and Experimental Verification

PubMed Central

Dougherty, Edward J.; Guo, Chunhua; Simons, S. Stoney; Chow, Carson C.

2012-01-01

Cofactors are intimately involved in steroid-regulated gene expression. Two critical questions are (1) the steps at which cofactors exert their biological activities and (2) the nature of that activity. Here we show that a new mathematical theory of steroid hormone action can be used to deduce the kinetic properties and reaction sequence position for the functioning of any two cofactors relative to a concentration limiting step (CLS) and to each other. The predictions of the theory, which can be applied using graphical methods similar to those of enzyme kinetics, are validated by obtaining internally consistent data for pair-wise analyses of three cofactors (TIF2, sSMRT, and NCoR) in U2OS cells. The analysis of TIF2 and sSMRT actions on GR-induction of an endogenous gene gave results identical to those with an exogenous reporter. Thus new tools to determine previously unobtainable information about the nature and position of cofactor action in any process displaying first-order Hill plot kinetics are now available. PMID:22272313

Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module.

PubMed

Skiba, Meredith A; Sikkema, Andrew P; Moss, Nathan A; Lowell, Andrew N; Su, Min; Sturgis, Rebecca M; Gerwick, Lena; Gerwick, William H; Sherman, David H; Smith, Janet L

2018-05-08

The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.

Synthesis of 9,9,9-trideutero-1,4-dihydroxynonane mercapturic acid (d3-DHN-MA), a useful internal standard for DHN-MA urinalysis.

PubMed

Chantegrel, B; Deshayes, C; Doutheau, A; Steghens, J P

2002-10-01

Racemic 1,4-dihydroxynonane mercapturic acid (DHN-MA) and 9,9,9-trideutero-1,4-dihydroxynonane mercapturic acid (d3-DHN-MA) are synthesized on a 400-mg scale (overall yield approximately 40%) by a two-step sequence involving Michael addition of N-acetyl-L-cysteine to methyl 4-hydroxynon-2(E)-enoate or methyl 9,9,9-trideutero-4-hydroxynon-2 (E)-enoate, followed by reduction of the intermediate adducts with lithium borohydride. The requisite starting methyl esters are obtained, respectively, from heptanal or 7,7,7-trideuteroheptanal and methyl 4-chlorophenylsulfinylacetate via a sulfoxide piperidine and carbonyl reaction described in the literature. The 7,7,7-trideuteroheptanal is easily prepared by classical methods in four steps from 6-bromo-1-hexanol. 13C NMR data indicate that DHN-MA as well as d3-DHN-MA are obtained as mixtures of four diastereomers. Preliminary results show that d3-DHN-MA could be used as an internal standard for mass spectrometric quantification of DHN-MA in human urine.

Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method

PubMed Central

Honda, Shozo; Morichika, Keisuke; Kirino, Yohei

2016-01-01

RNA digestions catalyzed by many ribonucleases generate RNA fragments containing a 2′,3′-cyclic phosphate (cP) at their 3′-termini. However, standard RNA-seq methods are unable to accurately capture cP-containing RNAs because the cP inhibits the adapter ligation reaction. We recently developed a method named “cP-RNA-seq” that is able to selectively amplify and sequence cP-containing RNAs. Here we describe the cP-RNA-seq protocol in which the 3′-termini of all RNAs, except those containing a cP, are cleaved through a periodate treatment after phosphatase treatment, hence subsequent adapter ligation and cDNA amplification steps are exclusively applied to cP-containing RNAs. cP-RNA-seq takes ~6 d, excluding the time required for sequencing and bioinformatics analyses, such downstream assays are not covered in detail in this protocol. Biochemical validation of the existence of cP in the identified RNAs takes ~3 d. Even though the cP-RNA-seq method was developed to identify angiogenin-generating 5′-tRNA halves as a proof of principle, the method should be applicable to global identification of cP-containing RNA repertoires in various transcriptomes. PMID:26866791

Appendix. Cloning and sequence of the gene encoding enzyme E-1 from the methionine salvage pathway of Klebsiella oxytoca.

PubMed

Balakrishnan, R; Frohlich, M; Rahaim, P T; Backman, K; Yocum, R R

1993-11-25

The methionine salvage pathway converts the methylthioribose moiety of 5'-(methylthio)-adenosine to methionine via a series of biochemical steps. One enzyme active in this pathway, a bifunctional enolase-phosphatase called E-1 that promotes oxidative cleavage of the synthetic substrate 2,3-diketo-1-phosphohexane to 2-keto-pentanoate, has been purified from Klebsiella pneumoniae and is characterized in the preceding paper (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides corresponding to portions of the amino terminus of E-1. These oligonucleotides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fragment that was used as a hybridization probe to obtain a clone of the E-1 gene from a K. oxytoca gene library. The DNA sequence of the E-1 coding region was determined, and the amino acid sequence of E-1 was deduced. E-1 appears to represent a novel class of enzymes since no homology to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has properties indistinguishable from those of the enzyme from K. pneumoniae.

Characterization of Sri Lanka rabies virus isolates using nucleotide sequence analysis of nucleoprotein gene.

PubMed

Arai, Y T; Takahashi, H; Kameoka, Y; Shiino, T; Wimalaratne, O; Lodmell, D L

2001-01-01

Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.

Identification of apple cultivars on the basis of simple sequence repeat markers.

PubMed

Liu, G S; Zhang, Y G; Tao, R; Fang, J G; Dai, H Y

2014-09-12

DNA markers are useful tools that play an important role in plant cultivar identification. They are usually based on polymerase chain reaction (PCR) and include simple sequence repeats (SSRs), inter-simple sequence repeats, and random amplified polymorphic DNA. However, DNA markers were not used effectively in the complete identification of plant cultivars because of the lack of known DNA fingerprints. Recently, a novel approach called the cultivar identification diagram (CID) strategy was developed to facilitate the use of DNA markers for separate plant individuals. The CID was designed whereby a polymorphic maker was generated from each PCR that directly allowed for cultivar sample separation at each step. Therefore, it could be used to identify cultivars and varieties easily with fewer primers. In this study, 60 apple cultivars, including a few main cultivars in fields and varieties from descendants (Fuji x Telamon) were examined. Of the 20 pairs of SSR primers screened, 8 pairs gave reproducible, polymorphic DNA amplification patterns. The banding patterns obtained from these 8 primers were used to construct a CID map. Each cultivar or variety in this study was distinguished from the others completely, indicating that this method can be used for efficient cultivar identification. The result contributed to studies on germplasm resources and the seedling industry in fruit trees.

The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product

PubMed Central

Dahlin, Paul; Srivastava, Vaibhav; Bulone, Vincent; McKee, Lauren S.

2016-01-01

The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture. PMID:27881978

Comprehensive molecular diagnosis of Epstein-Barr virus-associated lymphoproliferative diseases using next-generation sequencing.

PubMed

Ono, Shintaro; Nakayama, Manabu; Kanegane, Hirokazu; Hoshino, Akihiro; Shimodera, Saeko; Shibata, Hirofumi; Fujino, Hisanori; Fujino, Takahiro; Yunomae, Yuta; Okano, Tsubasa; Yamashita, Motoi; Yasumi, Takahiro; Izawa, Kazushi; Takagi, Masatoshi; Imai, Kohsuke; Zhang, Kejian; Marsh, Rebecca; Picard, Capucine; Latour, Sylvain; Ohara, Osamu; Morio, Tomohiro

2018-05-18

Epstein-Barr virus (EBV) is associated with several life-threatening diseases, such as lymphoproliferative disease (LPD), particularly in immunocompromised hosts. Some categories of primary immunodeficiency diseases (PIDs) including X-linked lymphoproliferative syndrome (XLP), are characterized by susceptibility and vulnerability to EBV infection. The number of genetically defined PIDs is rapidly increasing, and clinical genetic testing plays an important role in establishing a definitive diagnosis. Whole-exome sequencing is performed for diagnosing rare genetic diseases, but is both expensive and time-consuming. Low-cost, high-throughput gene analysis systems are thus necessary. We developed a comprehensive molecular diagnostic method using a two-step tailed polymerase chain reaction (PCR) and a next-generation sequencing (NGS) platform to detect mutations in 23 candidate genes responsible for XLP or XLP-like diseases. Samples from 19 patients suspected of having EBV-associated LPD were used in this comprehensive molecular diagnosis. Causative gene mutations (involving PRF1 and SH2D1A) were detected in two of the 19 patients studied. This comprehensive diagnosis method effectively detected mutations in all coding exons of 23 genes with sufficient read numbers for each amplicon. This comprehensive molecular diagnostic method using PCR and NGS provides a rapid, accurate, low-cost diagnosis for patients with XLP or XLP-like diseases.

The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product.

PubMed

Dahlin, Paul; Srivastava, Vaibhav; Bulone, Vincent; McKee, Lauren S

2016-01-01

The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica , a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture.

A hierarchical exact accelerated stochastic simulation algorithm

NASA Astrophysics Data System (ADS)

Orendorff, David; Mjolsness, Eric

2012-12-01

A new algorithm, "HiER-leap" (hierarchical exact reaction-leaping), is derived which improves on the computational properties of the ER-leap algorithm for exact accelerated simulation of stochastic chemical kinetics. Unlike ER-leap, HiER-leap utilizes a hierarchical or divide-and-conquer organization of reaction channels into tightly coupled "blocks" and is thereby able to speed up systems with many reaction channels. Like ER-leap, HiER-leap is based on the use of upper and lower bounds on the reaction propensities to define a rejection sampling algorithm with inexpensive early rejection and acceptance steps. But in HiER-leap, large portions of intra-block sampling may be done in parallel. An accept/reject step is used to synchronize across blocks. This method scales well when many reaction channels are present and has desirable asymptotic properties. The algorithm is exact, parallelizable and achieves a significant speedup over the stochastic simulation algorithm and ER-leap on certain problems. This algorithm offers a potentially important step towards efficient in silico modeling of entire organisms.

Copper-catalyzed decarboxylative trifluoromethylation of allylic bromodifluoroacetates.

PubMed

Ambler, Brett R; Altman, Ryan A

2013-11-01

The development of new synthetic fluorination reactions has important implications in medicinal, agricultural, and materials chemistries. Given the prevalence and accessibility of alcohols, methods to convert alcohols to trifluoromethanes are desirable. However, this transformation typically requires four-step processes, specialty chemicals, and/or stoichiometric metals to access the trifluoromethyl-containing product. A two-step copper-catalyzed decarboxylative protocol for converting allylic alcohols to trifluoromethanes is reported. Preliminary mechanistic studies distinguish this reaction from previously reported Cu-mediated reactions.

Step-by-Step Simulation of Radiation Chemistry Using Green Functions for Diffusion-Influenced Reactions

NASA Technical Reports Server (NTRS)

Plante, Ianik; Cucinotta, Francis A.

2011-01-01

Radiolytic species are formed approximately 1 ps after the passage of ionizing radiation through matter. After their formation, they diffuse and chemically react with other radiolytic species and neighboring biological molecules, leading to various oxidative damage. Therefore, the simulation of radiation chemistry is of considerable importance to understand how radiolytic species damage biological molecules [1]. The step-by-step simulation of chemical reactions is difficult, because the radiolytic species are distributed non-homogeneously in the medium. Consequently, computational approaches based on Green functions for diffusion-influenced reactions should be used [2]. Recently, Green functions for more complex type of reactions have been published [3-4]. We have developed exact random variate generators of these Green functions [5], which will allow us to use them in radiation chemistry codes. Moreover, simulating chemistry using the Green functions is which is computationally very demanding, because the probabilities of reactions between each pair of particles should be evaluated at each timestep [2]. This kind of problem is well adapted for General Purpose Graphic Processing Units (GPGPU), which can handle a large number of similar calculations simultaneously. These new developments will allow us to include more complex reactions in chemistry codes, and to improve the calculation time. This code should be of importance to link radiation track structure simulations and DNA damage models.

STEPS: Modeling and Simulating Complex Reaction-Diffusion Systems with Python

PubMed Central

Wils, Stefan; Schutter, Erik De

2008-01-01

We describe how the use of the Python language improved the user interface of the program STEPS. STEPS is a simulation platform for modeling and stochastic simulation of coupled reaction-diffusion systems with complex 3-dimensional boundary conditions. Setting up such models is a complicated process that consists of many phases. Initial versions of STEPS relied on a static input format that did not cleanly separate these phases, limiting modelers in how they could control the simulation and becoming increasingly complex as new features and new simulation algorithms were added. We solved all of these problems by tightly integrating STEPS with Python, using SWIG to expose our existing simulation code. PMID:19623245

Preferential amino acid sequences in alumina-catalyzed peptide bond formation.

PubMed

Bujdák, J; Rode, B M

2002-05-21

The catalytic effect of activated alumina on amino acid condensation was investigated. The readiness of amino acids to form peptide sequences was estimated on the basis of the yield of dipeptides and was found to decrease in the order glycine (Gly), alanine (Ala), leucine (Leu), valine (Val), proline (Pro). For example, approximately 15% Gly was converted to the dipeptide (Gly(2)), 5% to cyclic anhydride (cyc(Gly(2))) and small amounts of tri- (Gly(3)) and tetrapeptide (Gly(4)) were formed after 28 days. On the other hand, only trace amounts of Pro(2) were formed from proline under the same conditions. Preferential formation of certain sequences was observed in the mixed reaction systems containing two amino acids. For example, almost ten times more Gly-Val than Val-Gly was formed in the Gly+Val reaction system. The preferred sequences can be explained on the basis of an inductive effect that side groups have on the nucleophilicity and electrophilicity, respectively, of the amino and carboxyl groups. A comparison with published data of amino acid reactions in other reaction systems revealed that the main trends of preferential sequence formation were the same as those described for the salt-induced peptide formation (SIPF) reaction. The results of this work and other previously published papers show that alumina and related mineral surfaces might have played a crucial role in the prebiotic formation of the first peptides on the primitive earth.

Postural adjustment errors during lateral step initiation in older and younger adults

PubMed Central

Sparto, Patrick J.; Fuhrman, Susan I.; Redfern, Mark S.; Perera, Subashan; Jennings, J. Richard; Furman, Joseph M.

2016-01-01

The purpose was to examine age differences and varying levels of step response inhibition on the performance of a voluntary lateral step initiation task. Seventy older adults (70 – 94 y) and twenty younger adults (21 – 58 y) performed visually-cued step initiation conditions based on direction and spatial location of arrows, ranging from a simple choice reaction time task to a perceptual inhibition task that included incongruous cues about which direction to step (e.g. a left pointing arrow appearing on the right side of a monitor). Evidence of postural adjustment errors and step latencies were recorded from vertical ground reaction forces exerted by the stepping leg. Compared with younger adults, older adults demonstrated greater variability in step behavior, generated more postural adjustment errors during conditions requiring inhibition, and had greater step initiation latencies that increased more than younger adults as the inhibition requirements of the condition became greater. Step task performance was related to clinical balance test performance more than executive function task performance. PMID:25595953

Postural adjustment errors during lateral step initiation in older and younger adults

PubMed Central

Sparto, Patrick J.; Fuhrman, Susan I.; Redfern, Mark S.; Perera, Subashan; Jennings, J. Richard; Furman, Joseph M.

2014-01-01

The purpose was to examine age differences and varying levels of step response inhibition on the performance of a voluntary lateral step initiation task. Seventy older adults (70 – 94 y) and twenty younger adults (21 – 58 y) performed visually-cued step initiation conditions based on direction and spatial location of arrows, ranging from a simple choice reaction time task to a perceptual inhibition task that included incongruous cues about which direction to step (e.g. a left pointing arrow appearing on the right side of a monitor). Evidence of postural adjustment errors and step latencies were recorded from vertical ground reaction forces exerted by the stepping leg. Compared with younger adults, older adults demonstrated greater variability in step behavior, generated more postural adjustment errors during conditions requiring inhibition, and had greater step initiation latencies that increased more than younger adults as the inhibition requirements of the condition became greater. Step task performance was related to clinical balance test performance more than executive function task performance. PMID:25183162

A programming language for composable DNA circuits

PubMed Central

Phillips, Andrew; Cardelli, Luca

2009-01-01

Recently, a range of information-processing circuits have been implemented in DNA by using strand displacement as their main computational mechanism. Examples include digital logic circuits and catalytic signal amplification circuits that function as efficient molecular detectors. As new paradigms for DNA computation emerge, the development of corresponding languages and tools for these paradigms will help to facilitate the design of DNA circuits and their automatic compilation to nucleotide sequences. We present a programming language for designing and simulating DNA circuits in which strand displacement is the main computational mechanism. The language includes basic elements of sequence domains, toeholds and branch migration, and assumes that strands do not possess any secondary structure. The language is used to model and simulate a variety of circuits, including an entropy-driven catalytic gate, a simple gate motif for synthesizing large-scale circuits and a scheme for implementing an arbitrary system of chemical reactions. The language is a first step towards the design of modelling and simulation tools for DNA strand displacement, which complements the emergence of novel implementation strategies for DNA computing. PMID:19535415

Intra- and intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid.

PubMed

Lafontaine, D; Beaudry, D; Marquis, P; Perreault, J P

1995-10-01

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5'-hydroxyl and the 2',3'-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo. Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism. Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

A programming language for composable DNA circuits.

PubMed

Phillips, Andrew; Cardelli, Luca

2009-08-06

Recently, a range of information-processing circuits have been implemented in DNA by using strand displacement as their main computational mechanism. Examples include digital logic circuits and catalytic signal amplification circuits that function as efficient molecular detectors. As new paradigms for DNA computation emerge, the development of corresponding languages and tools for these paradigms will help to facilitate the design of DNA circuits and their automatic compilation to nucleotide sequences. We present a programming language for designing and simulating DNA circuits in which strand displacement is the main computational mechanism. The language includes basic elements of sequence domains, toeholds and branch migration, and assumes that strands do not possess any secondary structure. The language is used to model and simulate a variety of circuits, including an entropy-driven catalytic gate, a simple gate motif for synthesizing large-scale circuits and a scheme for implementing an arbitrary system of chemical reactions. The language is a first step towards the design of modelling and simulation tools for DNA strand displacement, which complements the emergence of novel implementation strategies for DNA computing.

Mechanistic insights into iron catalyzed dehydrogenation of formic acid: β-hydride elimination vs. direct hydride transfer.

PubMed

Yang, Xinzheng

2013-09-07

Density functional theory calculations reveal a complete reaction mechanism with detailed energy profiles and transition state structures for the dehydrogenation of formic acid catalyzed by an iron complex, [P(CH2CH2PPh2)3FeH](+). In the cationic reaction pathway, a β-hydride elimination process is confirmed to be the rate-determining step in this catalytic reaction. A potential reaction pathway starting with a direct hydride transfer from HCOO(-) to Fe is found to be possible, but slightly less favorable than the catalytic cycle with a β-hydride elimination step.

The structure of dimethylallyl tryptophan synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria

PubMed Central

Metzger, Ute; Schall, Christoph; Zocher, Georg; Unsöld, Inge; Stec, Edyta; Li, Shu-Ming; Heide, Lutz; Stehle, Thilo

2009-01-01

Ergot alkaloids are toxins and important pharmaceuticals that are produced biotechnologically on an industrial scale. The first committed step of ergot alkaloid biosynthesis is catalyzed by dimethylallyl tryptophan synthase (DMATS; EC 2.5.1.34). Orthologs of DMATS are found in many fungal genomes. We report here the x-ray structure of DMATS, determined at a resolution of 1.76 Å. A complex of DMATS from Aspergillus fumigatus with its aromatic substrate L-tryptophan and with an analogue of its isoprenoid substrate dimethylallyl diphosphate reveals the structural basis of this enzyme-catalyzed Friedel-Crafts reaction, which shows strict regiospecificity for position 4 of the indole nucleus of tryptophan as well as unusual independence of the presence of Mg2+ ions. The 3D structure of DMATS belongs to a rare β/α barrel fold, called prenyltransferase barrel, that was recently discovered in a small group of bacterial enzymes with no sequence similarity to DMATS. These bacterial enzymes catalyze the prenylation of aromatic substrates in the biosynthesis of secondary metabolites (i.e., a reaction similar to that of DMATS). PMID:19706516

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

PubMed Central

Gaur, R K; Valcárcel, J; Green, M R

1995-01-01

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 FIGURE 8 FIGURE 9 PMID:7493318

RIKEN Integrated Sequence Analysis (RISA) System—384-Format Sequencing Pipeline with 384 Multicapillary Sequencer

PubMed Central

Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Nagaoka, Sumiharu; Sasaki, Nobuya; Carninci, Piero; Konno, Hideaki; Akiyama, Junichi; Nishi, Katsuo; Kitsunai, Tokuji; Tashiro, Hideo; Itoh, Mari; Sumi, Noriko; Ishii, Yoshiyuki; Nakamura, Shin; Hazama, Makoto; Nishine, Tsutomu; Harada, Akira; Yamamoto, Rintaro; Matsumoto, Hiroyuki; Sakaguchi, Sumito; Ikegami, Takashi; Kashiwagi, Katsuya; Fujiwake, Syuji; Inoue, Kouji; Togawa, Yoshiyuki; Izawa, Masaki; Ohara, Eiji; Watahiki, Masanori; Yoneda, Yuko; Ishikawa, Tomokazu; Ozawa, Kaori; Tanaka, Takumi; Matsuura, Shuji; Kawai, Jun; Okazaki, Yasushi; Muramatsu, Masami; Inoue, Yorinao; Kira, Akira; Hayashizaki, Yoshihide

2000-01-01

The RIKEN high-throughput 384-format sequencing pipeline (RISA system) including a 384-multicapillary sequencer (the so-called RISA sequencer) was developed for the RIKEN mouse encyclopedia project. The RISA system consists of colony picking, template preparation, sequencing reaction, and the sequencing process. A novel high-throughput 384-format capillary sequencer system (RISA sequencer system) was developed for the sequencing process. This system consists of a 384-multicapillary auto sequencer (RISA sequencer), a 384-multicapillary array assembler (CAS), and a 384-multicapillary casting device. The RISA sequencer can simultaneously analyze 384 independent sequencing products. The optical system is a scanning system chosen after careful comparison with an image detection system for the simultaneous detection of the 384-capillary array. This scanning system can be used with any fluorescent-labeled sequencing reaction (chain termination reaction), including transcriptional sequencing based on RNA polymerase, which was originally developed by us, and cycle sequencing based on thermostable DNA polymerase. For long-read sequencing, 380 out of 384 sequences (99.2%) were successfully analyzed and the average read length, with more than 99% accuracy, was 654.4 bp. A single RISA sequencer can analyze 216 kb with >99% accuracy in 2.7 h (90 kb/h). For short-read sequencing to cluster the 3′ end and 5′ end sequencing by reading 350 bp, 384 samples can be analyzed in 1.5 h. We have also developed a RISA inoculator, RISA filtrator and densitometer, RISA plasmid preparator which can handle throughput of 40,000 samples in 17.5 h, and a high-throughput RISA thermal cycler which has four 384-well sites. The combination of these technologies allowed us to construct the RISA system consisting of 16 RISA sequencers, which can process 50,000 DNA samples per day. One haploid genome shotgun sequence of a higher organism, such as human, mouse, rat, domestic animals, and plants, can be revealed by seven RISA systems within one month. PMID:11076861

Probing the Rate-Determining Step of the Claisen-Schmidt Condensation by Competition Reactions

ERIC Educational Resources Information Center

Mak, Kendrew K. W.; Chan, Wing-Fat; Lung, Ka-Ying; Lam, Wai-Yee; Ng, Weng-Cheong; Lee, Siu-Fung

2007-01-01

Competition experiments are a useful tool for preliminary study of the linear free energy relationship of organic reactions. This article describes a physical organic experiment for upper-level undergraduates to identify the rate-determining step of the Claisen-Schmidt condensation of benzaldehyde and acetophenone by studying the linear free…

Stepping reaction time and gait adaptability are significantly impaired in people with Parkinson's disease: Implications for fall risk.

PubMed

Caetano, Maria Joana D; Lord, Stephen R; Allen, Natalie E; Brodie, Matthew A; Song, Jooeun; Paul, Serene S; Canning, Colleen G; Menant, Jasmine C

2018-02-01

Decline in the ability to take effective steps and to adapt gait, particularly under challenging conditions, may be important reasons why people with Parkinson's disease (PD) have an increased risk of falling. This study aimed to determine the extent of stepping and gait adaptability impairments in PD individuals as well as their associations with PD symptoms, cognitive function and previous falls. Thirty-three older people with PD and 33 controls were assessed in choice stepping reaction time, Stroop stepping and gait adaptability tests; measurements identified as fall risk factors in older adults. People with PD had similar mean choice stepping reaction times to healthy controls, but had significantly greater intra-individual variability. In the Stroop stepping test, the PD participants were more likely to make an error (48 vs 18%), took 715 ms longer to react (2312 vs 1517 ms) and had significantly greater response variability (536 vs 329 ms) than the healthy controls. People with PD also had more difficulties adapting their gait in response to targets (poorer stepping accuracy) and obstacles (increased number of steps) appearing at short notice on a walkway. Within the PD group, higher disease severity, reduced cognition and previous falls were associated with poorer stepping and gait adaptability performances. People with PD have reduced ability to adapt gait to unexpected targets and obstacles and exhibit poorer stepping responses, particularly in a test condition involving conflict resolution. Such impaired stepping responses in Parkinson's disease are associated with disease severity, cognitive impairment and falls. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure reactivity and thermodynamic analysis on the oxidation of ampicillin drug by copper(III) complex in aqueous alkaline medium (stopped-flow technique)

NASA Astrophysics Data System (ADS)

Shetti, Nagaraj P.; Hegde, Rajesh N.; Nandibewoor, Sharanappa T.

2009-07-01

Oxidation of penicillin derivative, ampicillin (AMP) by diperiodatocuprate(III) (DPC) in alkaline medium at a constant ionic strength of 0.01-mol dm -3 was studied spectrophotometrically. The reaction between DPC and ampicillin in alkaline medium exhibits 1:4 stoichiometry (ampicillin:DPC). Intervention of free radicals was observed in the reaction. Based on the observed orders and experimental evidences, a mechanism involving the protonated form of DPC as the reactive oxidant species has been proposed. The oxidation reaction in alkaline medium has been shown to proceed via a DPC-AMP complex, which decomposes slowly in a rate determining step to yield phenyl glycine (PG) and free radical species of 6-aminopenicillanic acid (6-APA), followed by other fast steps to give the products. The two major products were characterized by IR, NMR, LC-MS and Spot test. The reaction constants involved in the different steps of the mechanism were calculated. The activation parameters with respect to slow step of the mechanism were computed and discussed and thermodynamic quantities were also determined.

Gold-nanoparticle-catalyzed synthesis of propargylamines: the traditional A3-multicomponent reaction performed as a two-step flow process.

PubMed

Abahmane, Lahbib; Köhler, J Michael; Gross, G Alexander

2011-03-01

The alkyne, aldehyde, amine A(3)-coupling reaction, a traditional multicomponent reaction (MCR), has been investigated as a two-step flow process. The implicated aminoalkylation reaction of phenylacetylene with appropriate aldimine intermediates was catalyzed by gold nanoparticles impregnated on alumina. The aldimine formation was catalyzed by Montmorillonite K10 beforehand. The performance of the process has been investigated with respect to different reaction regimes. Usually, the A(3)-multicomponent reaction is performed as a "one-pot" process. Diversity-oriented syntheses using MCRs often have the shortcoming that only low selectivity and low yields are achieved. We have used a flow-chemistry approach to perform the A(3)-MCR in a sequential manner. In this way, the reaction performance was significantly enhanced in terms of shortened reaction time, and the desired propargylamines were obtained in high yields. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stereoselective Total Synthesis of Radiolabeled Artemisinin (Qinghaosu).

DTIC Science & Technology

Our previous total synthesis of (+)- artemisinin has been optimized from 18 to 11 steps. The final two steps in the sequence are: 1) alkylation of a...product (+)- artemisinin . The first step was repeated utilizing carbon-14 methyl iodide and the sequence completed as before to afford the desired...carbon-14labeled (+)- artemisinin . The label resides in the methyl group pendant from the lactone ring (ring D), the position of attachment being C-9, the carbon atom being C-16. Keywords: Antimalarials. (aw)

A gp41-based heteroduplex mobility assay provides rapid and accurate assessment of intrasubtype epidemiological linkage in HIV type 1 heterosexual transmission Pairs.

PubMed

Manigart, Olivier; Boeras, Debrah I; Karita, Etienne; Hawkins, Paulina A; Vwalika, Cheswa; Makombe, Nathan; Mulenga, Joseph; Derdeyn, Cynthia A; Allen, Susan; Hunter, Eric

2012-12-01

A critical step in HIV-1 transmission studies is the rapid and accurate identification of epidemiologically linked transmission pairs. To date, this has been accomplished by comparison of polymerase chain reaction (PCR)-amplified nucleotide sequences from potential transmission pairs, which can be cost-prohibitive for use in resource-limited settings. Here we describe a rapid, cost-effective approach to determine transmission linkage based on the heteroduplex mobility assay (HMA), and validate this approach by comparison to nucleotide sequencing. A total of 102 HIV-1-infected Zambian and Rwandan couples, with known linkage, were analyzed by gp41-HMA. A 400-base pair fragment within the envelope gp41 region of the HIV proviral genome was PCR amplified and HMA was applied to both partners' amplicons separately (autologous) and as a mixture (heterologous). If the diversity between gp41 sequences was low (<5%), a homoduplex was observed upon gel electrophoresis and the transmission was characterized as having occurred between partners (linked). If a new heteroduplex formed, within the heterologous migration, the transmission was determined to be unlinked. Initial blind validation of gp-41 HMA demonstrated 90% concordance between HMA and sequencing with 100% concordance in the case of linked transmissions. Following validation, 25 newly infected partners in Kigali and 12 in Lusaka were evaluated prospectively using both HMA and nucleotide sequences. Concordant results were obtained in all but one case (97.3%). The gp41-HMA technique is a reliable and feasible tool to detect linked transmissions in the field. All identified unlinked results should be confirmed by sequence analyses.

Organic syntheses employing supercritical carbon dioxide as a reaction solvent

NASA Technical Reports Server (NTRS)

Barstow, Leon E. (Inventor); Ward, Glen D. (Inventor); Bier, Milan (Inventor)

1991-01-01

Chemical reactions are readily carried out using supercritical carbon dioxide as the reaction medium. Supercritical carbon dioxide is of special value as a reaction medium in reactions for synthesizing polypeptides, for sequencing polypeptides, or for amino acid analysis.

Organic syntheses employing supercritical carbon dioxide as a reaction solvent

NASA Technical Reports Server (NTRS)

Barstow, Leon E. (Inventor); Ward, Glen D. (Inventor); Bier, Milan (Inventor)

1993-01-01

Chemical reactions are readily carried out using supercritical carbon dioxide as the reaction medium. Supercritical carbon dioxide is of special value as a reaction medium in reactions for synthesizing polypeptides, for sequencing polypeptides, or for amino acid analysis.

Development and integration of block operations for data invariant automation of digital preprocessing and analysis of biological and biomedical Raman spectra.

PubMed

Schulze, H Georg; Turner, Robin F B

2015-06-01

High-throughput information extraction from large numbers of Raman spectra is becoming an increasingly taxing problem due to the proliferation of new applications enabled using advances in instrumentation. Fortunately, in many of these applications, the entire process can be automated, yielding reproducibly good results with significant time and cost savings. Information extraction consists of two stages, preprocessing and analysis. We focus here on the preprocessing stage, which typically involves several steps, such as calibration, background subtraction, baseline flattening, artifact removal, smoothing, and so on, before the resulting spectra can be further analyzed. Because the results of some of these steps can affect the performance of subsequent ones, attention must be given to the sequencing of steps, the compatibility of these sequences, and the propensity of each step to generate spectral distortions. We outline here important considerations to effect full automation of Raman spectral preprocessing: what is considered full automation; putative general principles to effect full automation; the proper sequencing of processing and analysis steps; conflicts and circularities arising from sequencing; and the need for, and approaches to, preprocessing quality control. These considerations are discussed and illustrated with biological and biomedical examples reflecting both successful and faulty preprocessing.

Learning to predict chemical reactions.

PubMed

Kayala, Matthew A; Azencott, Chloé-Agathe; Chen, Jonathan H; Baldi, Pierre

2011-09-26

Being able to predict the course of arbitrary chemical reactions is essential to the theory and applications of organic chemistry. Approaches to the reaction prediction problems can be organized around three poles corresponding to: (1) physical laws; (2) rule-based expert systems; and (3) inductive machine learning. Previous approaches at these poles, respectively, are not high throughput, are not generalizable or scalable, and lack sufficient data and structure to be implemented. We propose a new approach to reaction prediction utilizing elements from each pole. Using a physically inspired conceptualization, we describe single mechanistic reactions as interactions between coarse approximations of molecular orbitals (MOs) and use topological and physicochemical attributes as descriptors. Using an existing rule-based system (Reaction Explorer), we derive a restricted chemistry data set consisting of 1630 full multistep reactions with 2358 distinct starting materials and intermediates, associated with 2989 productive mechanistic steps and 6.14 million unproductive mechanistic steps. And from machine learning, we pose identifying productive mechanistic steps as a statistical ranking, information retrieval problem: given a set of reactants and a description of conditions, learn a ranking model over potential filled-to-unfilled MO interactions such that the top-ranked mechanistic steps yield the major products. The machine learning implementation follows a two-stage approach, in which we first train atom level reactivity filters to prune 94.00% of nonproductive reactions with a 0.01% error rate. Then, we train an ensemble of ranking models on pairs of interacting MOs to learn a relative productivity function over mechanistic steps in a given system. Without the use of explicit transformation patterns, the ensemble perfectly ranks the productive mechanism at the top 89.05% of the time, rising to 99.86% of the time when the top four are considered. Furthermore, the system is generalizable, making reasonable predictions over reactants and conditions which the rule-based expert does not handle. A web interface to the machine learning based mechanistic reaction predictor is accessible through our chemoinformatics portal ( http://cdb.ics.uci.edu) under the Toolkits section.

RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

PubMed

Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

2006-11-21

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein

PubMed Central

Dilley, David R.; Wang, Zhenyong; Kadirjan-Kalbach, Deena K.; Ververidis, Fillipos; Beaudry, Randolph; Padmanabhan, Kallaithe

2013-01-01

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyses the final step in ethylene biosynthesis converting ACC to ethylene, cyanide, CO2, dehydroascorbate and water with inputs of Fe(II), ascorbate, bicarbonate (as activators) and oxygen. Cyanide activates ACCO. A ‘nest’ comprising several positively charged amino acid residues from the C-terminal α-helix 11 along with Lys158 and Arg299 are proposed as binding sites for ascorbate and bicarbonate to coordinately activate the ACCO reaction. The binding sites for ACC, bicarbonate and ascorbic acid for Malus domestica ACCO1 include Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. Glutamate 297, Phe300 and Glu301 in α-helix 11 are also important for the ACCO reaction. Our proposed reaction pathway incorporates cyanide as an ACCO/Fe(II) ligand after reaction turnover. The cyanide ligand is likely displaced upon binding of ACC and ascorbate to provide a binding site for oxygen. We propose that ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction. ACC oxidase has significant homology with Lycopersicon esculentum cysteine protease LeCp, which functions as a protease and as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase (Acs2) gene expression. ACC oxidase may play a similar role in signal transduction after post-translational processing. ACC oxidase becomes inactivated by fragmentation and apparently has intrinsic protease and transpeptidase activity. ACC oxidase contains several amino acid sequence motifs for putative protein–protein interactions, phosphokinases and cysteine protease. ACC oxidase is subject to autophosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner. PMID:24244837

The possible role of thiosulfate in the precipitation of 34S-rich barite in some Mississippi Valley-type deposits

USGS Publications Warehouse

Spirakis, C.S.

1991-01-01

The precipitation of extremely 34S-rich barite in the late stage of mineralization in the Mississippi Valleytype deposits of the Illinois-Kentucky district (U.S.A.) may be explained by reactions involving thiosulfate (S2O3=). Inorganic processes are known to concentrate 34S in the sulfonate site of thiosulfate and 32S in the sulfate site. In the mineralizing solution, these inorganic processes may have fractionated sulfur between the two sites by about 40 per mil. At the low temperatures of the late barite stage of mineralization, bacteria are known to metabolize thiosulfate by various reactions. In one of these, dissimilatory reduction, hydrogen sulfide and sulfite are produced. Isotopically light sulfite is preferentially reduced to sulfide by bacteria to leave a residual sulfite enriched in 34S. Part of the residual sulfite may be oxidized to form isotopically heavy sulfate; part may recombine with hydrogen sulfide to form thiosulfate. The recombination also enriches the sulfonate site in 34S and the sulfane site in 32S. Recycling the newly formed thiosulfate through the above steps further enriches sulfite and sulfate from oxidation of sulfite in 34S. During genesis of the ores, the aggregate effect of these reactions may have been the precipitation of extremely 34S-rich barite. The sequence of reactions suggested above requires the presence of organic matter. Previously proposed reactions to account for the precipitation of sulfide minerals and fluorite and for the carbonate paragenesis also require the presence of organic matter. Thus, organic matter in the host rocks may cause the various ore-zone reactions and account for the localization of the ores. ?? 1991 Springer-Verlag.

Quantitative comparison of catalytic mechanisms and overall reactions in convergently evolved enzymes: implications for classification of enzyme function.

PubMed

Almonacid, Daniel E; Yera, Emmanuel R; Mitchell, John B O; Babbitt, Patricia C

2010-03-12

Functionally analogous enzymes are those that catalyze similar reactions on similar substrates but do not share common ancestry, providing a window on the different structural strategies nature has used to evolve required catalysts. Identification and use of this information to improve reaction classification and computational annotation of enzymes newly discovered in the genome projects would benefit from systematic determination of reaction similarities. Here, we quantified similarity in bond changes for overall reactions and catalytic mechanisms for 95 pairs of functionally analogous enzymes (non-homologous enzymes with identical first three numbers of their EC codes) from the MACiE database. Similarity of overall reactions was computed by comparing the sets of bond changes in the transformations from substrates to products. For similarity of mechanisms, sets of bond changes occurring in each mechanistic step were compared; these similarities were then used to guide global and local alignments of mechanistic steps. Using this metric, only 44% of pairs of functionally analogous enzymes in the dataset had significantly similar overall reactions. For these enzymes, convergence to the same mechanism occurred in 33% of cases, with most pairs having at least one identical mechanistic step. Using our metric, overall reaction similarity serves as an upper bound for mechanistic similarity in functional analogs. For example, the four carbon-oxygen lyases acting on phosphates (EC 4.2.3) show neither significant overall reaction similarity nor significant mechanistic similarity. By contrast, the three carboxylic-ester hydrolases (EC 3.1.1) catalyze overall reactions with identical bond changes and have converged to almost identical mechanisms. The large proportion of enzyme pairs that do not show significant overall reaction similarity (56%) suggests that at least for the functionally analogous enzymes studied here, more stringent criteria could be used to refine definitions of EC sub-subclasses for improved discrimination in their classification of enzyme reactions. The results also indicate that mechanistic convergence of reaction steps is widespread, suggesting that quantitative measurement of mechanistic similarity can inform approaches for functional annotation.

Quantitative Comparison of Catalytic Mechanisms and Overall Reactions in Convergently Evolved Enzymes: Implications for Classification of Enzyme Function

PubMed Central

Almonacid, Daniel E.; Yera, Emmanuel R.; Mitchell, John B. O.; Babbitt, Patricia C.

2010-01-01

Functionally analogous enzymes are those that catalyze similar reactions on similar substrates but do not share common ancestry, providing a window on the different structural strategies nature has used to evolve required catalysts. Identification and use of this information to improve reaction classification and computational annotation of enzymes newly discovered in the genome projects would benefit from systematic determination of reaction similarities. Here, we quantified similarity in bond changes for overall reactions and catalytic mechanisms for 95 pairs of functionally analogous enzymes (non-homologous enzymes with identical first three numbers of their EC codes) from the MACiE database. Similarity of overall reactions was computed by comparing the sets of bond changes in the transformations from substrates to products. For similarity of mechanisms, sets of bond changes occurring in each mechanistic step were compared; these similarities were then used to guide global and local alignments of mechanistic steps. Using this metric, only 44% of pairs of functionally analogous enzymes in the dataset had significantly similar overall reactions. For these enzymes, convergence to the same mechanism occurred in 33% of cases, with most pairs having at least one identical mechanistic step. Using our metric, overall reaction similarity serves as an upper bound for mechanistic similarity in functional analogs. For example, the four carbon-oxygen lyases acting on phosphates (EC 4.2.3) show neither significant overall reaction similarity nor significant mechanistic similarity. By contrast, the three carboxylic-ester hydrolases (EC 3.1.1) catalyze overall reactions with identical bond changes and have converged to almost identical mechanisms. The large proportion of enzyme pairs that do not show significant overall reaction similarity (56%) suggests that at least for the functionally analogous enzymes studied here, more stringent criteria could be used to refine definitions of EC sub-subclasses for improved discrimination in their classification of enzyme reactions. The results also indicate that mechanistic convergence of reaction steps is widespread, suggesting that quantitative measurement of mechanistic similarity can inform approaches for functional annotation. PMID:20300652

QM/MM free-energy simulations of reaction in Serratia marcescens Chitinase B reveal the protonation state of Asp142 and the critical role of Tyr214.

PubMed

Jitonnom, Jitrayut; Limb, Michael A L; Mulholland, Adrian J

2014-05-08

Serratia marcescens Chitinase B (ChiB), belonging to the glycosidase family 18 (GH18), catalyzes the hydrolysis of β-1,4-glycosidic bond, with retention of configuration, via an unusual substrate-assisted mechanism, in which the substrate itself acts as an intramolecular nucleophile. Here, both elementary steps (glycosylation and deglycosylation) of the ChiB-catalyzed reaction are investigated by means of combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations at the SCC-DFTB/CHARMM22 level of theory. We examine the influence of the Asp142 protonation state on the reaction and the role that this residue performs in the reaction. Our simulations show that reaction with a neutral Asp142 is preferred and demonstrate that this residue provides electrostatic stabilization of the oxazolinium ion intermediate formed in the reaction. Insight into the conformational itinerary ((1,4)B↔(4)H5↔(4)C1) adopted by the substrate (bound in subsite -1) along the preferred reaction pathway is also provided by the simulations. The relative energies of the stationary points found along the reaction pathway calculated with SCC-DFTB and B3LYP were compared. The results suggest that SCC-DFTB is an accurate method for estimating the relative barriers for both steps of the reaction; however, it was found to overestimate the relative energy of an intermediate formed in the reaction when compared with the higher level of theory. Glycosylation is suggested to be a rate-determining step in the reaction with calculated overall reaction free-energy barrier of 20.5 kcal/mol, in a reasonable agreement with the 16.1 kcal/mol barrier derived from the experiment. The role of Tyr214 in catalysis was also investigated with the results, indicating that the residue plays a critical role in the deglycosylation step of the reaction. Simulations of the enzyme-product complex were also performed with an unbinding event suggested to have been observed, affording potential new mechanistic insight into the release of the product of ChiB.

Synthesis of phenanthridinones from N-methoxybenzamides and arenes by multiple palladium-catalyzed C-H activation steps at room temperature.

PubMed

Karthikeyan, Jaganathan; Cheng, Chien-Hong

2011-10-10

Many steps make light work: substituted phenanthridinones can be obtained with high regioselectivity and in very good yields by palladium-catalyzed cyclization reactions of N-methoxybenzamides with arenes. The reaction proceeds through multiple oxidative C-H activation and C-C/C-N formation steps in one pot at room temperature, and thus provides a simple method for generating bioactive phenanthridinones. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pressure modulates the self-cleavage step of the hairpin ribozyme

NASA Astrophysics Data System (ADS)

Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

2017-03-01

The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

Analysis of carbon-oxygen reactions by use of a square-input response technique and {sup 18}O isotope

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

Miura, Kouichi; Nakagawa, Hiroyuki

1996-12-31

Carbon gasification reaction has been investigated for decades including the pioneering works of Walker and his co-workers, but its mechanism has not been completely elucidated. The concept of the active surface area (ASA) was proposed by them, and its importance has been recognized. However, since ASA was measured by O{sub 2} chemisorption at below 300{degrees}C where carbon loss through gasification is negligible, it does not reflect the actual gasification situation. To overcome this weak point, measurements of ASA in a batch reactor and the so-called transient kinetic (TK) method were proposed. Ahmed and Back successfully measured the chemisorbed oxygen duringmore » the gasification using a batch reactor, and proposed a new mechanistic sequence for carbon-oxygen reaction which stresses the importance of the reaction between the gaseous oxygen and the chemisorbed oxygen. Radovic et al. proposed the concept of the reactive surface area (RSA), and reported excellent proportionality between the CO{sub 2} gasification rate and the RSA estimated by the TK and the TPD methods. Kapteijn et al. showed that the TK method with labeled molecules is more powerful to examine the mechanism. They found the presence of two types of surface oxygen complexes which desorb at different rates. A Square-input response (SIR) method is applied to the carbon-oxygen reaction. This method allows the observation of transient changes on two step changes. This method has been successfully applied to the analysis of a coal char gasification.« less

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

PubMed Central

Nika, Heinz; Hawke, David H.; Angeletti, Ruth Hogue

2014-01-01

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures. PMID:24688319

Paracoccus denitrificans PD1222 Utilizes Hypotaurine via Transamination Followed by Spontaneous Desulfination To Yield Acetaldehyde and, Finally, Acetate for Growth

PubMed Central

Felux, Ann-Katrin; Denger, Karin; Weiss, Michael; Cook, Alasdair M.

2013-01-01

Hypotaurine (HT; 2-aminoethane-sulfinate) is known to be utilized by bacteria as a sole source of carbon, nitrogen, and energy for growth, as is taurine (2-aminoethane-sulfonate); however, the corresponding HT degradation pathway has remained undefined. Genome-sequenced Paracoccus denitrificans PD1222 utilized HT (and taurine) quantitatively for heterotrophic growth and released the HT sulfur as sulfite (and sulfate) and HT nitrogen as ammonium. Enzyme assays with cell extracts suggested that an HT-inducible HT:pyruvate aminotransferase (Hpa) catalyzes the deamination of HT in an initial reaction step. Partial purification of the Hpa activity and peptide fingerprinting-mass spectrometry (PF-MS) identified the Hpa candidate gene; it encoded an archetypal taurine:pyruvate aminotransferase (Tpa). The same gene product was identified via differential PAGE and PF-MS, as was the gene of a strongly HT-inducible aldehyde dehydrogenase (Adh). Both genes were overexpressed in Escherichia coli. The overexpressed, purified Hpa/Tpa showed HT:pyruvate-aminotransferase activity. Alanine, acetaldehyde, and sulfite were identified as the reaction products but not sulfinoacetaldehyde; the reaction of Hpa/Tpa with taurine yielded sulfoacetaldehyde, which is stable. The overexpressed, purified Adh oxidized the acetaldehyde generated during the Hpa reaction to acetate in an NAD+-dependent reaction. Based on these results, the following degradation pathway for HT in strain PD1222 can be depicted. The identified aminotransferase converts HT to sulfinoacetaldehyde, which desulfinates spontaneously to acetaldehyde and sulfite; the inducible aldehyde dehydrogenase oxidizes acetaldehyde to yield acetate, which is metabolized, and sulfite, which is excreted. PMID:23603744

A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.

PubMed

Tan, Swee Jin; Phan, Huan; Gerry, Benjamin Michael; Kuhn, Alexandre; Hong, Lewis Zuocheng; Min Ong, Yao; Poon, Polly Suk Yean; Unger, Marc Alexander; Jones, Robert C; Quake, Stephen R; Burkholder, William F

2013-01-01

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps

PubMed Central

Tan, Swee Jin; Phan, Huan; Gerry, Benjamin Michael; Kuhn, Alexandre; Hong, Lewis Zuocheng; Min Ong, Yao; Poon, Polly Suk Yean; Unger, Marc Alexander; Jones, Robert C.; Quake, Stephen R.; Burkholder, William F.

2013-01-01

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation. PMID:23894273

Direct coal liquefaction process

DOEpatents

Rindt, John R.; Hetland, Melanie D.

1993-01-01

An improved multistep liquefaction process for organic carbonaceous mater which produces a virtually completely solvent-soluble carbonaceous liquid product. The solubilized product may be more amenable to further processing than liquid products produced by current methods. In the initial processing step, the finely divided organic carbonaceous material is treated with a hydrocarbonaceous pasting solvent containing from 10% and 100% by weight process-derived phenolic species at a temperature within the range of 300.degree. C. to 400.degree. C. for typically from 2 minutes to 120 minutes in the presence of a carbon monoxide reductant and an optional hydrogen sulfide reaction promoter in an amount ranging from 0 to 10% by weight of the moisture- and ash-free organic carbonaceous material fed to the system. As a result, hydrogen is generated via the water/gas shift reaction at a rate necessary to prevent condensation reactions. In a second step, the reaction product of the first step is hydrogenated.

Direct coal liquefaction process

DOEpatents

Rindt, J.R.; Hetland, M.D.

1993-10-26

An improved multistep liquefaction process for organic carbonaceous mater which produces a virtually completely solvent-soluble carbonaceous liquid product. The solubilized product may be more amenable to further processing than liquid products produced by current methods. In the initial processing step, the finely divided organic carbonaceous material is treated with a hydrocarbonaceous pasting solvent containing from 10% and 100% by weight process-derived phenolic species at a temperature within the range of 300 C to 400 C for typically from 2 minutes to 120 minutes in the presence of a carbon monoxide reductant and an optional hydrogen sulfide reaction promoter in an amount ranging from 0 to 10% by weight of the moisture- and ash-free organic carbonaceous material fed to the system. As a result, hydrogen is generated via the water/gas shift reaction at a rate necessary to prevent condensation reactions. In a second step, the reaction product of the first step is hydrogenated.

How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations

PubMed Central

Kirmizialtin, Serdal; Nguyen, Virginia; Johnson, Kenneth A.; Elber, Ron

2012-01-01

Summary Nearly every enzyme undergoes a significant change in structure after binding it’s substrate. New experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a ms timescale locks the correct nucleotide at the active site, but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, while the same steps diminish the reaction probability of an incorrect substrate. PMID:22483109

Process for conversion of lignin to reformulated hydrocarbon gasoline

DOEpatents

Shabtai, Joseph S.; Zmierczak, Wlodzimierz W.; Chornet, Esteban

1999-09-28

A process for converting lignin into high-quality reformulated hydrocarbon gasoline compositions in high yields is disclosed. The process is a two-stage, catalytic reaction process that produces a reformulated hydrocarbon gasoline product with a controlled amount of aromatics. In the first stage, a lignin material is subjected to a base-catalyzed depolymerization reaction in the presence of a supercritical alcohol as a reaction medium, to thereby produce a depolymerized lignin product. In the second stage, the depolymerized lignin product is subjected to a sequential two-step hydroprocessing reaction to produce a reformulated hydrocarbon gasoline product. In the first hydroprocessing step, the depolymerized lignin is contacted with a hydrodeoxygenation catalyst to produce a hydrodeoxygenated intermediate product. In the second hydroprocessing step, the hydrodeoxygenated intermediate product is contacted with a hydrocracking/ring hydrogenation catalyst to produce the reformulated hydrocarbon gasoline product which includes various desirable naphthenic and paraffinic compounds.

Rhenium/Oxygen Interactions at Elevated Temperatures

NASA Technical Reports Server (NTRS)

Jacobson, Nathan; Myers, Dwight; Zhu, Dong-Ming; Humphrey, Donald

2000-01-01

The oxidation of pure rhenium is examined from 600-1400 C in oxygen/argon mixtures. Linear weight loss kinetics are observed. Gas pressures, flow rates, and temperatures are methodically varied to determine the rate controlling steps. The reaction at 600 and 800 C appears to be controlled by a chemical reaction step at the surface; whereas the higher temperature reactions appear to be controlled by gas phase diffusion of oxygen to the rhenium surface. Attack of the rhenium appears to be along grain boundaries and crystallographic planes.

Design and Synthesis of a Library of Tetracyclic Hydroazulenoisoindoles

PubMed Central

Brummond, Kay M.; Mao, Shuli; Shinde, Sunita N.; Johnston, Paul J.; Day, Billy W.

2009-01-01

Forty-four tetracyclic hydroazulenoisoindoles were synthesized via a tandem cyclopropanation/Cope rearrangement followed by a Diels-Alder sequence from easily available five-membered cyclic cross-conjugated trienones. These trienones were obtained from two different routes depending upon whether R1 and R2 are alkyl or amino acid derived functional groups, via a rhodium(I)-catalyzed cycloisomerization reaction. In order to increase diversity, four maleimides and two 1,2,4-triazoline-3,5-diones were used as dienophiles in the Diels-Alder step. Several Diels-Alder adducts were further reacted under palladium-catalyzed hydrogenation conditions, leading to a diastereoselective reduction of the trisubstituted double bond. This library has demonstrated rapid access to a variety of structurally complex natural product-like compounds via stereochemical diversity and building block diversity approaches. PMID:19366169

SnAP reagents for the one-step synthesis of medium-ring saturated N-heterocycles from aldehydes

NASA Astrophysics Data System (ADS)

Vo, Cam-Van T.; Luescher, Michael U.; Bode, Jeffrey W.

2014-04-01

Interest in saturated N-heterocycles as scaffolds for the synthesis of bioactive molecules is increasing. Reliable and predictable synthetic methods for the preparation of these compounds, especially medium-sized rings, are limited. We describe the development of SnAP (Sn amino protocol) reagents for the transformation of aldehydes into seven-, eight- and nine-membered saturated N-heterocycles. This process occurs under mild, room-temperature conditions and offers exceptional substrate scope and functional-group tolerance. Air- and moisture-stable SnAP reagents are prepared on a multigram scale from inexpensive starting materials by simple reaction sequences. These new reagents and processes allow widely available aryl, heteroaryl and aliphatic aldehydes to be converted into diverse N-heterocycles, including diazepanes, oxazepanes, diazocanes, oxazocanes and hexahydrobenzoxazonines, by a single synthetic operation.

Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

PubMed

Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

2016-08-28

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

Population Based Assessment of MHC Class 1 Antigens Down Regulation as Marker in Increased Risk for Development and Progression of Breast Cancer From Benign Breast Lesions

DTIC Science & Technology

2006-01-01

isolated using a routine salting-out method (DNA E-Z Prepkit, Orchid Diagnostics Europe, St Katelijne Waver, Belgium). Sequence based typing In...electrophoresis using ethidiumbromide to show the single 2 KB band before sequencing. Next, sequencing reactions were performed separately for exons 2, 3...Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute

Designing a fixed-blade gang ripsaw arbor with a pencil

Treesearch

Charles J. Gatchell; Charles J. Gatchell

1996-01-01

This paper presents a step-by-step procedure for designing the "best" sequence of saw spacings for a fixed-blade gang ripsaw arbor. Using the information contained in a cutting bill and knowledge of the lumber width distributions to be processed, thousands of possible saw spacing sequences can be reduced to a few good ones.

Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.).

PubMed

Allen, Alexandra M; Barker, Gary L A; Berry, Simon T; Coghill, Jane A; Gwilliam, Rhian; Kirby, Susan; Robinson, Phil; Brenchley, Rachel C; D'Amore, Rosalinda; McKenzie, Neil; Waite, Darren; Hall, Anthony; Bevan, Michael; Hall, Neil; Edwards, Keith J

2011-12-01

Food security is a global concern and substantial yield increases in cereal crops are required to feed the growing world population. Wheat is one of the three most important crops for human and livestock feed. However, the complexity of the genome coupled with a decline in genetic diversity within modern elite cultivars has hindered the application of marker-assisted selection (MAS) in breeding programmes. A crucial step in the successful application of MAS in breeding programmes is the development of cheap and easy to use molecular markers, such as single-nucleotide polymorphisms. To mine selected elite wheat germplasm for intervarietal single-nucleotide polymorphisms, we have used expressed sequence tags derived from public sequencing programmes and next-generation sequencing of normalized wheat complementary DNA libraries, in combination with a novel sequence alignment and assembly approach. Here, we describe the development and validation of a panel of 1114 single-nucleotide polymorphisms in hexaploid bread wheat using competitive allele-specific polymerase chain reaction genotyping technology. We report the genotyping results of these markers on 23 wheat varieties, selected to represent a broad cross-section of wheat germplasm including a number of elite UK varieties. Finally, we show that, using relatively simple technology, it is possible to rapidly generate a linkage map containing several hundred single-nucleotide polymorphism markers in the doubled haploid mapping population of Avalon × Cadenza. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Expression, Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

PubMed Central

Tanaka, Takeshi; Shima, Yasuyuki; Ogawa, Naoki; Nagayama, Koki; Yoshida, Takashi; Ohmachi, Tetsuo

2011-01-01

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH2)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase. PMID:21209787

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

PubMed Central

Belin, D; Bost, S; Vassalli, J D; Strub, K

1996-01-01

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery. Images PMID:8599930

A two-step recognition of signal sequences determines the translocation efficiency of proteins.

PubMed

Belin, D; Bost, S; Vassalli, J D; Strub, K

1996-02-01

The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery.

Accessory stimulus modulates executive function during stepping task

PubMed Central

Watanabe, Tatsunori; Koyama, Soichiro; Tanabe, Shigeo

2015-01-01

When multiple sensory modalities are simultaneously presented, reaction time can be reduced while interference enlarges. The purpose of this research was to examine the effects of task-irrelevant acoustic accessory stimuli simultaneously presented with visual imperative stimuli on executive function during stepping. Executive functions were assessed by analyzing temporal events and errors in the initial weight transfer of the postural responses prior to a step (anticipatory postural adjustment errors). Eleven healthy young adults stepped forward in response to a visual stimulus. We applied a choice reaction time task and the Simon task, which consisted of congruent and incongruent conditions. Accessory stimuli were randomly presented with the visual stimuli. Compared with trials without accessory stimuli, the anticipatory postural adjustment error rates were higher in trials with accessory stimuli in the incongruent condition and the reaction times were shorter in trials with accessory stimuli in all the task conditions. Analyses after division of trials according to whether anticipatory postural adjustment error occurred or not revealed that the reaction times of trials with anticipatory postural adjustment errors were reduced more than those of trials without anticipatory postural adjustment errors in the incongruent condition. These results suggest that accessory stimuli modulate the initial motor programming of stepping by lowering decision threshold and exclusively under spatial incompatibility facilitate automatic response activation. The present findings advance the knowledge of intersensory judgment processes during stepping and may aid in the development of intervention and evaluation tools for individuals at risk of falls. PMID:25925321

Sequence verification of synthetic DNA by assembly of sequencing reads

PubMed Central

Wilson, Mandy L.; Cai, Yizhi; Hanlon, Regina; Taylor, Samantha; Chevreux, Bastien; Setubal, João C.; Tyler, Brett M.; Peccoud, Jean

2013-01-01

Gene synthesis attempts to assemble user-defined DNA sequences with base-level precision. Verifying the sequences of construction intermediates and the final product of a gene synthesis project is a critical part of the workflow, yet one that has received the least attention. Sequence validation is equally important for other kinds of curated clone collections. Ensuring that the physical sequence of a clone matches its published sequence is a common quality control step performed at least once over the course of a research project. GenoREAD is a web-based application that breaks the sequence verification process into two steps: the assembly of sequencing reads and the alignment of the resulting contig with a reference sequence. GenoREAD can determine if a clone matches its reference sequence. Its sophisticated reporting features help identify and troubleshoot problems that arise during the sequence verification process. GenoREAD has been experimentally validated on thousands of gene-sized constructs from an ORFeome project, and on longer sequences including whole plasmids and synthetic chromosomes. Comparing GenoREAD results with those from manual analysis of the sequencing data demonstrates that GenoREAD tends to be conservative in its diagnostic. GenoREAD is available at www.genoread.org. PMID:23042248

Double-deprotected chemically amplified photoresists (DD-CAMP): higher-order lithography

NASA Astrophysics Data System (ADS)

Earley, William; Soucie, Deanna; Hosoi, Kenji; Takahashi, Arata; Aoki, Takashi; Cardineau, Brian; Miyauchi, Koichi; Chun, Jay; O'Sullivan, Michael; Brainard, Robert

2017-03-01

The synthesis and lithographic evaluation of 193-nm and EUV photoresists that utilize a higher-order reaction mechanism of deprotection is presented. Unique polymers utilize novel blocking groups that require two acid-catalyzed steps to be removed. When these steps occur with comparable reaction rates, the overall reaction can be higher order (<= 1.85). The LWR of these resists is plotted against PEB time for a variety of compounds to acquire insight into the effectiveness of the proposed higher-order mechanisms. Evidence acquired during testing of these novel photoresist materials supports the conclusion that higher-order reaction kinetics leads to improved LWR vs. control resists.

Recent advances in the chemistry of Rh carbenoids: multicomponent reactions of diazocarbonyl compounds

NASA Astrophysics Data System (ADS)

Medvedev, J. J.; Nikolaev, V. A.

2015-07-01

Multicomponent reactions of diazo compounds catalyzed by RhII complexes become a powerful tool for organic synthesis. They enable three- or four-step processes to be carried out as one-pot procedures (actually as one step) with high stereoselectivity to give complex organic molecules, including biologically active compounds. This review addresses recent results in the chemistry of Rh-catalyzed multicomponent reactions of diazocarbonyl compounds with the intermediate formation of N-, O- and C=O-ylides. The diastereo- and enantioselectivity of these reactions and the possibility of using various co-catalysts to increase the efficiency of the processes under consideration are discussed. The bibliography includes 120 references.

Detailed mechanism of the NO + CO reaction on Rh(1 0 0) and Rh(1 1 1): A first-principles study

NASA Astrophysics Data System (ADS)

Tan, Lu; Huang, Liangliang; Liu, Yingchun; Wang, Qi

2018-06-01

Through DFT calculations, the detailed mechanism of the catalytic NO + CO reaction, a prototypical system with great practical applications especially in the automobile-exhaust aftertreatment, was determined on Rh(1 0 0) and Rh(1 1 1). The elementary steps and their energy evolution were revealed. These steps include NO dissociation, N2 formation through N recombination, CO2 formation, and N2O formation, transformation, and dissociation. The reaction steps of NO2 formation and direct reaction between NO and CO were also studied, and were verified to be relatively insignificant in this reaction system. Results shed light on the atomic-level origin why Rh(1 0 0) is more active for this reaction system and more selective for the production of N2 versus N2O compared with Rh(1 1 1). Meanwhile, the preference between the two routes for N2 production, i.e., N atoms recombination and N2O as intermediate, was found to be dependent on the distribution of surface species and the interaction among them intricately. This work provides a basis for further kinetic modeling to investigate the catalytic properties on a realistic scale.

Elementary reaction modeling of reversible CO/CO2 electrochemical conversion on patterned nickel electrodes

NASA Astrophysics Data System (ADS)

Luo, Yu; Shi, Yixiang; Li, Wenying; Cai, Ningsheng

2018-03-01

CO/CO2 are the major gas reactant/product in the fuel electrode of reversible solid oxide cells (RSOC). This study proposes a two-charge-transfer-step mechanism to describe the reaction and transfer processes of CO-CO2 electrochemical conversion on a patterned Ni electrode of RSOC. An elementary reaction model is developed to couple two charge transfer reactions, C(Ni)+O2-(YSZ) ↔ CO(Ni)+(YSZ) +2e- and CO(Ni)+O2-(YSZ) ↔ CO2(Ni)+(YSZ)+2e-, with adsorption/desorption, surface chemical reactions and surface diffusion. This model well validates in both solid oxide electrolysis cell (SOEC) and solid oxide fuel cell (SOFC) modes by the experimental data from a patterned Ni electrode with 10 μm stripe width at different pCO (0-0.25 atm), pCO2 (0-0.35 atm) and operating temperature (600-700 °C). This model indicates SOEC mode is dominated by charge transfer step C(Ni)+O2-(YSZ)↔CO(Ni)+(YSZ) +2e-, while SOFC mode by CO(Ni)+ O2-(YSZ)↔CO2(Ni)+(YSZ)+2e- on the patterned Ni electrode. The sensitivity analysis shows charge transfer step is the major rate-determining step for RSOC, besides, surface diffusion of CO and CO2 as well as CO2 adsorption also plays a significant role in the electrochemical reaction of SOEC while surface diffusion of CO and CO2 desorption could be co-limiting in SOFC.