AntiHunter 2.0: increased speed and sensitivity in searching BLAST output for EST antisense transcripts.

PubMed

Lavorgna, Giovanni; Triunfo, Riccardo; Santoni, Federico; Orfanelli, Ugo; Noci, Sara; Bulfone, Alessandro; Zanetti, Gianluigi; Casari, Giorgio

2005-07-01

An increasing number of eukaryotic and prokaryotic genes are being found to have natural antisense transcripts (NATs). There is also growing evidence to suggest that antisense transcription could play a key role in many human diseases. Consequently, there have been several recent attempts to set up computational procedures aimed at identifying novel NATs. Our group has developed the AntiHunter program for the identification of expressed sequence tag (EST) antisense transcripts from BLAST output. In order to perform an analysis, the program requires a genomic sequence plus an associated list of transcript names and coordinates of the genomic region. After masking the repeated regions, the program carries out a BLASTN search of this sequence in the selected EST database, reporting via email the EST entries that reveal an antisense transcript according to the user-supplied list. Here, we present the newly developed version 2.0 of the AntiHunter tool. Several improvements have been added to this version of the program in order to increase its ability to detect a larger number of antisense ESTs. As a result, AntiHunter can now detect, on average, >45% more antisense ESTs with little or no increase in the percentage of the false positives. We also raised the maximum query size to 3 Mb (previously 1 Mb). Moreover, we found that a reasonable trade-off between the program search sensitivity and the maximum allowed size of the input-query sequence could be obtained by querying the database with the MEGABLAST program, rather than by using the BLAST one. We now offer this new opportunity to users, i.e. if choosing the MEGABLAST option, users can input a query sequence up to 30 Mb long, thus considerably improving the possibility to analyze longer query regions. The AntiHunter tool is freely available at http://bioinfo.crs4.it/AH2.0.

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

PubMed

Prykhozhij, Sergey V; Fuller, Charlotte; Steele, Shelby L; Veinotte, Chansey J; Razaghi, Babak; Robitaille, Johane M; McMaster, Christopher R; Shlien, Adam; Malkin, David; Berman, Jason N

2018-06-14

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

Expression of Antisense Long Noncoding RNAs as Potential Regulators in Rainbow Trout with Different Tolerance to Plant-Based Diets.

PubMed

Abernathy, Jason; Overturf, Ken

2018-01-04

Reformulation of aquafeeds in salmonid diets to include more plant proteins is critical for sustainable aquaculture. However, increasing plant proteins can lead to stunted growth and enteritis. Toward an understanding of the regulatory mechanisms behind plant protein utilization, directional RNA sequencing of liver tissues from a rainbow trout strain selected for growth on an all plant-protein diet and a control strain, both fed a plant diet for 12 weeks, were utilized to construct long noncoding RNAs. Antisense long noncoding RNAs were selected for differential expression and functional analyses since they have been shown to have regulatory actions within a genome. A total of 142 unique antisense long noncoding RNAs were differentially expressed between strains, 60 of which could be mapped to a gene. Genes underlying these noncoding RNAs are indicated in lipid metabolism and immunity. Six noncoding transcripts were also found to overlap with differentially expressed protein-coding genes, all of which were co-expressed. Associating variation in regulatory elements between rainbow trout strains with differing tolerance to plant-protein diets will assist in future studies toward increased gains throughout carnivorous aquaculture.

Cellular uptake mediated by epidermal growth factor receptor facilitates the intracellular activity of phosphorothioate-modified antisense oligonucleotides

PubMed Central

Wang, Shiyu; Allen, Nickolas; Vickers, Timothy A; Revenko, Alexey S; Sun, Hong; Liang, Xue-hai; Crooke, Stanley T

2018-01-01

Abstract Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs. PMID:29514240

Affimer proteins are versatile and renewable affinity reagents

PubMed Central

Tiede, Christian; Bedford, Robert; Heseltine, Sophie J; Smith, Gina; Wijetunga, Imeshi; Ross, Rebecca; AlQallaf, Danah; Roberts, Ashley PE; Balls, Alexander; Curd, Alistair; Hughes, Ruth E; Martin, Heather; Needham, Sarah R; Zanetti-Domingues, Laura C; Sadigh, Yashar; Peacock, Thomas P; Tang, Anna A; Gibson, Naomi; Kyle, Hannah; Platt, Geoffrey W; Ingram, Nicola; Taylor, Thomas; Coletta, Louise P; Manfield, Iain; Knowles, Margaret; Bell, Sandra; Esteves, Filomena; Maqbool, Azhar; Prasad, Raj K; Drinkhill, Mark; Bon, Robin S; Patel, Vikesh; Goodchild, Sarah A; Martin-Fernandez, Marisa; Owens, Ray J; Nettleship, Joanne E; Webb, Michael E; Harrison, Michael; Lippiat, Jonathan D; Ponnambalam, Sreenivasan; Peckham, Michelle; Smith, Alastair; Ferrigno, Paul Ko; Johnson, Matt; McPherson, Michael J; Tomlinson, Darren Charles

2017-01-01

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001 PMID:28654419

Antisense oligonucleotides as therapeutics for hyperlipidaemias.

PubMed

Crooke, Rosanne M

2005-07-01

Hyperlipidaemia, due to elevations of low-density lipoprotein cholesterol (LDL-C) or triglycerides (TGs), is recognised as a significant risk factor contributing to the development of coronary heart disease (CHD), the leading cause of morbidity and mortality in the Western world. Even though a variety of established antihyperlipidaemic agents are available, the majority of high-risk patients do not reach their lipid goals, indicating the need for new and more effective therapeutics to be used alone or as combination agents with existing drugs. Antisense oligonucleotides (ASOs), designed to specifically and selectively inhibit novel targets involved in cholesterol/TG homeostasis, represent a new class of agents that may prove beneficial for the treatment of hyperlipidaemias resulting from various genetic, metabolic or behavioural factors. This article describes the antisense technology platform, highlights the advantages of these novel drugs for the treatment of hyperlipidaemia and reviews the current research in this area.

SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

PubMed Central

Poulsen, Line Dahl; Kielpinski, Lukasz Jan; Salama, Sofie R.; Krogh, Anders; Vinther, Jeppe

2015-01-01

Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA–RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing. PMID:25805860

Waveguide-based optical chemical sensor

DOEpatents

Grace, Karen M [Ranchos de Taos, NM; Swanson, Basil I [Los Alamos, NM; Honkanen, Seppo [Tucson, AZ

2007-03-13

The invention provides an apparatus and method for highly selective and sensitive chemical sensing. Two modes of laser light are transmitted through a waveguide, refracted by a thin film host reagent coating on the waveguide, and analyzed in a phase sensitive detector for changes in effective refractive index. Sensor specificity is based on the particular species selective thin films of host reagents which are attached to the surface of the planar optical waveguide. The thin film of host reagents refracts laser light at different refractive indices according to what species are forming inclusion complexes with the host reagents.

Stereospecific Nickel-Catalyzed Cross-Coupling Reactions of Alkyl Grignard Reagents and Identification of Selective Anti-Breast Cancer Agents**

PubMed Central

Osborne, Charlotte A.; Moore, Curtis E.; Morrissette, Naomi S.; Jarvo, Elizabeth R.

2014-01-01

β-Hydrogen-containing alkyl Grignard reagents were used in a stereospecific nickel-catalyzed cross-coupling reaction to form sp3–sp3 carbon–carbon bonds. Aryl Grignard reagents were also utilized to synthesize 1,1-diarylalkanes. Several compounds synthesized by this method exhibited selective inhibition of proliferation of MCF-7 breast cancer cells. PMID:24478275

Widespread antisense transcription of Populus genome under drought.

PubMed

Yuan, Yinan; Chen, Su

2018-06-06

Antisense transcription is widespread in many genomes and plays important regulatory roles in gene expression. The objective of our study was to investigate the extent and functional relevance of antisense transcription in forest trees. We employed Populus, a model tree species, to probe the antisense transcriptional response of tree genome under drought, through stranded RNA-seq analysis. We detected nearly 48% of annotated Populus gene loci with antisense transcripts and 44% of them with co-transcription from both DNA strands. Global distribution of reads pattern across annotated gene regions uncovered that antisense transcription was enriched in untranslated regions while sense reads were predominantly mapped in coding exons. We further detected 1185 drought-responsive sense and antisense gene loci and identified a strong positive correlation between the expression of antisense and sense transcripts. Additionally, we assessed the antisense expression in introns and found a strong correlation between intronic expression and exonic expression, confirming antisense transcription of introns contributes to transcriptional activity of Populus genome under drought. Finally, we functionally characterized drought-responsive sense-antisense transcript pairs through gene ontology analysis and discovered that functional groups including transcription factors and histones were concordantly regulated at both sense and antisense transcriptional level. Overall, our study demonstrated the extensive occurrence of antisense transcripts of Populus genes under drought and provided insights into genome structure, regulation pattern and functional significance of drought-responsive antisense genes in forest trees. Datasets generated in this study serve as a foundation for future genetic analysis to improve our understanding of gene regulation by antisense transcription.

Nucleic acids--genes, drugs, molecular lego and more.

PubMed

Häner, Robert

2010-01-01

Chemically modified nucleic acids find widespread use as tools in research, as diagnostic reagents and even as pharmaceutical compounds. On the background of antisense research and development, the synthesis and evaluation of modified oligonucleotides was intensively pursued in the early to mid nineties in corporate research of former Ciba. Most of these efforts concentrated on the development of sugar and/or backbone-modified derivatives for pharmaceutical applications. Additionally, oligonucleotide metal conjugates were investigated with the goal to develop artificial ribonucleases. Since the turn of the millennium also the potential of non-nucleosidic and non-hydrogen bonding building blocks has increasingly been recognized. Such derivatives possess unique properties that may have an impact in the fields of materials and genetic research. In this brief account, we take a personal look back on some past as well as some recent results.

In vitro optimization of antisense oligodeoxynucleotide design: an example using the connexin gene family.

PubMed

Law, Lee Yong; Zhang, Wei V; Stott, N Susan; Becker, David L; Green, Colin R

2006-09-01

The completion of the human and mouse genomes has identified at least 20 connexin isomers in this family of intercellular channel proteins. However, there are no specific gap junction blockers or channel-blocking mimetic peptides available for the study of specific connexins. We designed antisense oligodeoxynucleotides that functionally reduce targeted connexin protein expression and can be used to reveal the biological function of individual connexins in vivo. Connexin mRNA was firstly exposed in vitro to deoxyribozymes complementing the sense coding sequence. Those that cleaved the target connexin mRNA in defined regions were used as the basis to design oligodeoxynucleotides to the accessible sites, thus taking into account tertiary mRNA configurations rather than relying on computed predictions. Antisense oligodeoxynucleotides designed to bind to accessible mRNA sites selectively reduced connexin26 and -43 mRNA expression in a corneal epithelium ex vivo model. Connexin43 protein levels were reduced correlating with the knockdown in mRNA and the protein's rapid turnover; protein levels of connexin26 did not alter, supporting lower turnover rates reported for that protein. We show, for the first time, an inexpensive and empirical approach to the preparation of specific and functional antisense oligodeoxynucleotides against known gene targets in the post-genomic era.

CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription

PubMed Central

Carlson, Jonathan; Yan, Jiyu; Akinsiku, Olusimidele T.; Schaefer, Malinda; Sabbaj, Steffanie; Bet, Anne; Levy, David N.; Heath, Sonya; Tang, Jianming; Kaslow, Richard A.; Walker, Bruce D.; Ndung’u, Thumbi; Goulder, Philip J.; Heckerman, David; Hunter, Eric; Goepfert, Paul A.

2010-01-01

Retroviruses pack multiple genes into relatively small genomes by encoding several genes in the same genomic region with overlapping reading frames. Both sense and antisense HIV-1 transcripts contain open reading frames for known functional proteins as well as numerous alternative reading frames (ARFs). At least some ARFs have the potential to encode proteins of unknown function, and their antigenic properties can be considered as cryptic epitopes (CEs). To examine the extent of active immune response to virally encoded CEs, we analyzed human leukocyte antigen class I–associated polymorphisms in HIV-1 gag, pol, and nef genes from a large cohort of South Africans with chronic infection. In all, 391 CEs and 168 conventional epitopes were predicted, with the majority (307; 79%) of CEs derived from antisense transcripts. In further evaluation of CD8 T cell responses to a subset of the predicted CEs in patients with primary or chronic infection, both sense- and antisense-encoded CEs were immunogenic at both stages of infection. In addition, CEs often mutated during the first year of infection, which was consistent with immune selection for escape variants. These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity. PMID:20065064

CONTINUOUS GAS ANALYZER

DOEpatents

Katz, S.; Weber, C.W.

1960-02-16

A reagent gas and a sample gas are chemically combined on a continuous basis in a reaction zone maintained at a selected temperature. The reagent gas and the sample gas are introduced to the reaction zone at preselected. constant molar rates of flow. The reagent gas and the selected gas in the sample mixture combine in the reaction zone to form a product gas having a different number of moles from the sum of the moles of the reactants. The difference in the total molar rates of flow into and out of the reaction zone is measured and indicated to determine the concentration of the selected gas.

Stereospecific nickel-catalyzed cross-coupling reactions of alkyl Grignard reagents and identification of selective anti-breast-cancer agents.

PubMed

Yonova, Ivelina M; Johnson, A George; Osborne, Charlotte A; Moore, Curtis E; Morrissette, Naomi S; Jarvo, Elizabeth R

2014-02-24

Alkyl Grignard reagents that contain β-hydrogen atoms were used in a stereospecific nickel-catalyzed cross-coupling reaction to form C(sp(3))-C(sp(3)) bonds. Aryl Grignard reagents were also utilized to synthesize 1,1-diarylalkanes. Several compounds synthesized by this method exhibited selective inhibition of proliferation of MCF-7 breast cancer cells. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Situ formation of apatite for sequestering radionuclides and heavy metals

DOEpatents

Moore, Robert C.

2003-07-15

Methods for in situ formation in soil of a permeable reactive barrier or zone comprising a phosphate precipitate, such as apatite or hydroxyapatite, which is capable of selectively trapping and removing radionuclides and heavy metal contaminants from the soil, while allowing water or other compounds to pass through. A preparation of a phosphate reagent and a chelated calcium reagent is mixed aboveground and injected into the soil. Subsequently, the chelated calcium reagent biodegrades and slowly releases free calcium. The free calcium reacts with the phosphate reagent to form a phosphate precipitate. Under the proper chemical conditions, apatite or hydroxyapatite can form. Radionuclide and heavy metal contaminants, including lead, strontium, lanthanides, and uranium are then selectively sequestered by sorbing them onto the phosphate precipitate. A reducing agent can be added for reduction and selective sequestration of technetium or selenium contaminants.

Concave Reagents. 20. Sterically Shielded m-Terphenyls as Selective Agents in General Protonations(1).

PubMed

Lüning, U.; Baumgartner, H.; Manthey, C.; Meynhardt, B.

1996-11-01

New m-terphenyls with acidic substituents in the 2'-position have been used in general protonations leading to reagent-controlled selectivity enhancements: up to 96:4 for the gamma/alpha-protonation of unsymmetrically substituted allyl anions, up to 97:3 for the protonation of cyclohexyl anions generating preferentially the thermodynamically less stable cis-products. In order to allow a general, reagent-controlled protonation the acidity of the protonating agent should be as low as possible.

C-fos mediates antipsychotic-induced neurotensin gene expression in the rodent striatum.

PubMed

Robertson, G S; Tetzlaff, W; Bedard, A; St-Jean, M; Wigle, N

1995-07-01

The ubiquitous inducibility of the immediate-early gene c-fos in the central nervous system has led to the search for downstream genes which are regulated by its product, Fos. Recent evidence suggests that c-fos induction by a single injection of the classical antipsychotic haloperidol may contribute to the subsequent increase in neurotensin gene expression in the rodent striatum. Consistent with this proposal, in the present study haloperidol-induced Fos-like immunoreactivity and neurotensin/neuromedin N messenger RNA were found to be expressed by the same population of striatal neurons. Moreover, inhibition of haloperidol-induced c-fos expression by intrastriatal injection of antisense phosphorothioate oligodeoxynucleotides complimentary either to bases 109-126 or 127-144 of c-fos attenuated the subsequent increase in neurotensin/neuromedin N messenger RNA. However, injection of a sense phosphorothioate oligodeoxynucleotide corresponding to bases 127-144 of c-fos did not reduce haloperidol-induced c-fos or neurotensin/neuromedin N expression. Furthermore, constitutive expression of Jun-like immunoreactivity in the striatum was not reduced by either the sense or antisense phosphorothioate oligodeoxynucleotides. Similarly, the sense and antisense phosphorothioate oligodeoxynucleotide failed to reduce proenkephalin messenger RNA, which is located in the same striatal neurons that express haloperidol-induced neurotensin/neuromedin N messenger RNA, which is located in the same striatal neurons that express haloperidol-induced neurotensin/neuromedin N messenger RNA. Lastly, haloperidol-induced increases in nerve growth factor I-A-, JunB- and FosB-like immunoreactivity and fosB messenger RNA were not decreased by intrastriatal injection of either the sense or antisense phosphorothioate oligodeoxynucleotides. These results indicate that the antisense phosphorothioate oligodeoxynucleotides attenuated haloperidol-induced neurotensin/neuromedin N expression by selectively reducing c-fos expression and emphasize the potential importance of immediate-early gene induction in the mechanism of action of this antipsychotic drug.

Identification of sequence motifs significantly associated with antisense activity.

PubMed

McQuisten, Kyle A; Peek, Andrew S

2007-06-07

Predicting the suppression activity of antisense oligonucleotide sequences is the main goal of the rational design of nucleic acids. To create an effective predictive model, it is important to know what properties of an oligonucleotide sequence associate significantly with antisense activity. Also, for the model to be efficient we must know what properties do not associate significantly and can be omitted from the model. This paper will discuss the results of a randomization procedure to find motifs that associate significantly with either high or low antisense suppression activity, analysis of their properties, as well as the results of support vector machine modelling using these significant motifs as features. We discovered 155 motifs that associate significantly with high antisense suppression activity and 202 motifs that associate significantly with low suppression activity. The motifs range in length from 2 to 5 bases, contain several motifs that have been previously discovered as associating highly with antisense activity, and have thermodynamic properties consistent with previous work associating thermodynamic properties of sequences with their antisense activity. Statistical analysis revealed no correlation between a motif's position within an antisense sequence and that sequences antisense activity. Also, many significant motifs existed as subwords of other significant motifs. Support vector regression experiments indicated that the feature set of significant motifs increased correlation compared to all possible motifs as well as several subsets of the significant motifs. The thermodynamic properties of the significantly associated motifs support existing data correlating the thermodynamic properties of the antisense oligonucleotide with antisense efficiency, reinforcing our hypothesis that antisense suppression is strongly associated with probe/target thermodynamics, as there are no enzymatic mediators to speed the process along like the RNA Induced Silencing Complex (RISC) in RNAi. The independence of motif position and antisense activity also allows us to bypass consideration of this feature in the modelling process, promoting model efficiency and reducing the chance of overfitting when predicting antisense activity. The increase in SVR correlation with significant features compared to nearest-neighbour features indicates that thermodynamics alone is likely not the only factor in determining antisense efficiency.

HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period

PubMed Central

2012-01-01

Background Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection. Results Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4–3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4–3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3′ LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-κΒ binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication. Conclusions The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4–3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle. PMID:22569184

In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes.

PubMed

Bey, E A; Cathcart, M K

2000-03-01

We previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion (O(2)(.-)) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O(2)(.-). This study definitively addresses the involvement of O(2)(.-) in monocyte oxidation of LDL. Using an antisense ODN designed to target p47phox mRNA, we found that treatment of monocytes with antisense ODN caused a substantial and selective decrease in expression of p47phox protein, whereas sense ODN was without effect. Corresponding functional assays demonstrated that antisense ODN inhibited production of O(2)(.-). As sense ODN caused no inhibition of O(2)(.-) production, these results suggested that inhibition of p47phox expression caused reduction in O(2)(.-) production. Evaluation of the contribution of O(2)(.-) production to monocyte-mediated oxidation of LDL lipids confirmed that O(2)(.-) production is required for LDL lipid oxidation as antisense ODN treatment significantly inhibited LDL oxidation whereas sense ODN treatment caused no inhibition. This is the first report of the reduction of NADPH oxidase activity in intact human monocytes by directly targeting the mRNA of a significant member of this enzyme complex. Our results provide convincing data that O(2)(.-) is indeed required for monocyte-mediated LDL oxidation.

Antisense technology: an emerging platform for cardiovascular disease therapeutics.

PubMed

Lee, Richard G; Crosby, Jeff; Baker, Brenda F; Graham, Mark J; Crooke, Rosanne M

2013-12-01

Antisense oligonucleotides and small interfering RNAs, which suppress the translation of specific mRNA target proteins, are emerging as important therapeutic modalities for the treatment of cardiovascular disease. Over the last 25 years, the advances in all aspects of antisense technology, as well as a detailed understanding of the mechanism of action of antisense drugs, have enabled their use as therapeutic agents. These advancements culminated in the FDA approval of the first chronically administered cardiovascular antisense therapeutic, mipomersen, which targets hepatic apolipoprotein B mRNA. This review provides a brief history of antisense technology, highlights the progression of mipomersen from preclinical studies to multiple Phase III registration trials, and gives an update on the status of other cardiovascular antisense therapeutics currently in the clinic.

Oriented and covalent immobilization of target molecules to solid supports: synthesis and application of a light-activatable and thiol-reactive cross-linking reagent.

PubMed

Collioud, A; Clémence, J F; Sänger, M; Sigrist, H

1993-01-01

Light-dependent oriented and covalent immobilization of target molecules has been achieved by combining two modification procedures: light-dependent coupling of target molecules to inert surfaces and thiol-selective reactions occurring at macromolecule or substrate surfaces. For immobilization purposes the heterobifunctional reagent N-[m-[3-(trifluoromethyl)diazirin-3-yl]phenyl]-4-maleimidobutyr amide was synthesized and chemically characterized. The photosensitivity of the carbene-generating reagent and its reactivity toward thiols were ascertained. Light-induced cross-linking properties of the reagent were documented (i) by reacting first the maleimide function with a thiolated surface, followed by carbene insertion into applied target molecules, (ii) by photochemical coupling of the reagent to an inert support followed by thermochemical reactions with thiol functions, and (iii) by thermochemical modification of target molecules prior to carbene-mediated insertion into surface materials. Procedures mentioned led to light-dependent covalent immobilization of target molecules including amino acids, a synthetic peptide, and antibody-derived F(ab') fragments. Topically selective, light-dependent immobilization was attained with the bifunctional reagent by irradiation of coated surfaces through patterned masks. Glass and polystyrene served as substrates. Molecular orientation is asserted by inherently available or selectively introduced terminal thiol functions in F(ab') fragments and synthetic polypeptides, respectively.

Antisense transcription is pervasive but rarely conserved in enteric bacteria.

PubMed

Raghavan, Rahul; Sloan, Daniel B; Ochman, Howard

2012-01-01

Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell's transcription machinery. IMPORTANCE Application of high-throughput methods has revealed the expression throughout bacterial genomes of transcripts encoded on the strand complementary to protein-coding genes. Because transcription is costly, it is usually assumed that these transcripts, termed antisense RNAs (asRNAs), serve some function; however, the role of most asRNAs is unclear, raising questions about their relevance in cellular processes. Because natural selection conserves functional elements, comparisons between related species provide a method for assessing functionality genome-wide. Applying such an approach, we assayed all transcripts in two closely related bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, and demonstrate that, although the levels of genome-wide antisense transcription are similarly high in both bacteria, only a small fraction of asRNAs are shared across species. Moreover, the promoters associated with asRNAs show no evidence of sequence conservation between, or even within, species. These findings indicate that despite the genome-wide transcription of asRNAs, many of these transcripts are likely nonfunctional.

Hitting bacteria at the heart of the central dogma: sequence-specific inhibition.

PubMed

Rasmussen, Louise Carøe Vohlander; Sperling-Petersen, Hans Uffe; Mortensen, Kim Kusk

2007-08-10

An important objective in developing new drugs is the achievement of high specificity to maximize curing effect and minimize side-effects, and high specificity is an integral part of the antisense approach. The antisense techniques have been extensively developed from the application of simple long, regular antisense RNA (asRNA) molecules to highly modified versions conferring resistance to nucleases, stability of hybrid formation and other beneficial characteristics, though still preserving the specificity of the original nucleic acids. These new and improved second- and third-generation antisense molecules have shown promising results. The first antisense drug has been approved and more are in clinical trials. However, these antisense drugs are mainly designed for the treatment of different human cancers and other human diseases. Applying antisense gene silencing and exploiting RNA interference (RNAi) are highly developed approaches in many eukaryotic systems. But in bacteria RNAi is absent, and gene silencing by antisense compounds is not nearly as well developed, despite its great potential and the intriguing possibility of applying antisense molecules in the fight against multiresistant bacteria. Recent breakthrough and current status on the development of antisense gene silencing in bacteria including especially phosphorothioate oligonucleotides (PS-ODNs), peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) will be presented in this review.

Methods and apparatuses for reagent delivery, reactive barrier formation, and pest control

DOEpatents

Gilmore, Tyler [Pasco, WA; Kaplan, Daniel I [Aiken, SC; Last, George [Richland, WA

2002-07-09

A reagent delivery method includes positioning reagent delivery tubes in contact with soil. The tubes can include a wall that is permeable to a soil-modifying reagent. The method further includes supplying the reagent in the tubes, diffusing the reagent through the permeable wall and into the soil, and chemically modifying a selected component of the soil using the reagent. The tubes can be in subsurface contact with soil, including groundwater, and can be placed with directional drilling equipment independent of groundwater well casings. The soil-modifying reagent includes a variety of gases, liquids, colloids, and adsorbents that may be reactive or non-reactive with soil components. The method may be used inter alia to form reactive barriers, control pests, and enhance soil nutrients for microbes and plants.

Hemorrhagic Shock-Induced Vascular Hyporeactivity in the Rat: Relationship to Gene Expression of Nitric Oxide Synthase, Endothelin-1, and Select Cytokines in Corresponding Organs

DTIC Science & Technology

2005-01-01

the Selected Genes Sense Antisense Product length (bp) G3PDH 5=-TCCTGCACCACCAACTGCTTAG-3= 5=-TGCTTCACCACCTTCTTGATGTC-3= 341 iNOS 5...GAPDH, as a housekeeping gene, was not affected significantly by the hemorrhage protocol. The results showed that mRNA levels of all enzymes and

Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers

PubMed Central

Latgé, Guillaume; Poulet, Christophe; Bours, Vincent; Jerusalem, Guy

2018-01-01

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers. PMID:29301303

Natural Antisense Transcripts: Molecular Mechanisms and Implications in Breast Cancers.

PubMed

Latgé, Guillaume; Poulet, Christophe; Bours, Vincent; Josse, Claire; Jerusalem, Guy

2018-01-02

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers.

Protein crystallography prescreen kit

DOEpatents

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

2007-10-02

A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

Protein crystallography prescreen kit

DOEpatents

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

2005-07-12

A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

Phthalocyaninatoruthenium(II), Hexakis(dimethylsulfoxide)Phthalocyaninatoruthenium(II), and Hexadis(dimethylsulfoxide-d6)phthalocyanin-atoruthenium(II), Three Highly Selective NMR Shift Reagents.

DTIC Science & Technology

1977-07-18

dimethylsu lfoxi de-d6) phthalocyanin — atoruthenium (II), Three Highly Selective NMR Shift Reagents ( ~‘ iby j \\ / Clement K. Choy and F•lalcolm E. Kenney...Running head : Phthalocyaninatorut henium( II) Shift Reagents I NTRODUCT ION Previously, work on FePc (Pc = phthalocyanine li gand , C32H16N8) show- ing...RuPc and dimethylsulfoxide -d 6 were re cted together and the product isolated, An nmr spectrum of the product showed only phthalocyanine resorar.ces

Silyl Glyoxylates. Conception and Realization of Flexible Conjunctive Reagents for Multicomponent Coupling

PubMed Central

Boyce, Gregory R.; Greszler, Stephen N.; Linghu, Xin; Malinowski, Justin T.; Nicewicz, David A.; Satterfield, Andrew D.; Schmitt, Daniel C.; Steward, Kimberly M.

2012-01-01

This Perspective describes the discovery and development of silyl glyoxylates, a new family of conjunctive reagents for use in multicomponent coupling reactions. The selection of the nucleophilic and electrophilic components determines whether the silyl glyoxylate reagent will function as a synthetic equivalent to the dipolar glycolic acid synthon, the glyoxylate anion synthon, or the α-keto ester homoenolate synthon. The ability to select for any of these reaction modes has translated to excellent structural diversity in the derived three- and four-component coupling adducts. Preliminary findings on the development of catalytic reactions using these reagents are detailed, as are the design and discovery of new reactions directed toward particular functional group arrays embedded within bioactive natural products. PMID:22414181

New reagent for extraction photomeric determination of anionic surface-active substances

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

Chernova, R.K.; Yastrebova, N.I.; Pankratov, A.N.

1995-02-01

The new reagent 2,6-diphenyl-4-(4-dimethylamino)styrylpyryl chloride is suggested for extraction photometric determination of anionic surface-active substances (SAS). This reagent possesses high sensitivity and selectivity, and can be used for the determination of both individual SAS of any kind and the total amount of anionic SAS. The reagent was used in analysis of highly mineralized statal waters and for the determination of sulfated products in polyoxyethylated alkylphenols.

Antisense oligonucleotides for the treatment of dyslipidaemia.

PubMed

Visser, Maartje E; Witztum, Joseph L; Stroes, Erik S G; Kastelein, John J P

2012-06-01

Antisense oligonucleotides (ASOs) are short synthetic analogues of natural nucleic acids designed to specifically bind to a target messenger RNA (mRNA) by Watson-Crick hybridization, inducing selective degradation of the mRNA or prohibiting translation of the selected mRNA into protein. Antisense technology has the ability to inhibit unique targets with high specificity and can be used to inhibit synthesis of a wide range of proteins that could influence lipoprotein levels and other targets. A number of different classes of antisense agents are under development. To date, mipomersen, a 2'-O-methoxyethyl phosphorothioate 20-mer ASO, is the most advanced ASO in clinical development. It is a second-generation ASO developed to inhibit the synthesis of apolipoprotein B (apoB)-100 in the liver. In Phase 3 clinical trials, mipomersen has been shown to significantly reduce plasma low-density lipoprotein cholesterol (LDL-c) as well as other atherogenic apoB containing lipoproteins such as lipoprotein (a) [Lp(a)] and small-dense LDL particles. Although concerns have been raised because of an increase in intrahepatic triglyceride content, preliminary data from long-term studies suggest that with continued treatment, liver fat levels tend to stabilize or decline. Further studies are needed to evaluate potential clinical relevance of these changes. Proprotein convertase subtilisin/kexin-9 (PCSK9) is another promising novel target for lowering LDL-c by ASOs. Both second-generation ASOs and ASOs using locked nucleic acid technology have been developed to inhibit PCSK9 and are under clinical development. Other targets currently being addressed include apoC-III and apo(a) or Lp(a). By directly inhibiting the synthesis of specific proteins, ASO technology offers a promising new approach to influence the metabolism of lipids and to control lipoprotein levels. Its application to a wide variety of potential targets can be expected if these agents prove to be clinically safe and effective.

Presidential Green Chemistry Challenge: 2007 Academic Award

EPA Pesticide Factsheets

Presidential Green Chemistry Challenge 2007 award winner, Professor Michael J. Krische, developed selective C-C bond-forming hydrogenation without organometallic reagents, eliminating hazardous reagents and hazardous waste.

Antisense Therapy in Neurology

PubMed Central

Lee, Joshua J.A.; Yokota, Toshifumi

2013-01-01

Antisense therapy is an approach to fighting diseases using short DNA-like molecules called antisense oligonucleotides. Recently, antisense therapy has emerged as an exciting and promising strategy for the treatment of various neurodegenerative and neuromuscular disorders. Previous and ongoing pre-clinical and clinical trials have provided encouraging early results. Spinal muscular atrophy (SMA), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy (including limb-girdle muscular dystrophy 2B; LGMD2B, Miyoshi myopathy; MM, and distal myopathy with anterior tibial onset; DMAT), and myotonic dystrophy (DM) are all reported to be promising targets for antisense therapy. This paper focuses on the current progress of antisense therapies in neurology. PMID:25562650

The development of a neutralizing amines based reagent for maintaining the water chemistry for medium and high pressures steam boilers

NASA Astrophysics Data System (ADS)

Butakova, M. V.; Orlov, K. A.; Guseva, O. V.

2017-11-01

An overview of the development for neutralizing amine based reagent for water chemistry of steam boilers for medium and high pressures was given. Total values of the neutralization constants and the distribution coefficients of the compositions selected as a main criteria for reagent composition. Experimental results of using this new reagent for water chemistry in HRSG of power plant in oil-production company are discussed.

Cross-reactions of reagents from streptococcal grouping kits with Streptococcus porcinus.

PubMed Central

Thompson, T; Facklam, R

1997-01-01

Streptococcus porcinus is usually associated with swine. Because we have received several isolates from human sources that had cross-reacted with commercial group B streptococcal reagents, we examined several commercial kits to determine the extent of this cross-reaction. Fifteen reference and 15 clinical strains of S. porcinus were tested for cross-reactions with group B streptococcal reagents from 12 different commercial kits. Cross-reactions were detected with all group B reagents, but the number of cross-reactions varied with each kit. We recommend that manufacturers of reagents designed to identify group B streptococci by serologic methods test their reagents for cross-reactions with selected S. porcinus cultures or antigens. PMID:9196216

Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

PubMed Central

Homann, M; Tzortzakaki, S; Rittner, K; Sczakiel, G; Tabler, M

1993-01-01

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1. Images PMID:8332489

Targeted nanoparticle delivery overcomes off-target immunostimulatory effects of oligonucleotides and improves therapeutic efficacy in chronic lymphocytic leukemia

PubMed Central

Yu, Bo; Mao, Yicheng; Bai, Li-Yuan; Herman, Sarah E. M.; Wang, Xinmei; Ramanunni, Asha; Jin, Yan; Mo, Xiaokui; Cheney, Carolyn; Chan, Kenneth K.; Jarjoura, David; Marcucci, Guido; Lee, Robert J.; Byrd, John C.

2013-01-01

Several RNA-targeted therapeutics, including antisense oligonucleotides (ONs), small interfering RNAs, and miRNAs, constitute immunostimulatory CpG motifs as an integral part of their design. The limited success with free antisense ONs in hematologic malignancies in recent clinical trials has been attributed to the CpG motif–mediated, TLR-induced prosurvival effects and inefficient target modulation in desired cells. In an attempt to diminish their off-target prosurvival and proinflammatory effects and specific delivery, as a proof of principle, in the present study, we developed an Ab-targeted liposomal delivery strategy using a clinically relevant CD20 Ab (rituximab)–conjugated lipopolyplex nanoparticle (RIT-INP)– and Bcl-2–targeted antisense G3139 as archetypical antisense therapeutics. The adverse immunostimulatory responses were abrogated by selective B cell–targeted delivery and early endosomal compartmentalization of G3139-encapsulated RIT-INPs, resulting in reduced NF-κB activation, robust Bcl-2 down-regulation, and enhanced sensitivity to fludarabine-induced cytotoxicity. Furthermore, significant in vivo therapeutic efficacy was noted after RIT-INP–G3139 administration in a disseminated xenograft leukemia model. The results of the present study demonstrate that CD20-targeted delivery overcomes the immunostimulatory properties of CpG-containing ON therapeutics and improves efficient gene silencing and in vivo therapeutic efficacy for B-cell malignancies. The broader implications of similar approaches in overcoming immunostimulatory properties of RNA-directed therapeutics in hematologic malignancies are also discussed. PMID:23165478

Reactive Derivatives of Nucleic Acids and Their Components as Affinity Reagents

NASA Astrophysics Data System (ADS)

Knorre, Dmitrii G.; Vlasov, Valentin V.

1985-09-01

The review is devoted to derivatives of nucleic acids and their components — nucleotides, nucleoside triphosphates, and oligonucleotides carrying reactive groups. Such derivatives are important tools for the investigation of protein-nucleic acid interactions and the functional topography of complex protein and nucleoprotein structures and can give rise to the prospect of being able to influence in a highly selective manner living organisms, including the nucleic acids and the nucleoproteins of the genetic apparatus. The review considers the principal groups of such reagents, the methods of their synthesis, and their properties which determine the possibility of their use for the selective (affinity) modification of biopolymers. The general principles of the construction of affinity reagents and their applications are analysed in relation to nucleotide affinity reagents. The bibliography includes 121 references.

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

PubMed Central

Ludwig, Linda B; Ambrus, Julian L; Krawczyk, Kristie A; Sharma, Sanjay; Brooks, Stephen; Hsiao, Chiu-Bin; Schwartz, Stanley A

2006-01-01

Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Results Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development. PMID:17090330

Automated macromolecular crystallization screening

DOEpatents

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

2005-03-01

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

E- or Z-Selective synthesis of 4-fluorovinyl-1,2,3-triazoles with fluorinated second-generation Julia-Kocienski reagents.

PubMed

Kumar, Rakesh; Singh, Govindra; Todaro, Louis J; Yang, Lijia; Zajc, Barbara

2015-02-07

A highly modular approach to N-substituted 4-(1-fluorovinyl)triazoles is described. In situ desilylation and Cu-catalyzed ligation reaction of TMS-protected α-fluoropropargyl benzothiazole sulfone with aryl, alkyl, and metallocenyl azides furnished second-generation Julia-Kocienski reagents in good to excellent yields. Condensation reactions of these reagents with aldehydes can be tuned to yield E or Z-alkenes selectively. Under mild conditions with DBU as the base, reactions of aldehydes furnished E-alkenes as the major isomer. On the other hand, in condensation reactions with LHMDS as the base and in appropriate solvents, both aldehydes and ketones reacted to yield fluoroalkenes with Z-selectivity. Stereochemical assignment of E/Z olefins obtained in the reaction of a ketone with two Julia reagents was performed via X-ray crystallographic analysis and comparisons of NMR data. The method allows efficient and ready diversification of the N1-substituent and substituents at the double bond.

E- or Z-selective synthesis of 4-fluorovinyl-1,2,3-triazoles with fluorinated second-generation Julia-Kocienski reagents

PubMed Central

Kumar, Rakesh; Singh, Govindra; Todaro, Louis J.; Yang, Lijia; Zajc, Barbara

2016-01-01

A highly modular approach to N-substituted 4-(1-fluorovinyl)triazoles is described. In situ desilylation and Cu-catalyzed ligation reaction of TMS-protected α-fluoropropargyl benzothiazole sulfone with aryl, alkyl, and metallocenyl azides furnished second-generation Julia-Kocienski reagents in good to excellent yields. Condensation reactions of these reagents with aldehydes can be tuned to yield E or Z-alkenes selectively. Under mild conditions with DBU as base, reactions of aldehydes furnished E-alkenes as the major isomer. On the other hand, in condensations with LHMDS as base and in appropriate solvents, both aldehydes and ketones reacted to yield fluoroalkenes with Z-selectivity. Stereochemical assignment to E/Z olefins obtained in the reaction of a ketone with two Julia reagents was performed via X-ray crystallographic analysis and comparisons of NMR data. The method allows efficient and ready diversification of N1-substituent and substituents at the double bond. PMID:25491086

High-performance reagent modes for flotation recovery of platiniferous copper and nickel sulfides from hard-to-beneficiate ores

NASA Astrophysics Data System (ADS)

Matveeva, T. N.; Chanturiya, V. A.

2017-07-01

The paper presents the results of the recent research performed in IPKON Russian Academy of Sciences that deals with development and substantiation of new selective reagents for effective flotation recovery of non-ferrous and noble metals from refractory ores. The choice and development of new selective reagents PTTC, OPDTC, modified butylxanthate (BXm) and modified diethyl-dithiocarbamate (DEDTCm) to float platiniferous copper and nickel sulfide minerals from hard-to-beneficiate ores is substantiated. The mechanism of reagents adsorption and regulation of minerals floatability is discussed. The study of reagent modes indicates that by combining PTTC with the modified xanthate results in 6 - 7 % increase in the recovery of copper, nickel and PGM in the flotation of the low-sulfide platiniferous Cu-Ni ore from the Fedorovo-Panskoye deposit. The substitution of OPDTC for BX makes it possible to increase recovery of Pt by 13 %, Pd by 9 % and 2 - 4 times the noble metal content in the flotation concentrate.

Microfluidics in the selection of affinity reagents for the detection of cancer: paving a way towards future diagnostics.

PubMed

Hung, Lien-Yu; Wang, Chih-Hung; Fu, Chien-Yu; Gopinathan, Priya; Lee, Gwo-Bin

2016-08-07

Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.

A multi-model approach to nucleic acid-based drug development.

PubMed

Gautherot, Isabelle; Sodoyer, Regís

2004-01-01

With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies. Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency. Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic. Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product. Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites. Individually, each strategy has its drawbacks from a drug development perspective. Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment. In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis. Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications. Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response. For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such approaches also have limitations. To streamline nucleic acid drug discovery, we propose a multi-model strategy integrating high-throughput-adapted bacterial screening, followed by reporter-based and/or natural cellular models and potentially also in vitro assays for characterization of the most promising candidate sequences, before final in vivo testing.

Caspase selective reagents for diagnosing apoptotic mechanisms.

PubMed

Poreba, Marcin; Groborz, Katarzyna; Navarro, Mario; Snipas, Scott J; Drag, Marcin; Salvesen, Guy S

2018-05-10

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.

A New Colorimetric Assay of Tabletop Sweeteners Using a Modified Biuret Reagent: An Analytical Chemistry Experiment for the Undergraduate Curriculum

ERIC Educational Resources Information Center

Fenk, Christopher J.; Kaufman, Nathan; Gerbig, Donald G., Jr.

2007-01-01

A new, fast and effective colorimetric analysis of the artificial sweetener aspartame is presented for application in undergraduate laboratory courses. This new method incorporates the use of a modified biuret reagent for selective detection and analysis of aspartame in aqueous solutions. The modified reagent is less caustic than the traditional…

Flotation selectivity of novel alkyl dicarboxylate reagents for apatite-calcite separation.

PubMed

Karlkvist, Tommy; Patra, Anuttam; Rao, Kota Hanumantha; Bordes, Romain; Holmberg, Krister

2015-05-01

The investigation aims to demonstrate the conceptual thoughts behind developing mineral specific reagents for use in flotation of calcium containing ores. For this purpose, a series of dicarboxylate-based surfactants with varying distance between the carboxylate groups (one, two or three methylene groups) was synthesized. A surfactant with the same alkyl chain length but with only one carboxylate group was also synthesized and evaluated. The adsorption behavior of these new reagents on pure apatite and pure calcite surfaces was studied using Hallimond tube flotation, FTIR and ζ potential measurements. The relation between the adsorption behavior of a given surfactant at a specific mineral surface and its molecular structure over a range of concentrations and pH values, as well as the region of maximum recovery, was established. It was found that one of the reagents, with a specific distance between the carboxylate groups, was much more selective for a particular mineral surface than the other homologues. For example, out of the four compounds synthesized, only the one where the carboxylate groups were separated by a single methylene group floated apatite but not calcite, whereas calcite was efficiently floated with the monocarboxylic reagent, but not with the other reagents synthesized. This selective adsorption of a given surfactant to a particular mineral surface relative to other mineral surfaces as evidenced in the flotation studies was substantiated by ζ potential and infra-red spectroscopy data. Copyright © 2014 Elsevier Inc. All rights reserved.

Selective oxidation of steroidal allylic alcohols using pyrazole and pyridinium chlorochoromate.

PubMed

Parish, E J; Chitrakorn, S; Lowery, S

1984-07-01

ABASTRACT: This paper presents a modified method for the selective oxidation of allylic alchols. Pyrazole, when used with pyridinium chlorochromate, is a mild and useful reagent system for the rapid and selective oxidation of steroidal allylic alcohols to the corresponding α, β-unsaturated ketones. The reaction of each substrate was carried out by adding the oxidant to a dry methylene chloride solution containing pyrazole and an allylic alchol. This report is the first on the use of pyrazole to augment selective oxidation by a chronium (VI) reagent.

Central and peripheral administration of antisense oligonucleotide targeting amyloid-β protein precursor improves learning and memory and reduces neuroinflammatory cytokines in Tg2576 (AβPPswe) mice.

PubMed

Farr, Susan A; Erickson, Michelle A; Niehoff, Michael L; Banks, William A; Morley, John E

2014-01-01

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Currently, there are no therapies to stop or reverse the symptoms of AD. We have developed an antisense oligonucleotide (OL-1) against the amyloid-β protein precursor (AβPP) that can decrease AβPP expression and amyloid-β protein (Aβ) production. This antisense rapidly crosses the blood-brain barrier, reverses learning and memory impairments, reduces oxidative stress, and restores brain-to-blood efflux of Aβ in SAMP8 mice. Here, we examined the effects of this AβPP antisense in the Tg2576 mouse model of AD. We administered the OL-1 antisense into the lateral ventricle 3 times at 2week intervals. Seventy-two hours after the third injection, we tested learning and memory in T-maze foot shock avoidance. In the second study, we injected the mice with OL-1 antisense 3 times at 2-week intervals via the tail vein. Seventy-two hours later, we tested learning and memory T-maze, novel object recognition, and elevated plus maze. At the end of behavioral testing, brain tissue was collected. OL-1 antisense administered centrally improved acquisition and retention of T-maze foot shock avoidance. OL-1 antisense administered via tail vein improved learning and memory in both T-maze foot shock avoidance and novel object-place recognition. In the elevated plus maze, the mice which received OL-1 antisense spent less time in the open arms and had fewer entries into the open arms indicating reduced disinhibitation. Biochemical analyses reveal significant reduction of AβPP signal and a reduction of measures of neuroinflammation. The current findings support the therapeutic potential of OL-1 AβPP antisense.

Peripheral administration of antisense oligonucleotides targeting the amyloid-β protein precursor reverses AβPP and LRP-1 overexpression in the aged SAMP8 mouse brain.

PubMed

Erickson, Michelle A; Niehoff, Michael L; Farr, Susan A; Morley, John E; Dillman, Lucy A; Lynch, Kristin M; Banks, William A

2012-01-01

The senescence accelerated mouse-prone 8 (SAMP8) mouse model of Alzheimer's disease has a natural mutation leading to age-related increases in the amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) in the brain, memory impairment, and deficits in Aβ removal from the brain. Previous studies show that centrally administered antisense oligonucleotide directed against AβPP can decrease AβPP expression and Aβ production in the brains of aged SAMP8 mice, and improve memory. The same antisense crosses the blood-brain barrier and reverses memory deficits when injected intravenously. Here, we give 6 μg of AβPP or control antisense 3 times over 2 week intervals to 12 month old SAMP8 mice. Object recognition test was done 48 hours later, followed by removal of whole brains for immunoblot analysis of AβPP, low-density lipoprotein-related protein-1 (LRP-1), p-glycoprotein (Pgp), receptor for advanced glycation endproducts (RAGE), or ELISA of soluble Aβ(40). Our results show that AβPP antisense completely reverses a 30% age-associated increase in AβPP signal (p < 0.05 versus untreated 4 month old SAMP8). Soluble Aβ(40) increased with age, but was not reversed by antisense. LRP-1 large and small subunits increased significantly with age (147.7%, p < 0.01 and 123.7%, p < 0.05 respectively), and AβPP antisense completely reversed these increases (p < 0.05). Pgp and RAGE were not significantly altered with age or antisense. Antisense also caused improvements in memory (p < 0.001). Together, these data support the therapeutic potential of AβPP antisense and show a unique association between AβPP and LRP-1 expression in the SAMP8 mouse.

Regulation of the LDL receptor gene expression by hormones.

PubMed

Streicher, R; Kotzka, J; Müller-Wieland, D; Krone, W

1998-01-01

Promoter activity of the LDL receptor gene is stimulated by insulin and estradiol and mediated by SRE-1, which acts as a hormone sensitive cis-elemente. Using the antisense technique we reveal that SREBP-1 is selectively involved in the signal transduction pathway of insulin and IGF-I.

Development of Novel Environmentally Sustainable Binders for Energetic Formulations

DTIC Science & Technology

2015-06-01

Senenayake, C. H., Addition of Grignard reagents to aryl acid chlorides: an efficient synthesis of aryl ketones, Org. Lett. 2005, 7, 5593- 5595. (b) Gowda...M. S.; Pande, S. S.; Ramakrishna, R. A.; Prabhu, K. R., Acylation of Grignard reagents mediated by N-methylpyrrolidinone: a remarkable selectivity...acid moiety to introduce the necessary linker. Although the standard isocyanates- based reagents induce significant toxicity, the isocyanate function

Epithelin/Granulin Precursor Expression in Human Breast Carcinoma

DTIC Science & Technology

1998-09-01

antisense RNA as an inhibitor of oncogenic protein production (13). The development of stable transfected clones with antisense cDNA is advantageous...in that it allows a continuous supply of antisense RNA to disrupt protein synthesis, and it is well suited for in vivo tumorigenic assays. Our...processed form epithelin 1 in normal mammary epithelial cells and mammary carcinoma cells. 3- Effect of inhibition of PCDGF expression ( antisense

Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.

PubMed Central

Nato, F; Mazie, J C; Fournier, J M; Slizewicz, B; Sagot, N; Guibourdenche, M; Postic, D; Riou, J Y

1991-01-01

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents. PMID:1909346

Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

PubMed Central

Perez, J R; Higgins-Sochaski, K A; Maltese, J Y; Narayanan, R

1994-01-01

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion. Images PMID:8035811

An in vivo and in silico approach to study cis-antisense: a short cut to higher order response

NASA Astrophysics Data System (ADS)

Courtney, Colleen; Varanasi, Usha; Chatterjee, Anushree

2014-03-01

Antisense interactions are present in all domains of life. Typically sense, antisense RNA pairs originate from overlapping genes with convergent face to face promoters, and are speculated to be involved in gene regulation. Recent studies indicate the role of transcriptional interference (TI) in regulating expression of genes in convergent orientation. Modeling antisense, TI gene regulation mechanisms allows us to understand how organisms control gene expression. We present a modeling and experimental framework to understand convergent transcription that combines the effects of transcriptional interference and cis-antisense regulation. Our model shows that combining transcriptional interference and antisense RNA interaction adds multiple-levels of regulation which affords a highly tunable biological output, ranging from first order response to complex higher-order response. To study this system we created a library of experimental constructs with engineered TI and antisense interaction by using face-to-face inducible promoters separated by carefully tailored overlapping DNA sequences to control expression of a set of fluorescent reporter proteins. Studying this gene expression mechanism allows for an understanding of higher order behavior of gene expression networks.

miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA

PubMed Central

Hansen, Thomas B; Wiklund, Erik D; Bramsen, Jesper B; Villadsen, Sune B; Statham, Aaron L; Clark, Susan J; Kjems, Jørgen

2011-01-01

MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels. PMID:21964070

Antisense protein kinase A RIalpha inhibits 7,12-dimethylbenz(a)anthracene-induction of mammary cancer: blockade at the initial phase of carcinogenesis.

PubMed

Nesterova, Maria V; Cho-Chung, Yoon S

2004-07-01

There are two types of cyclic AMP (cAMP)-dependent protein kinase (PKA), type I (PKA-I) and type II (PKA-II), which share a common catalytic (C) subunit but contain distinct regulatory (R) subunits, RI versus RII, respectively. Evidence suggests that increased expression of PKA-I and its regulatory subunit (RIalpha) correlates with tumorigenesis and tumor growth. We investigated the effect of sequence-specific inhibition of RIalpha gene expression at the initial phase of 7,12-dimethylbenz(alphaa)anthracene (DMBA)-induced mammary carcinogenesis. Antisense RIalpha oligodeoxynucleotide (ODN) targeted against PKA RIalpha was administered (0.1 mg/day/rat, i.p.) 1 day before DMBA intubation and during the first 9 days post-DMBA intubation to determine the anticarcinogenic effects. Antisense RIalpha, in a sequence-specific manner, inhibited the tumor production. At 90 days after DMBA intubation, untreated controls and RIalpha-antisense-treated rats exhibited an average mean number of tumors per rat of 4.2 and 1.8, respectively, and 90% of control and 45% of antisense-treated animals had tumors. The antisense also delayed the first tumor appearance. An increase in RIalpha and PKA-I levels in the mammary gland and liver preceded DMBA-induced tumor production, and antisense down-regulation of RIalpha restored normal levels of PKA-I and PKA-II in these tissues. Antisense RIalpha in the liver induced the phase II enzymes, glutathione S-transferase and quinone oxidoreductase, c-fos protein, and activator protein 1 (AP-1)- and cAMP response element (CRE)-directed transcription. In the mammary glands, antisense RIalpha promoted DNA repair processes. In contrast, the CRE transcription-factor decoy could not mimic these effects of antisense RIalpha. The results demonstrate that RIalpha antisense produces dual anticarcinogenic effects: (a) increasing DMBA detoxification in the liver by increasing phase II enzyme activities, increasing CRE-binding-protein phosphorylation and enhancing CRE- and Ap-1-directed transcription; and (b) activating DNA repair processes in the mammary gland by down-regulating PKA-I.

Analysis of Antisense Expression by Whole Genome Tiling Microarrays and siRNAs Suggests Mis-Annotation of Arabidopsis Orphan Protein-Coding Genes

PubMed Central

Richardson, Casey R.; Luo, Qing-Jun; Gontcharova, Viktoria; Jiang, Ying-Wen; Samanta, Manoj; Youn, Eunseog; Rock, Christopher D.

2010-01-01

Background MicroRNAs (miRNAs) and trans-acting small-interfering RNAs (tasi-RNAs) are small (20–22 nt long) RNAs (smRNAs) generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs) are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery. Principal Findings We explored rice (Oryza sativa) sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans) and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis ‘orphan’ hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM) was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the “ancient” (deeply conserved) class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for “new” rapidly-evolving MIRNA genes. Conclusions Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other kingdoms, which can provide insight into antisense transcription, miRNA evolution, and post-transcriptional gene regulation. PMID:20520764

The Role of CREB in CML

DTIC Science & Technology

2008-02-01

Feng YQ et al. Anti-beta s- ribozyme reduces beta s mRNA levels in transgenic mice: Potential application to the gene therapy of sickle cell anemia... ribozymes . RNA 2003;9:1254–1263. 13 Pace BS, Qian X, Ofori-Acquah SF. Selective inhibition of beta-globin RNA transcripts by antisense RNA molecules. Cell

Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.

PubMed

Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

2016-09-20

Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species.

In situ formation of phosphate barriers in soil

DOEpatents

Moore, Robert C.

2002-01-01

Reactive barriers and methods for making reactive barriers in situ in soil for sequestering soil ontaminants including actinides and heavy metals. The barrier includes phosphate, and techniques are disclosed for forming specifically apatite barriers. The method includes injecting dilute reagents into soil in proximity to a contamination plume or source such as a waste drum to achieve complete or partial encapsulation of the waste. Controlled temperature and pH facilitates rapid formation of apatite, for example, where dilute aqueous calcium chloride and dilute aqueous sodium phosphate are the selected reagents. Mixing of reagents to form precipitate is mediated and enhanced through movement of reagents in soil as a result of phenomena including capillary action, movement of groundwater, soil washing and reagent injection pressure.

Voltage-gated calcium channel and antisense oligonucleotides thereto

NASA Technical Reports Server (NTRS)

Friedman, Peter A. (Inventor); Duncan, Randall L. (Inventor); Hruska, Keith A. (Inventor); Barry, Elizabeth L. R. (Inventor)

1998-01-01

An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

PubMed

Cantin, E M; Podsakoff, G; Willey, D E; Openshaw, H

1992-01-01

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

RNAi triggered by symmetrically transcribed transgenes in Drosophila melanogaster.

PubMed Central

Giordano, Ennio; Rendina, Rosaria; Peluso, Ivana; Furia, Maria

2002-01-01

Specific silencing of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA molecules. In vivo, these molecules can be generated either by transcription of sequences having an inverted-repeat (IR) configuration or by simultaneous transcription of sense-antisense strands. Since IR constructs are difficult to prepare and can stimulate genomic rearrangements, we investigated the silencing potential of symmetrically transcribed sequences. We report that Drosophila transgenes whose sense-antisense transcription was driven by two convergent arrays of Gal4-dependent UAS sequences can induce specific, dominant, and heritable repression of target genes. This effect is not dependent on a mechanism based on homology-dependent DNA/DNA interactions, but is directly triggered by transcriptional activation and is accompanied by specific depletion of the endogenous target RNA. Tissue-specific induction of these transgenes restricts the target gene silencing to selected body domains, and spreading phenomena described in other cases of post-transcriptional gene silencing (PTGS) were not observed. In addition to providing an additional tool useful for Drosophila functional genomic analysis, these results add further strength to the view that events of sense-antisense transcription may readily account for some, if not all, PTGS-cosuppression phenomena and can potentially play a relevant role in gene regulation. PMID:11861567

Antisense reduction of tau in adult mice protects against seizures.

PubMed

DeVos, Sarah L; Goncharoff, Dustin K; Chen, Guo; Kebodeaux, Carey S; Yamada, Kaoru; Stewart, Floy R; Schuler, Dorothy R; Maloney, Susan E; Wozniak, David F; Rigo, Frank; Bennett, C Frank; Cirrito, John R; Holtzman, David M; Miller, Timothy M

2013-07-31

Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS--brain and spinal cord tissue, interstitial fluid, and CSF--while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability.

Study of Highly Selective and Efficient Thiol Derivatization using Selenium Reagents by Mass Spectrometry

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

Xu, Kehua; Zhang, Yun W.; Tang, Bo

2010-08-15

Biological thiols are critical physiological components and their detection often involves derivatization. This paper reports a systemic mass spectrometry (MS) investigation of the cleavage of Se-N bond by thiol to form a new Se-S bond, the new selenium chemistry for thiol labeling. Our data shows that the reaction is highly selective, rapid, reversible and efficient. For instance, among twenty amino acids, only cysteine was found to be reactive with Se-N containing reagents and the reaction takes place in seconds. By adding dithiothreitol (DTT), the newly formed Se-S bond of peptides/proteins can be reduced back to free thiol. The high selectivitymore » and excellent reversibility of the reaction provide potential of using this chemistry for selective identification of thiol compounds or enriching and purifying thiol peptides/proteins. In addition, the derivatized thiol peptides have interesting dissociation behavior, which is tunable using different selenium reagents. For example, by introducing an adjacent nucleophilic group into the selenium reagent in the case of using ebselen, the reaction product of ebselen with glutathione (GSH) is easy to lose the selenium tag upon collision-induced dissociation (CID), which is useful to "fish out" those peptides containing free cysteine residues by precursor ion scan. By contrast, the selenium tag of N-(phenylseleno) phthalimide reagent can be stable and survive in CID process, which would be of value in pinpointing thiol location using a top-down proteomic approach. Also, the high conversion yield of the reaction allows the counting of total number of thiol in proteins. We believe that ebselen or N-(phenylseleno) phthalimide as tagging thiol-protein reagents will have important applications in both qualitative and quantitative analysis of different thiol-proteins derived from living cells by MS method.« less

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

PubMed

Tang, Danming; Lam, Cynthia; Louie, Salina; Hoi, Kam Hon; Shaw, David; Yim, Mandy; Snedecor, Brad; Misaghi, Shahram

2018-01-01

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting Cancer with Antisense Oligomers

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

Hnatowich, DJ

With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their nativemore » and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes radiotherapy rather than imaging, the delivery problem may be obviated by attaching the antisense oligomer to an antitumor antibody to improve delivery following intravenous administration. Since many antibodies are trapped in endosomes following internalization, a cell penetrating peptide such as tat will also be included to ensure transport of the complex without entrapment. Rather than covalent conjugation of the three entities, we are using streptavidin as linker after biotinylated each component. Our recent efforts have concentrated on establishing the influence of the streptavidin linker on the properties of each component within the delivery nanoparticle. Thus, we have shown that the Herceptin antibody, when linked to a labeled oligomer via streptavidin, remains capable of directing the label oligomer to Her2+ tumor cells in vitro and Her2+ tumor xenografts in mice. In addition, we have demonstrated that a labeled antisense oligomer within the nanoparticle remains capable of migrating to the nucleus and binding to its target mRNA in vitro and in vivo. We have shown that the tat peptide also preserves its properties of cell transport when incubated as one component of the nanoparticle. Most recently, we have addressed another of our concerns, namely whether the streptavidin would adversely effect the biodistribution of the antisense oligomer. We were pleased to find that the 99mTc-labeled antisense MORF within the Herceptin three component and two component nanoparticles accumulated and was retained in tumor in a manner suggestive of radiolabeled Herceptin itself. Thus the preserved properties within the streptavidin delivery nanoparticle of the Herceptin antibody, the tat peptide and the 111In labeled antisense MORF oligomer will explain why we have successfully demonstrated an Auger electron-mediated, antisense-mediated radiotherapy in cells in culture. One remaining concern is that the delivery nanoparticle may deliver the Auger electron emitting radionuclide to the nucleus of normal cells as well as tumor cells. We have now performed tumored mice studies of the three component delivery nanoparticle with the antisense MORF labeled with Cy3 so that tissue slices could be examined by immunohistology for evidence of MORF accumulations in the nuclei of both tumor and normal tissues. Microscopic examination shows nuclear staining in approximately 20% of the tumor cells in animals injected with the antisense nanoparticle and 10% of the tumor cells in animals receiving the sense nanoparticle, whereas no nuclear staining is seen in the tumor cells of mice given the PBS injection as another control. No nuclear staining was observed in all sections from all normal organs. Finally, my colleagues and I wish to express our gratitude to the DOE for their generous support of our research at a time when the NIH was unwilling to fund what they believed to be a risky« less

Growth inhibition of N1E-115 mouse neuroblastoma cells by c-myc or N-myc antisense oligodeoxynucleotides causes limited differentiation but is not coupled to neurite formation.

PubMed

Larcher, J C; Basseville, M; Vayssiere, J L; Cordeau-Lossouarn, L; Croizat, B; Gros, F

1992-06-30

Antisense oligodeoxynucleotides were found to be stable in the culture medium containing fetal calf serum (heat-inactivated 30 minutes at 65 degrees C) and in cells. Antisense oligomer treatment causes cessation of mitoses, but does not lead to morphological differentiation. Under antisense conditions, we have observed an increase in the amount of two neurospecific protein, namely peripherin and gamma-enolase. Comparison of the results obtained with chemical inducers and antisense oligodeoxynucleotides allows us to postulate three phases in N1E-115 differentiation: the first correspond to the arrest of mitosis, the second to the expression of a limited neuronal program, and the third to the morphological and electrophysiological differentiation.

Antisense therapy and emerging applications for the management of dyslipidemia.

PubMed

Toth, Peter P

2011-01-01

Because a significant percentage of patients who require high-dose statin therapy for dyslipidemia experience treatment-related muscle symptoms and an inconsistent clinical response, alternative or adjunctive approaches to the management of dyslipidemia are needed. One alternative approach, antisense therapy, may offer an effective and well-tolerated option for patients not satisfactorily responsive to or intolerant to standard pharmacologic dyslipidemia therapies. This review provides an overview of antisense technology and its potential role in the management of dyslipidemia. Source material was obtained primarily from the published literature identified through a search of the PubMed database. Antisense technology is an evolving approach to therapy that has gone through a series of refinements to enhance molecular stability, potency, and tolerability. Mipomersen is an antisense molecule capable of producing clinically meaningful reductions in low-density lipoprotein cholesterol in patients with severe familial hypercholesterolemia. Further long-term clinical studies are required to more clearly quantify its impact on risk for cardiovascular events and establish whether it increases risk for hepatosteatosis. Antisense therapy represents a potentially effective and well-tolerated emerging treatment modality for numerous diseases. In the treatment of hypercholesterolemia, the antisense therapy mipomersen may provide a possible treatment option for patients with treatment-resistant dyslipidemia. Copyright © 2011 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Apparatus for reduction of selected ion intensities in confined ion beams

DOEpatents

Eiden, Gregory C.; Barinaga, Charles J.; Koppenaal, David W.

2001-01-01

An apparatus for producing an ion beam having an increased proportion of analyte ions compared to carrier gas ions is disclosed. Specifically, the apparatus has an ion trap or a collision cell containing a reagent gas wherein the reagent gas accepts charge from the analyte ions thereby selectively neutralizing the carrier gas ions. Also disclosed is the collision cell as employed in various locations within analytical instruments including an inductively coupled plasma mass spectrometer.

Surface chemistry control for selective fossil resin flotation

DOEpatents

Miller, Jan D.; Yi, Ye; Yu, Qiang

1994-01-01

A froth flotation method is disclosed for separating fine particles of fossil resin from by use of frothing reagents which include an aliphatic organic compound having a polar group and containing not more than four carbon atoms. Butanol is an effective frothing reagent in this method.

Surface chemistry control for selective fossil resin flotation

DOEpatents

Miller, J.D.; Yi, Y.; Yu, Q.

1994-06-07

A froth flotation method is disclosed for separating fine particles of fossil resin by use of frothing reagents which include an aliphatic organic compound having a polar group and containing not more than four carbon atoms. Butanol is an effective frothing reagent in this method. 12 figs.

PLGA-PEG-PLGA microspheres as a delivery vehicle for antisense oligonucleotides to CTGF: Implications on post-surgical peritoneal adhesion prevention

NASA Astrophysics Data System (ADS)

Azeke, John Imuetinyan-Jesu, Jr.

Abdominal adhesions are the aberrant result of peritoneal wound healing commonly associated with surgery and inflammation. A subject of a large number of studies since the first half of the last century, peritoneal adhesion prevention has, for the most part, evaded the scientific community and continues to cost Americans an estimated $2-4 billion annually. It is known that transforming growth factor-beta (TGF-beta) plays a key role in the wound healing cascade; however, suppression of this multifunctional growth factor's activity may have more harmful consequences than can be tolerated. As a result, much attention has fallen on connective tissue growth factor (CTGF), a downstream mediator of TGF-beta's fibrotic action. It has been demonstrated in several in vitro models, that the suppression of CTGF hinders fibroblast proliferation, a necessary condition for fibrosis. Furthermore, antisense oligonucleotides (antisense oligos, AO) to CTGF have been shown to knock down CTGF mRNA levels by specifically hindering the translation of CTGF protein. Antisense technologies have met with a great deal of excitement as a viable means of preventing diseases such as adhesions by hindering protein translation at the mRNA level. However, the great challenge associated with the use of these drugs lies in the short circulation time when administered "naked". Viral delivery systems, although excellent platforms in metabolic studies, are not ideal for diagnostic use because of the inherent danger associated with viral vectors. Microparticles made of biodegradable polymers have therefore presented themselves as a viable means of delivering these drugs to target cells over extended periods. Herein, we present two in vivo studies confirming the up-regulation of TGF-beta protein and CTGF mRNA following injury to the uterine tissues of female rats. We were able to selectively knockdown post-operative CTGF protein levels following surgery, however, our observations led us to conclude that, while both cytokines are over-expressed within the first day following injury, CTGF protein levels could not be correlated with observed adhesion development. In addition, we synthesized linear triblock copolymers of polyethylene glycol (PEG) and poly(D,L-lactide-co-glycolide) (PLGA), two of the most widely studied biodegradable polymers in use today. Bulk gels and microparticles of the copolymers were then evaluated for gelling behavior, temperature stability, and drug loading and release kinetics in order assess their suitability as potential carriers of antisense therapeutics. A novel approach to affecting the antisense oligonucleotide release kinetics by varying the relative concentrations of co-encapsulated cationic lipid transfection agents was also presented.

Identification and Characterization of a Cis Antisense RNA of the rpoH Gene of Salmonella enterica Serovar Typhi.

PubMed

Xiong, Changyan; Li, Xuejiao; Liu, Juanli; Zhao, Xin; Xu, Shungao; Huang, Xinxiang

2018-01-01

Antisense RNAs from complementary strands of protein coding genes regulate the expression of genes involved in many cellular processes. Using deep sequencing analysis of the Salmonella enterica serovar Typhi ( S. Typhi) transcriptome, a novel antisense RNA encoded on the strand complementary to the rpoH gene was revealed. In this study, the molecular features of this antisense RNA were assessed using northern blotting and rapid amplification of cDNA ends. The 3,508 nt sequence of RNA was identified as the antisense RNA of the rpoH gene and was named ArpH. ArpH was found to attenuate the invasion of HeLa cells by S. Typhi by regulating the expression of SPI-1 genes. In an rpoH mutant strain, the invasive capacity of S. Typhi was increased, whereas overexpression of ArpH positively regulates rpoH mRNA levels. Results of this study suggest that the cis -encoded antisense RNA ArpH is likely to affect the invasive capacity of S. Typhi by regulating the expression of rpoH .

Identification and Characterization of a Cis-Encoded Antisense RNA Associated with the Replication Process of Salmonella enterica Serovar Typhi

PubMed Central

Dadzie, Isaac; Xu, Shungao; Ni, Bin; Zhang, Xiaolei; Zhang, Haifang; Sheng, Xiumei; Xu, Huaxi; Huang, Xinxiang

2013-01-01

Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise whiles others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned. PMID:23637809

Chemical Amplification with Encapsulated Reagents

NASA Technical Reports Server (NTRS)

Chen, Jian; Koemer, Steffi; Craig, Stephen; Lin, Shirley; Rudkevich, Dmitry M.; Rebek, Julius, Jr.

2002-01-01

Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.

Cis-encoded non-coding antisense RNAs in streptococci and other low GC Gram (+) bacterial pathogens

PubMed Central

Cho, Kyu Hong; Kim, Jeong-Ho

2015-01-01

Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory non-coding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small non-coding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded non-coding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+) bacteria to provide a guide for future studies. PMID:25859258

Improved therapeutic effectiveness by combining recombinant p14(ARF) with antisense complementary DNA of EGFR in laryngeal squamous cell carcinoma.

PubMed

Liu, Feng; Du, JinTao; Xian, Junming; Liu, Yafeng; Liu, Shixi; Lin, Yan

2015-01-01

The tumor suppressor p14(ARF) and proto-oncogene epidermal growth factor receptor (EGFR) play important roles in the development of laryngeal squamous cell carcinoma (LSCC). This study was aimed to determine whether combining recombinant p14(ARF) with antisense complementary DNA of EGFR could improve the therapeutic effectiveness in LSCC. After human larynx cancer cells (Hep-2) were infected with recombinant adenoviruses (Ad-p14(ARF) and Ad-antisense EGFR) together or alone in vitro, the proliferation and cell cycle distribution of Hep-2 cells were detected by MTT assay and flow cytometer analysis, respectively. Furthermore, the antitumor effects of recombinant adenoviruses together or alone on Hep-2 xenografts were examined in vivo. The levels of p14(ARF) and EGFR expressed in Hep-2 cells and xenografts were determined by western blot assay. Ad-p14(ARF) combining with Ad-antisense EGFR markedly inhibited the Hep-2 proliferation compared with alone (P=0.001, P=0.002 respectively). Combination of Ad-p14(ARF) and Ad-antisense EGFR led to the proportion of Hep-2 cells in G0/G1 phases increased by up to 86.9%. The down-expression of EGFR protein and overexpression of p14(ARF) protein were observed in vitro and in vivo, and this effect was preserved when Ad-p14(ARF) was combined with Ad-antisense EGFR. Besides, Ad-p14(ARF) plus Ad-antisense EGFR significantly (P<0.05) increased the antitumor activity against Hep-2 tumor xenografts comparing with Ad-p14(ARF) or Ad-antisense EGFR alone. Combination Ad-p14(ARF) with Ad-antisense EGFR significantly increased the antitumor responses in LSCC. An effectively potential gene therapy to prevent proliferation of LSCC was provided. Copyright © 2015 Elsevier Inc. All rights reserved.

Natural antisense RNAs as mRNA regulatory elements in bacteria: a review on function and applications.

PubMed

Saberi, Fatemeh; Kamali, Mehdi; Najafi, Ali; Yazdanparast, Alavieh; Moghaddam, Mehrdad Moosazadeh

2016-01-01

Naturally occurring antisense RNAs are small, diffusible, untranslated transcripts that pair to target RNAs at specific regions of complementarity to control their biological function by regulating gene expression at the post-transcriptional level. This review focuses on known cases of antisense RNA control in prokaryotes and provides an overview of some natural RNA-based mechanisms that bacteria use to modulate gene expression, such as mRNA sensors, riboswitches and antisense RNAs. We also highlight recent advances in RNA-based technology. The review shows that studies on both natural and synthetic systems are reciprocally beneficial.

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

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

Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.

The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport inmore » sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.« less

Angubindin-1 opens the blood-brain barrier in vivo for delivery of antisense oligonucleotide to the central nervous system.

PubMed

Zeniya, Satoshi; Kuwahara, Hiroya; Daizo, Kaiichi; Watari, Akihiro; Kondoh, Masuo; Yoshida-Tanaka, Kie; Kaburagi, Hidetoshi; Asada, Ken; Nagata, Tetsuya; Nagahama, Masahiro; Yagi, Kiyohito; Yokota, Takanori

2018-05-17

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Noncoding transcripts in sense and antisense orientation regulate the epigenetic state of ribosomal RNA genes.

PubMed

Bierhoff, H; Schmitz, K; Maass, F; Ye, J; Grummt, I

2010-01-01

Alternative transcription of the same gene in sense and antisense orientation regulates expression of protein-coding genes. Here we show that noncoding RNA (ncRNA) in sense and antisense orientation also controls transcription of rRNA genes (rDNA). rDNA exists in two types of chromatin--a euchromatic conformation that is permissive to transcription and a heterochromatic conformation that is transcriptionally silent. Silencing of rDNA is mediated by NoRC, a chromatin-remodeling complex that triggers heterochromatin formation. NoRC function requires RNA that is complementary to the rDNA promoter (pRNA). pRNA forms a DNA:RNA triplex with a regulatory element in the rDNA promoter, and this triplex structure is recognized by DNMT3b. The results imply that triplex-mediated targeting of DNMT3b to specific sequences may be a common pathway in epigenetic regulation. We also show that rDNA is transcribed in antisense orientation. The level of antisense RNA (asRNA) is down-regulated in cancer cells and up-regulated in senescent cells. Ectopic asRNA triggers trimethylation of histone H4 at lysine 20 (H4K20me3), suggesting that antisense transcripts guide the histone methyltransferase Suv4-20 to rDNA. The results reveal that noncoding RNAs in sense and antisense orientation are important determinants of the epigenetic state of rDNA.

Detection Progress of Selected Drugs in TLC

PubMed Central

Pyka, Alina

2014-01-01

This entry describes applications of known indicators and dyes as new visualizing reagents and various visualizing systems as well as photocatalytic reactions and bioautography method for the detection of bioactive compounds including drugs and compounds isolated from herbal extracts. Broadening index, detection index, characteristics of densitometric band, modified contrast index, limit of detection, densitometric visualizing index, and linearity range of detected compounds were used for the evaluation of visualizing effects of applied visualizing reagents. It was shown that visualizing effect depends on the chemical structure of the visualizing reagent, the structure of the substance detected, and the chromatographic adsorbent applied. The usefulness of densitometry to direct detection of some drugs was also shown. Quoted papers indicate the detection progress of selected drugs investigated by thin-layer chromatography (TLC). PMID:24551853

Antisense long non-coding RNAs in rainbow trout: Discovery and potential role in muscle growth and quality traits

USDA-ARS?s Scientific Manuscript database

Endogenous mRNA-antisense transcripts are involved in regulation of a wide range of biological processes including muscle development and quality traits of farm animals. Standard RNA-Seq can be used to identify sense-antisense transcripts. However, strand-specific RNA-Seq is required to resolve ambi...

Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

ERIC Educational Resources Information Center

Canal, Clinton E.; Chang, Qing; Gold, Paul E.

2008-01-01

Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

Methodical aspects of blood coagulation measurements in birds applying commercial reagents--a pilot study.

PubMed

Guddorf, Vanessa; Kummerfeld, Norbert; Mischke, Reinhard

2014-01-01

The aim of this study was to examine the suitability of commercially available reagents for measurements of coagulation parameters in citrated plasma from birds. Therefore, plasma samples of 17 healthy donor birds of different species were used to determine prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin time (TT) applying various commercial reagents which are routinely used in coagulation diagnostics in humans and mammals. A PT reagent based on human placental thromboplastin yielded not only shorter clotting times than a reagent containing recombinant human tissue factor (median 49 vs. 84 s), but also showed a minor range of distribution of values (43-55 s vs. 30-147 s, minimum-maximum, n = 5 turkeys). An aPTT reagent containing kaolin and phospholipids of animal origin delivered the shortest clotting times and the lowest range of variation in comparison to three other reagents of different composition. However, even when this reagent was used, aPTTs were partially extremely long (> 200 s). Thrombin time was 38 s (28-57 s, n = 5 chicken) when measured with bovine thrombin at a final concentration of 2 IU thrombin/ ml. Coefficients of variation for within-run precision analysis (20 repetitions) of PT was 8.0% and 4.7% for aPTT measurements using selected reagents of mammalian origin. In conclusion, of the commercially available reagents tested, a PT reagent based on human placental thromboplastin and an aPTT reagent including rabbit brain phospholipid and kaolin, show some promise for potential use in birds.

Green acetylation of solketal and glycerol formal by heterogeneous acid catalysts to form a biodiesel fuel additive.

PubMed

Dodson, Jennifer R; Leite, Thays d C M; Pontes, Nathália S; Peres Pinto, Bianca; Mota, Claudio J A

2014-09-01

A glut of glycerol has formed from the increased production of biodiesel, with the potential to integrate the supply chain by using glycerol additives to improve biodiesel properties. Acetylated acetals show interesting cold flow and viscosity effects. Herein, a solventless heterogeneously catalyzed process for the acetylation of both solketal and glycerol formal to new products is demonstrated. The process is optimized by studying the effect of acetylating reagent (acetic acid and acetic anhydride), reagent molar ratios, and a variety of commercial solid acid catalysts (Amberlyst-15, zeolite Beta, K-10 Montmorillonite, and niobium phosphate) on the conversion and selectivities. High conversions (72-95%) and selectivities (86-99%) to the desired products results from using acetic anhydride as the acetylation reagent and a 1:1 molar ratio with all catalysts. Overall, there is a complex interplay between the solid catalyst, reagent ratio, and acetylating agent on the conversion, selectivities, and byproducts formed. The variations are discussed and explained in terms of reactivity, thermodynamics, and reaction mechanisms. An alternative and efficient approach to the formation of 100% triacetin involves the ring-opening, acid-catalyzed acetylation from solketal or glycerol formal with excesses of acetic anhydride. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Constructing New Bioorthogonal Reagents and Reactions.

PubMed

Row, R David; Prescher, Jennifer A

2018-05-15

Chemical tools are transforming our understanding of biomolecules and living systems. Included in this group are bioorthogonal reagents-functional groups that are inert to most biological species, but can be selectively ligated with complementary probes, even in live cells and whole organisms. Applications of these tools have revealed fundamental new insights into biomolecule structure and function-information often beyond the reach of genetic approaches. In many cases, the knowledge gained from bioorthogonal probes has enabled new questions to be asked and innovative research to be pursued. Thus, the continued development and application of these tools promises to both refine our view of biological systems and facilitate new discoveries. Despite decades of achievements in bioorthogonal chemistry, limitations remain. Several reagents are too large or insufficiently stable for use in cellular environments. Many bioorthogonal groups also cross-react with one another, restricting them to singular tasks. In this Account, we describe our work to address some of the voids in the bioorthogonal toolbox. Our efforts to date have focused on small reagents with a high degree of tunability: cyclopropenes, triazines, and cyclopropenones. These motifs react selectively with complementary reagents, and their unique features are enabling new pursuits in biology. The Account is organized by common themes that emerged in our development of novel bioorthogonal reagents and reactions. First, natural product structures can serve as valuable starting points for probe design. Cyclopropene, triazine, and cyclopropenone motifs are all found in natural products, suggesting that they would be metabolically stable and compatible with a variety of living systems. Second, fine-tuning bioorthogonal reagents is essential for their successful translation to biological systems. Different applications demand different types of probes; thus, generating a collection of tools that span a continuum of reactivities and stabilities remains an important goal. We have used both computational analyses and mechanistic studies to guide the optimization of various cyclopropene and triazine probes. Along the way, we identified reagents that are chemoselective but best suited for in vitro work. Others are selective and robust enough for use in living organisms. The last section of this Account highlights the need for the continued pursuit of new reagents and reactions. Challenges exist when bioorthogonal chemistries must be used in concert, given that many exploit similar mechanisms and cannot be used simultaneously. Such limitations have precluded certain multicomponent labeling studies and other biological applications. We have relied on mechanistic and computational insights to identify mutually orthogonal sets of reactions, in addition to exploring unique genres of reactivity. The continued development of mechanistically distinct, biocompatible reactions will further diversify the bioorthogonal reaction portfolio for examining biomolecules.

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

PubMed

Nakashima, N; Tamura, T

2013-06-01

Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-β-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose. © 2013 The Society for Applied Microbiology.

Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.

PubMed Central

Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.

1997-01-01

On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential. PMID:12223765

Characterization of Antisense Transformed Plants Deficient in the Tobacco Anionic Peroxidase.

PubMed

Lagrimini, L. M.; Gingas, V.; Finger, F.; Rothstein, S.; Liu, TTY.

1997-08-01

On the basis of the biological compounds that they metabolize, plant peroxidases have long been implicated in plant growth, cell wall biogenesis, lignification, and host defenses. Transgenic tobacco (Nicotiana tabacum L.) plants that underexpress anionic peroxidase were generated using antisense RNA. The antisense RNA was found to be specific for the anionic isoenzyme and highly effective, reducing endogenous transcript levels and total peroxidase activity by as much as 1600-fold. Antisense-transformed plants appeared normal at initial observation; however, growth studies showed that plants with reduced peroxidase activity grow taller and flower sooner than control plants. In contrast, previously transformed plants overproducing anionic peroxidase were shorter and flowered later than controls. Axillary buds were more developed in antisense-transformed plants and less developed in plants overproducing this enzyme. It was found that the lignin content in leaf, stem, and root was unchanged in antisense-transformed plants, which does not support a role for anionic peroxidase in the lignification of secondary xylem vessels. However, studies of wounded tissue show some reduction in wound-induced deposition of lignin-like polymers. The data support a possible role for tobacco anionic peroxidase in host defenses but not without a reduction in growth potential.

Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line.

PubMed

Leonetti, C; Biroccio, A; Benassi, B; Stringaro, A; Stoppacciaro, A; Semple, S C; Zupi, G

2001-06-01

Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

Inhibition of aac(6′)-Ib-mediated amikacin resistance by nuclease-resistant external guide sequences in bacteria

PubMed Central

Soler Bistué, Alfonso J. C.; Martín, Fernando A.; Vozza, Nicolás; Ha, Hongphuc; Joaquín, Jonathan C.; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2009-01-01

Inhibition of bacterial gene expression by RNase P-directed cleavage is a promising strategy for the development of antibiotics and pharmacological agents that prevent expression of antibiotic resistance. The rise in multiresistant bacteria harboring AAC(6′)-Ib has seriously limited the effectiveness of amikacin and other aminoglycosides. We have recently shown that recombinant plasmids coding for external guide sequences (EGS), short antisense oligoribonucleotides (ORN) that elicit RNase P-mediated cleavage of a target mRNA, induce inhibition of expression of aac(6′)-Ib and concomitantly induce a significant decrease in the levels of resistance to amikacin. However, since ORN are rapidly degraded by nucleases, development of a viable RNase P-based antisense technology requires the design of nuclease-resistant RNA analog EGSs. We have assayed a variety of ORN analogs of which selected LNA/DNA co-oligomers elicited RNase P-mediated cleavage of mRNA in vitro. Although we found an ideal configuration of LNA/DNA residues, there seems not to be a correlation between number of LNA substitutions and level of activity. Exogenous administration of as low as 50 nM of an LNA/DNA co-oligomer to the hyperpermeable E. coli AS19 harboring the aac(6′)-Ib inhibited growth in the presence of amikacin. Our experiments strongly suggest an RNase P-mediated mechanism in the observed antisense effect. PMID:19666539

Protocols | Office of Cancer Clinical Proteomics Research

Cancer.gov

Each reagent on the Antibody Portal has been characterized by a combination of methods specific for that antibody. To view the customized antibody methods and protocols (Standard Operating Procedures) used to generate and characterize each reagent, select an antibody of interest and open the protocols associated with their respective characterization methods along with characterization data.

Selected heavy metals speciation in chemically stabilised sewage sludge

NASA Astrophysics Data System (ADS)

Wiśniowska, Ewa; Włodarczyk-Makuła, Marła

2017-11-01

Selected heavy metals (Pb, Ni, Cd) were analysed in soil, digested sewage sludge as well as in the sludge stabilised with CaO or Fenton's reagent. The dose of Fenton's reagent was as follows: Fe2+ = 1g.L-1, Fe2+/H2O2=1:100; stabilisation lasted for 2 h. Dose of CaO was equal to 1 g CaO.g d.m.-1 Total concentration of all metals in the digested sewage sludge was higher than in the soil. Chemical stabilisation of sludge with Fenton's reagent increased total metal content in the sludge as a result of total solids removal. Opposite effect was stated when the sludge was mixed with CaO. Also chemical fractions of heavy metals were identified (exchangeable, carbonate bound, iron oxides bound, organic and residual). The results indicate that stabilisation of the sludge with Fenton's reagent increased mobility of heavy metals compared to the digested sludge. Amendment of CaO increased percent share of examined metals in residual fraction, thus immobilised them and decreased their bioavailability.

Diazo Reagents with Small Steric Footprints for Simultaneous Arming/SAR Studies of Alcohol-Containing Natural Products via O–H Insertion

PubMed Central

Chamni, Supakarn; He, Qing-Li; Dang, Yongjun; Bhat, Shridhar; Liu, Jun O.; Romo, Daniel

2011-01-01

Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O–H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors. PMID:21894934

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

PubMed Central

Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674

Antisense imaging of gene expression in the brain in vivo

NASA Astrophysics Data System (ADS)

Shi, Ningya; Boado, Ruben J.; Pardridge, William M.

2000-12-01

Antisense radiopharmaceuticals could be used to image gene expression in the brain in vivo, should these polar molecules be made transportable through the blood-brain barrier. The present studies describe an antisense imaging agent comprised of an iodinated peptide nucleic acid (PNA) conjugated to a monoclonal antibody to the rat transferrin receptor by using avidin-biotin technology. The PNA was a 16-mer antisense to the sequence around the methionine initiation codon of the luciferase mRNA. C6 rat glioma cells were permanently transfected with a luciferase expression plasmid, and C6 experimental brain tumors were developed in adult rats. The expression of the luciferase transgene in the tumors in vivo was confirmed by measurement of luciferase enzyme activity in the tumor extract. The [125I]PNA conjugate was injected intravenously in anesthetized animals with brain tumors and killed 2 h later for frozen sectioning of brain and film autoradiography. No image of the luciferase gene expression was obtained after the administration of either the unconjugated antiluciferase PNA or a PNA conjugate that was antisense to the mRNA of a viral transcript. In contrast, tumors were imaged in all rats administered the [125I]PNA that was antisense to the luciferase sequence and was conjugated to the targeting antibody. In conclusion, these studies demonstrate gene expression in the brain in vivo can be imaged with antisense radiopharmaceuticals that are conjugated to a brain drug-targeting system.

Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

[Study on composite stabilization of arsenic (As) contaminated soil].

PubMed

Wang, Hao; Pan, Li-xiang; Zhang, Xiang-yu; Li, Meng; Song, Bao-hua

2013-09-01

Since the contaminated soil may contain various kinds of heavy metals, use of single chemical reagent leads to poor remediation and high cost. In this study, soil containing As, Zn, Cd was sampled, and different reagents were selected to carry out the rapid stabilization of contaminated soil. The TCLP (toxicity characteristic leaching procedure) was used to evaluate the leachate toxicity of heavy metals and the results indicated that calcium-containing, sulphur-containing and iron-containing reagents had good performance in reducing the metal mobility. The stabilization efficiency of the six reagents tested ranked in the order of CaO > Na2S > organic sulfur > Chitosan > FeSO4 > (C2H5)2NCS2Na. Two types of reagents (six reagents) were combined based on the target properties of different reagents and the stabilization efficiency was evaluated and analyzed. The results indicated that the composite reagents had higher stabilization efficiency: the efficiency of 3% FeSO4 + 5% CaO was 81.7%, 97.2% and 68.2% for As, Cd and Zn, respectively, and the efficiency of 3% CaO + 5% organic sulfur was 76.6%, 95.7% and 93.8% for these three metals, respectively. Speciation analysis was carried out in this study and the results suggested that it was the change of metals from the exchangeable state to the reduction (for inorganic reagent) or oxidation state (for organic reagent) that caused the soil stabilization and the degree of change determined the stabilization efficiency.

Direct catalytic cross-coupling of organolithium compounds

NASA Astrophysics Data System (ADS)

Giannerini, Massimo; Fañanás-Mastral, Martín; Feringa, Ben L.

2013-08-01

Catalytic carbon-carbon bond formation based on cross-coupling reactions plays a central role in the production of natural products, pharmaceuticals, agrochemicals and organic materials. Coupling reactions of a variety of organometallic reagents and organic halides have changed the face of modern synthetic chemistry. However, the high reactivity and poor selectivity of common organolithium reagents have largely prohibited their use as a viable partner in direct catalytic cross-coupling. Here we report that in the presence of a Pd-phosphine catalyst, a wide range of alkyl-, aryl- and heteroaryl-lithium reagents undergo selective cross-coupling with aryl- and alkenyl-bromides. The process proceeds quickly under mild conditions (room temperature) and avoids the notorious lithium halogen exchange and homocoupling. The preparation of key alkyl-, aryl- and heterobiaryl intermediates reported here highlights the potential of these cross-coupling reactions for medicinal chemistry and material science.

Role of CREB in CML

DTIC Science & Technology

2007-02-01

antisense RNA for suppressing gene expression in nematode worms (Caenorhabditis elegans) 2. This was followed by the introduction of dsRNA into worms...When single-stranded antisense RNA and double stranded RNA was introduced into worms, they found that dsRNA was more effective than either strand...RISC ( RNA -induced silencing complex), which contains helicase activity that unwinds the two strands 3 of RNA molecules, allowing the antisense

Sense-antisense (complementary) peptide interactions and the proteomic code; potential opportunities in biology and pharmaceutical science.

PubMed

Miller, Andrew D

2015-02-01

A sense peptide can be defined as a peptide whose sequence is coded by the nucleotide sequence (read 5' → 3') of the sense (positive) strand of DNA. Conversely, an antisense (complementary) peptide is coded by the corresponding nucleotide sequence (read 5' → 3') of the antisense (negative) strand of DNA. Research has been accumulating steadily to suggest that sense peptides are capable of specific interactions with their corresponding antisense peptides. Unfortunately, although more and more examples of specific sense-antisense peptide interactions are emerging, the very idea of such interactions does not conform to standard biology dogma and so there remains a sizeable challenge to lift this concept from being perceived as a peripheral phenomenon if not worse, into becoming part of the scientific mainstream. Specific interactions have now been exploited for the inhibition of number of widely different protein-protein and protein-receptor interactions in vitro and in vivo. Further, antisense peptides have also been used to induce the production of antibodies targeted to specific receptors or else the production of anti-idiotypic antibodies targeted against auto-antibodies. Such illustrations of utility would seem to suggest that observed sense-antisense peptide interactions are not just the consequence of a sequence of coincidental 'lucky-hits'. Indeed, at the very least, one might conclude that sense-antisense peptide interactions represent a potentially new and different source of leads for drug discovery. But could there be more to come from studies in this area? Studies on the potential mechanism of sense-antisense peptide interactions suggest that interactions may be driven by amino acid residue interactions specified from the genetic code. If so, such specified amino acid residue interactions could form the basis for an even wider amino acid residue interaction code (proteomic code) that links gene sequences to actual protein structure and function, even entire genomes to entire proteomes. The possibility that such a proteomic code should exist is discussed. So too the potential implications for biology and pharmaceutical science are also discussed were such a code to exist.

Diazo compounds in continuous-flow technology.

PubMed

Müller, Simon T R; Wirth, Thomas

2015-01-01

Diazo compounds are very versatile reagents in organic chemistry and meet the challenge of selective assembly of structurally complex molecules. Their leaving group is dinitrogen; therefore, they are very clean and atom-efficient reagents. However, diazo compounds are potentially explosive and extremely difficult to handle on an industrial scale. In this review, it is discussed how continuous flow technology can help to make these powerful reagents accessible on large scale. Microstructured devices can improve heat transfer greatly and help with the handling of dangerous reagents safely. The in situ formation and subsequent consumption of diazo compounds are discussed along with advances in handling diazomethane and ethyl diazoacetate. The potential large-scale applications of a given methodology is emphasized. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs

PubMed Central

Shen, Xiulong; Corey, David R

2018-01-01

Abstract RNA plays a central role in the expression of all genes. Because any sequence within RNA can be recognized by complementary base pairing, synthetic oligonucleotides and oligonucleotide mimics offer a general strategy for controlling processes that affect disease. The two primary antisense approaches for regulating expression through recognition of cellular RNAs are single-stranded antisense oligonucleotides and duplex RNAs. This review will discuss the chemical modifications and molecular mechanisms that make synthetic nucleic acid drugs possible. Lessons learned from recent clinical trials will be summarized. Ongoing clinical trials are likely to decisively test the adequacy of our current generation of antisense nucleic acid technologies and highlight areas where more basic research is needed. PMID:29240946

Selective Androgen Receptor Downregulators (SARDs): A New Prostate Cancer Therapy

DTIC Science & Technology

2006-10-01

of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol, 12: 1558...cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol...used to down-regulate the AR include antisense oligonucleotides (9, 10), ribozyme treatments (11, 12), AR dominant negatives (13) and small

Colorimetric chemical analysis sampler for the presence of explosives

DOEpatents

Nunes, Peter J [Danville, CA; Del Eckels, Joel [Livermore, CA; Reynolds, John G [San Ramon, CA; Pagoria, Philip F [Livermore, CA; Simpson, Randall L [Livermore, CA

2011-09-27

A tester for testing for explosives comprising a body, a lateral flow swab unit operably connected to the body, a explosives detecting reagent contained in the body, and a dispenser operatively connected to the body and the lateral flow swab unit. The dispenser selectively allows the explosives detecting reagent to be delivered to the lateral flow swab unit.

Colorimetric chemical analysis sampler for the presence of explosives

DOEpatents

Nunes, Peter J.; Eckels, Joel Del; Reynolds, John G.; Pagoria, Philip F.; Simpson, Randall L.

2014-07-01

A tester for testing for explosives comprising a body, a lateral flow swab unit operably connected to the body, a explosives detecting reagent contained in the body, and a dispenser operatively connected to the body and the lateral flow swab unit. The dispenser selectively allows the explosives detecting reagent to be delivered to the lateral flow swab unit.

Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells

PubMed Central

Wu, Li; Wang, Yuan; Wu, Junzhou; Lv, Cong; Wang, Jie; Tang, Xinjing

2013-01-01

We synthesized three 20mer caged circular antisense oligodeoxynucleotides (R20, R20B2 and R20B4) with a photocleavable linker and an amide bond linker between two 10mer oligodeoxynucleotides. With these caged circular antisense oligodeoxynucleotides, RNA-binding affinity and its digestion by ribonuclease H were readily photomodulated. RNA cleavage rates were upregulated ∼43-, 25- and 15-fold for R20, R20B2 and R20B4, respectively, upon light activation in vitro. R20B2 and R20B4 with 2- or 4-nt gaps in the target RNA lost their ability to bind the target RNA even though a small amount of RNA digestion was still observed. The loss of binding ability indicated promising gene photoregulation through a non-enzymatic strategy. To test this strategy, three caged circular antisense oligonucleotides (PS1, PS2 and PS3) with 2′-OMe RNA and phosphorothioate modifications were synthesized to target GFP expression. Upon light activation, photomodulation of target hybridization and GFP expression in cells was successfully achieved with PS1, PS2 and PS3. These caged circular antisense oligonucleotides show promising applications of photomodulating gene expression through both ribonuclease H and non-enzyme involved antisense strategies. PMID:23104375

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

PubMed

Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

2016-09-01

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

The nanostructural characterization of strawberry pectins in pectate lyase or polygalacturonase silenced fruits elucidates their role in softening.

PubMed

Posé, Sara; Kirby, Andrew R; Paniagua, Candelas; Waldron, Keith W; Morris, Victor J; Quesada, Miguel A; Mercado, José A

2015-11-05

To ascertain the role of pectin disassembly in fruit softening, chelated- (CSP) and sodium carbonate-soluble (SSP) pectins from plants with a pectate lyase, FaplC, or a polygalacturonase, FaPG1, downregulated by antisense transformation were characterized at the nanostructural level. Fruits from transgenic plants were firmer than the control, although FaPG1 suppression had a greater effect on firmness. Size exclusion chromatography showed that the average molecular masses of both transgenic pectins were higher than that of the control. Atomic force microscopy analysis of pectins confirmed the higher degree of polymerization as result of pectinase silencing. The mean length values for CSP chains increased from 84 nm in the control to 95.5 and 101 nm, in antisense FaplC and antisense FaPG1 samples, respectively. Similarly, SSP polyuronides were longer in transgenic fruits (61, 67.5 and 71 nm, in the control, antisense FaplC and antisense FaPG1 samples, respectively). Transgenic pectins showed a more complex structure, with a higher percentage of branched chains than the control, especially in the case of FaPG1 silenced fruits. Supramolecular pectin aggregates, supposedly formed by homogalacturonan and rhamnogalacturonan I, were more frequently observed in antisense FaPG1 samples. The larger modifications in the nanostructure of pectins in FaPG1 silenced fruits when compared with antisense pectate lyase plants correlate with the higher impact of polygalacturonase silencing on reducing strawberry fruit softening. Copyright © 2015 Elsevier Ltd. All rights reserved.

Complementation of a Fanconi anemia group A cell line by UbA{sup 52}

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

Moses, R.E.; Heina, J.A.; Jakobs, P.M.

1994-09-01

Cells from patients with Fanconi anemia (FA) display chromosomal instability and increased sensitivity to mitomycin C (MMC) and diepoxybutane (DEB) relative to normal cells. Several genes act in this pathway of DNA damage processing based upon four known complementation groups in FA. We have made a cDNA expression library in a vector with a G418 selectable marker to identify FA genes other than the FA-C group. Approximately 1 x 10{sup 6} independent cDNA clones were isolated with an average cDNA size of 1.5 kb. Five cell lines resistant to MMC and DEB were isolated from 6 x 10{sup 6} G418-resistantmore » transfectants from 65 individual transfections of the FA-A fibroblast line GM6914. The isolated cell lines also showed normal chromosome stability. The same cDNA (600 bp) was recovered from three independent cell lines by PCR using flanking sequence primers. The gene has sequence identity with a known gene, the ubiquitin fusion gene, UbA{sub 52}. Interestingly, each of the cDNAs were inserted in antisense orientation relative to the cytomegalovirus (CMV) promoter as determined by sequencing and PCR using UbA{sub 52}-specific internal primers. Southern blot analysis indicated the cell lines had distinct chromosomal insertion sites. Mutation analysis by chemical cleavage showed no reading frame mutations, indicating that UbA{sub 52} is not the FA-A gene. Re-transfection with the UbA{sub 52} gene in antisense gave complementation for MMC, DEB and chromosome stability to varying degrees. Re-transfection of the antisense construct with the CMV promotor removed or with a sense construct did not alter the MMC sensitivity. We conclude that the antisense UbA{sub 52} gene has a non-specific effect, perhaps acting by altering the cell cycle or susceptibility to apoptosis.« less

JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis1[OPEN

PubMed Central

Xiao, Jun; Li, Chunhua; Xu, Shujuan; Xing, Lijing; Xu, Yunyuan; Chong, Kang

2015-01-01

Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1’s function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1’s regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis. PMID:26392261

Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

PubMed

Bhatti, L; Behle, K; Stevens, R H

1992-10-01

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number following incubation with anti-sense oligonucleotides, surface expression of Fc epsilon R II was consistent as measured over different time points. PCR analysis revealed that while most cells expressed either the alpha or the beta form of Fc epsilon R II, EBV-transformed cell lines, particularly RPMI 8866, were found to express both alpha and beta forms simultaneously. This may constitute a mechanism whereby EBV infection confers an immortal state to the cell, resulting in its uncontrolled proliferation. Cell lines expressing only one receptor form, either alpha or beta, were unaffected after incubation with anti-sense oligonucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)

Slow Off-Rate Modified Aptamer (SOMAmer) as a Novel Reagent in Immunoassay Development for Accurate Soluble Glypican-3 Quantification in Clinical Samples.

PubMed

Duo, Jia; Chiriac, Camelia; Huang, Richard Y-C; Mehl, John; Chen, Guodong; Tymiak, Adrienne; Sabbatini, Peter; Pillutla, Renuka; Zhang, Yan

2018-04-17

Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.

Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection

DTIC Science & Technology

2013-07-01

4175) model system REPORTABLE OUTCOMES 1. Lee Spraggon and Luca Cartegni; Antisense Modulation of RNA Processing as a Therapeutic Approach in...modulation. Proc Natl Acad Sci U S A 108: 17779-17784. 26. Spraggon L, Cartegni L (2013) Antisense modulation of RNA processing as a therapeutic...pre-print copy 1 Antisense Modulation of RNA Processing as a Therapeutic Approach in Cancer Therapy Lee Spraggon and Luca Cartegni Molecular

International reference reagents: antihuman globulin. An ISBT/ICSH joint working party report. International Society of Blood Transfusion. International Committee for Standardization in Haematology.

PubMed

Case, J; Ford, D S; Chung, A; Collins, R; Kochman, S; Mazda, T; Overbeeke, M; Perera, R; Sakuldamrongpanich, T; Scott, M; Voak, D; Zupańska, B

1999-01-01

An international working party has conducted a study designed to select a suitable reference reagent for antihuman globulin, to replace those first made available in 1987. The chosen preparation contains levels of anti-IgG and anti-C3 (anti-C3c and anti-C3d) potency that are considered suitable to serve for reference when evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components. The reference material is available in 2-ml freeze-dried aliquots from seven assigned distribution centres.

Microwave-induced electrostatic etching: generation of highly reactive magnesium for application in Grignard reagent formation.

PubMed

van de Kruijs, Bastiaan H P; Dressen, Mark H C L; Meuldijk, Jan; Vekemans, Jef A J M; Hulshof, Lumbertus A

2010-04-07

A detailed study regarding the influence of microwave irradiation on the formation of a series of Grignard reagents in terms of rates and selectivities has revealed that these heterogeneous reactions may display a beneficial microwave effect. The interaction between microwaves and magnesium turnings generates violent electrostatic discharges. These discharges on magnesium lead to melting of the magnesium surface, thus generating highly active magnesium particles. As compared to conventional operation the microwave-induced discharges on the magnesium surface lead to considerably shorter initiation times for the insertion of magnesium in selected substrates (i.e. halothiophenes, halopyridines, octyl halides, and halobenzenes). Thermographic imaging and surface characterization by scanning electron microscopy showed that neither selective heating nor a "specific" microwave effect was causing the reduction in initiation times. This novel and straightforward initiation method eliminates the use of toxic and environmentally adverse initiators. Thus, this initiation method limits the formation of by-products. We clearly demonstrated that microwave irradiation enables fast Grignard reagent formation. Therefore, microwave technology is promising for process intensification of Grignard based coupling reactions.

Copper-Catalyzed Oxy-Alkynylation of Diazo Compounds with Hypervalent Iodine Reagents.

PubMed

Hari, Durga Prasad; Waser, Jerome

2016-02-24

Alkynes have found widespread applications in synthetic chemistry, biology, and materials sciences. In recent years, methods based on electrophilic alkynylation with hypervalent iodine reagents have made acetylene synthesis more flexible and efficient, but they lead to the formation of one equivalent of an iodoarene as side-product. Herein, a more efficient strategy involving a copper-catalyzed oxy-alkynylation of diazo compounds with ethynylbenziodoxol(on)e (EBX) reagents is described, which proceeds with generation of nitrogen gas as the only waste. This reaction is remarkable for its broad scope in both EBX reagents and diazo compounds. In addition, vinyl diazo compounds gave enynes selectively as single geometric isomers. The functional groups introduced during the transformation served as easy handles to access useful building blocks for synthetic and medicinal chemistry.

Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals

NASA Astrophysics Data System (ADS)

Paulmurugan, Ramasamy; Sekar, Narayana M.; Sekar, Thillai V.

2012-03-01

MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve therapeutic effect in living animals.

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

PubMed Central

Sierant, Malgorzata; Paduszynska, Alina; Kazmierczak-Baranska, Julia; Nacmias, Benedetta; Sorbi, Sandro; Bagnoli, Silvia; Sochacka, Elzbieta; Nawrot, Barbara

2011-01-01

RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide. PMID:21559198

[Influence of antisense RNA and sequences of viral transactivators traps on RNA synthesis of HTLV-1 virus].

PubMed

Borisenko, A S; Kotus, E V; Kaloshin, A A

2008-01-01

Significant number of scientific publications devoted to inhibition of viral replication by antisense RNA (asRNA) genes shows that this approach is useful for gene therapy of viral infections. To investigate the possibility of suppression of HTLV-1 virus reproduction by asRNA we constructed recombinant plasmids containing asRNA genes against U3 long terminal repeats region and X gene under the control of promoter of myeloproliferative sarcoma virus (MPSV) or without such promoter. Using stable calcium-phosphate transfection method with subsequent selection in the presence of G-418, RaHOS line-based cell clones carrying both asRNA genes and sequences able to bind HTLV-1 transactivator proteins (i.e. "traps" of viral transactivators, TVT) were obtained. Data from dot-hybridization analysis of viral RNA extracted from RaHOS cell clones showed that TVT sequences are able to suppress the viral RNA synthesis on 90% and asRNA against X gene synthesis--on 50%.

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design.

PubMed Central

Tabler, M; Homann, M; Tzortzakaki, S; Sczakiel, G

1994-01-01

Trans-cleaving hammerhead ribozymes with long target-specific antisense sequences flanking the catalytic domain share some features with conventional antisense RNA and are therefore termed 'catalytic antisense RNAs'. Sequences 5' to the catalytic domain form helix I and sequences 3' to it form helix III when complexed with the target RNA. A catalytic antisense RNA of more than 400 nucleotides, and specific for the human immunodeficiency virus type 1 (HIV-1), was systematically truncated within the arm that constituted originally a helix I of 128 base pairs. The resulting ribozymes formed helices I of 13, 8, 5, 3, 2, 1 and 0 nucleotides, respectively, and a helix III of about 280 nucleotides. When their in vitro cleavage activity was compared with the original catalytic antisense RNA, it was found that a helix I of as little as three nucleotides was sufficient for full endonucleolytic activity. The catalytically active constructs inhibited HIV-1 replication about four-fold more effectively than the inactive ones when tested in human cells. A conventional hammerhead ribozyme having helices of just 8 nucleotides on either side failed to cleave the target RNA in vitro when tested under the conditions for catalytic antisense RNA. Cleavage activity could only be detected after heat-treatment of the ribozyme substrate mixture which indicates that hammerhead ribozymes with short arms do not associate as efficiently to the target RNA as catalytic antisense RNA. The requirement of just a three-nucleotide helix I allows simple PCR-based generation strategies for asymmetric hammerhead ribozymes. Advantages of an asymmetric design will be discussed. Images PMID:7937118

Down-regulation of the IbEXP1 gene enhanced storage root development in sweetpotato

PubMed Central

Bae, Jung Myung

2013-01-01

The role of an expansin gene (IbEXP1) in the formation of the storage root (SR) was investigated by expression pattern analysis and characterization of IbEXP1-antisense sweetpotato (Ipomoea batatas cv. Yulmi) plants in an attempt to elucidate the molecular mechanism underlying SR development in sweetpotato. The transcript level of IbEXP1 was high in the fibrous root (FR) and petiole at the FR stage, but decreased significantly at the young storage root (YSR) stage. IbEXP1-antisense plants cultured in vitro produced FRs which were both thicker and shorter than those of wild-type (WT) plants. Elongation growth of the epidermal cells was significantly reduced, and metaxylem and cambium cell proliferation was markedly enhanced in the FRs of IbEXP1-antisense plants, resulting in an earlier thickening growth in these plants relative to WT plants. There was a marked reduction in the lignification of the central stele of the FRs of the IbEXP1-antisense plants, suggesting that the FRs of the mutant plants possessed a higher potential than those of WT plants to develop into SRs. IbEXP1-antisense plants cultured in soil produced a larger number of SRs and, consequently, total SR weight per IbEXP1-antisense plant was greater than that per WT plant. These results demonstrate that SR development was accelerated in IbEXP1-antisense plants and suggest that IbEXP1 plays a negative role in the formation of SR by suppressing the proliferation of metaxylem and cambium cells to inhibit the initial thickening growth of SRs. IbEXP1 is the first sweetpotato gene whose role in SR development has been directly identified in soil-grown transgenic sweetpotato plants. PMID:22945944

Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

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

Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile

Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25more » nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen-signaling proteins in pollen tubes from the lilly Agapanthus umbellatus. For the uptake of DNA pollen tubes represent a unique system since the growing tip is surrounded by a loose matrix of hemicellulose and pectins, exposing the plasma membrane7 and the first uptake of ODNs by pollen tubes was reported as early as 1994. A breakthrough in the employment of antisense ODN inhibition as a powerful approach in plant biology was recently presented through our work on intact barley leaves. As was illustrated by confocal microscopy and fluorescently labeled ODNs, naked ODNs were taken up through the leaf petiole and efficiently imported into the plant cell and the nucleus. The work portrayed in that study demonstrate the applicability of antisense ODN inhibition in plant biology, e.g. as a rapid antecedent to time-consuming transgenic studies, and that it operates through RNase H degradation. We employed the antisense ODN strategy to demonstrate the importance of the SUSIBA2 transcription factor in regulation of starch synthesis, and to depict a possible mechanism for sugar signaling in plants and how it might confer endosperm-specific gene expression during seed development. We also described the employment of the antisense ODN strategy for studies on in vitro spike cultures of barley. Here we present further evidence as to the value of the antisense ODN approach in plant biology by following the effects on starch branching enzyme (SBE) accumulation in barley leaves after suppression of individual SBE genes. In agreement with transcript analyses of SBE expression in barley leaves, a zymogram assay (Fig. 1) revealed that sucrose treatment of barley leaves increased the number of SBE activity bands as compared to sorbitol treatment. In the presence of antisense SBEI or SBEIIA ODNs, zymograms of sucrose-treated leaves displayed only a subset of these activities with bands in the top portion of the zymogram gel missing or diminished. With antisense SBEIIB ODN, all activity bands in the top portion of the gel as well as the lowest band were absent. Based on these data we provide a tentative annotation for the various SBE activity bands. In animal experiments, naked ODNs are usually not taken up by the cells since both the ODNs and the outside of the plasma membrane carry a net negative charge. Thus the uptake of naked ODNs into barley leaf cells was surprising and called for an explanation. As demonstrated in our subsequent paper, the answer seems to be that the ODNs slip into the cells through sugar translocators as they are activated in the presence of the appropriate sugar (Fig. 2). Whether it is the structural resemblance between the sugar (deoxyribose) backbone of the ODNs and the transported sugars that allows for the ODNs to be transferred, or if other mechanisms are involved, remains to be elucidated.« less

Reduction of methylviologen-mediated oxidative stress tolerance in antisense transgenic tobacco seedlings through restricted expression of StAPX.

PubMed

Sun, Wei-Hong; Wang, Yong; He, Hua-Gang; Li, Xue; Song, Wan; Du, Bin; Meng, Qing-Wei

2013-07-01

Ascorbate peroxidases are directly involved in reactive oxygen species (ROS) scavenging by reducing hydrogen peroxide to water. The tomato thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco. RNA gel blot analysis confirmed that StAPX in tomato leaves was induced by methylviologen-mediated oxidative stress. The sense transgenic seedlings exhibited higher tAPX activity than that of the wild type (WT) plants under oxidative stress conditions, while the antisense seedlings exhibited lower tAPX activity. Lower APX activities of antisense transgenic seedlings caused higher malondialdehyde contents and relative electrical conductivity. The sense transgenic seedlings with higher tAPX activity maintained higher chlorophyll content and showed the importance of tAPX in maintaining the optimal chloroplast development under methylviologen stress conditions, whereas the antisense lines maintained lower chlorophyll content than WT seedlings. Results indicated that the over-expression of StAPX enhanced tolerance to methylviologen-mediated oxidative stress in sense transgenic tobacco early seedlings, whereas the suppression of StAPX in antisense transgenic seedlings showed high sensitivity to oxidative stress.

Highly sensitive derivatization reagents possessing positively charged structures for the determination of oligosaccharides in glycoproteins by high-performance liquid chromatography electrospray ionization tandem mass spectrometry.

PubMed

Min, Jun Zhe; Nagai, Keisuke; Shi, Qing; Zhou, Wenjun; Todoroki, Kenichiro; Inoue, Koichi; Lee, Yong-Ill; Toyo'oka, Toshimasa

2016-09-23

We have developed three kinds of novel derivatization reagents (4-CEBTPP, 4-CBBTPP, 5-COTPP) with triphenylphosphine (TPP) as a basic structure carrying a permanent positive charge for resolution of the oligosaccharides in glycoprotein using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The synthesized reagents reacted with the sialylglycosylamine of the sialylglycopeptide after treatment by PNGase F. The final derivatives were analyzed by ESI-MS and sensitively detected in the selected reaction monitoring (SRM) mode. Furthermore, the limits of detection (S/N=3) on the SRM chromatograms were at the fmol level (30fmol). Therefore, we used the limit of detection of the reagent products detected by the SRM and evaluated the utility of each reagent. Among the reagents, the positively charged 4-CEBTPP derivative's peak area was the highest; 4-CEBTPP with a positively charged structure showed about a 20 times greater sensitivity for the glycosylamine of the SGP product compared to the conventional fluorescence reagent, Fmoc-Cl. In addition, various fragment ions based on the carbohydrate units also appeared in the MS/MS spectra. Among the fragment ions, m/z 627.37 (CE=40eV) corresponding to 4-CEBTPP-GlcNAc and m/z 120.09 (CE=100eV) corresponding to 4-CEBTPP are the most important ones for identifying the oligosaccharide. 4-CEBTPP-SGA was easily identified by the selected-ion chromatogram in the product ion scan (m/z 120.09) and in the precursor ion scan (m/z 627.37) by MS/MS detection. The derivatized analytes have a high ionization efficiency and they are detected with a high sensitivity in the electrospray ionization. The novel derivatization reagent with a multi-function provided a higher sensitivity for the oligosaccharide analysis, as well as a better specificity and feasibility. Furthermore, several oligosaccharides in fetuin and ribonuclease B were successfully identified by the proposed procedure. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection*

PubMed Central

Gold, Matthew G.; Fowler, Douglas M.; Means, Christopher K.; Pawson, Catherine T.; Stephany, Jason J.; Langeberg, Lorene K.; Fields, Stanley; Scott, John D.

2013-01-01

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces. PMID:23625929

Effect of injection of antisense oligodeoxynucleotides of GAD isozymes into rat ventromedial hypothalamus on food intake and locomotor activity.

PubMed

Bannai, M; Ichikawa, M; Nishihara, M; Takahashi, M

1998-02-16

In the ventromedial hypothalamus (VMH), gamma-aminobutyric acid (GABA) plays a role in regulating feeding and running behaviors. The GABA synthetic enzyme, glutamic acid decarboxylase (GAD), consists of two isozymes, GAD65 and GAD67. In the present study, the phosphorothioated antisense oligodeoxynucleotides (ODNs) of each GAD isozyme were injected bilaterally into the VMH of male rats, and food intake, body weight and locomotor activity were monitored. ODNs were incorporated in the water-absorbent polymer (WAP, 0.2 nmol/microliter) so that ODNs were retained at the injection site. Each antisense ODN of GAD65 or GAD67 tended to reduce food intake on day 1 (day of injection=day 0) though not significantly. An injection combining both antisense ODNs significantly decreased food intake only on day 1, but body weight remained significantly lower than the control for 5 days. This suppression of body weight gain could be attributed to a significant increase in locomotor activity between days 3 and 5. Individual treatment with either ODNs did not change locomotor activity. The increase in daily locomotor activity in the group receiving the combined antisense ODNs occurred mainly during the light phase. Neither vehicle (WAP) nor control ODN affected food intake, body weight and locomotor activity. Histological studies indicated that antisense ODN distributed within 800 micron from the edge of the area where WAP was located 24 h after the injection gradually disappeared within days, but still remained within 300 micron m distance even 7 days after the injection. Antisense ODN was effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias. Further, HPLC analysis revealed that antisense ODNs of GAD isozymes, either alone or combined, decreased the content of GABA by 50% in VMH 24 h after the injection. These results indicate that suppression of GABA synthesis by either of the GAD isozymes is synergistically involved in suppressing food intake and enhancing locomotor activity in rat VMH. Copyright 1998 Elsevier Science B.V.

Selective sequential precipitation of dissolved metals in mine drainage from coal mine

NASA Astrophysics Data System (ADS)

Yim, Giljae; Bok, Songmin; Ji, Sangwoo; Oh, Chamteut; Cheong, Youngwook; Han, Youngsoo; Ahn, Joosung

2017-04-01

In abandoned mines in Korea, a large amount of mine drainage continues to flow out and spread pollution. In purification of the mine drainage a massive amount of sludge is generated as waste. Since this metal sludge contains high Fe, Al and Mn oxides, developing the treatment method to recover homogeneous individual metal with high purity may beneficial to recycle waste metals as useful resources and reduce the amount of sludge production. In this regard, we established a dissolved metals selective precipitation process to treat Waryong Industry's mine drainage. The process that selectively precipitates metals dissolved in mine drainage is a continuous Fe-buffer-Al process, and each process consists of the neutralization tank, the coagulation tank, and the settling tank. Based on this process, this study verified the operational applicability of the Fe and Al selective precipitation. Our previous study revealed that high-purity Fe and Al precipitates could be recovered at a flow rate of 1.5 ton/day, while the lower purity was achieved when the rate was increased to about 3 ton/day due to the difficulty in reagent dosage control. In the current study was conducted to increase the capacity of the system to recover Fe and Al as high-purity precipitates at a flow rate of 10 ton/day with the ensured continuous operations by introducing an automatic reagent injection system. The previous study had a difficulty in controlling the pH and operating system continuously due to the manually controlled reagent injection system. To upgrade this and ensure the optimal pH in a stable way, a continuous reagent injection system was installed. The result of operation of the 10 ton/day system confirmed that the scaled-up process could maintain the stable recovery rates and purities of precipitates on site.

Flowering time control: another window to the connection between antisense RNA and chromatin.

PubMed

Ietswaart, Robert; Wu, Zhe; Dean, Caroline

2012-09-01

A high proportion of all eukaryotic genes express antisense RNA (asRNA), which accumulates to varying degrees at different loci. Whether there is a general function for asRNA is unknown, but its widespread occurrence and frequent regulation by stress suggest an important role. The best-characterized plant gene exhibiting a complex antisense transcript pattern is the Arabidopsis floral regulator FLOWERING LOCUS C (FLC). Changes occur in the accumulation, splicing, and polyadenylation of this antisense transcript, termed COOLAIR, in different environments and genotypes. These changes are associated with altered chromatin regulation and differential FLC expression, provoking mechanistic comparisons with many well-studied loci in yeast and mammals. Detailed analysis of these specific examples may shed light on the complex interplay between asRNA and chromatin modifications in different genomes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inhibition of bone resorption in vitro by antisense RNA and DNA molecules targeted against carbonic anhydrase II or two subunits of vacuolar H(+)-ATPase.

PubMed Central

Laitala, T; Väänänen, H K

1994-01-01

The bone resorbing cells, osteoclasts, express high levels of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) during bone resorption. We have used antisense RNA and DNA molecules targeted against CA II, and against 16- and 60-kD subunits of vacuolar H(+)-ATPase (V-ATPase), to block the expression of these proteins in vitro. Osteoclastic bone resorption was studied in two in vitro culture systems: release of 45Calcium from prelabeled newborn mouse calvaria cultures, and resorption pit assays performed with rat osteoclasts cultured on bovine bone slices. Both antisense RNA and DNA against CA II and the V-ATPase were used to compare their specificities as regards inhibiting bone resorption in vitro. The antisense molecules inhibited the synthesis of these proteins by decreasing the amounts of mRNA in the cells in a highly specific manner. In osteoclast cultures treated with the 16-kD V-ATPase antisense RNA, acidification of an unknown population of intracellular vesicles was highly stimulated. The acidification of these vesicles was not sensitive to amiloride or bafilomycin A1. This suggests the existence of a back-up system for acidification of intracellular vesicles, when the expression of the V-ATPase is blocked. Our results further indicate that blocking the expression of CA II and V-ATPase with antisense RNA or DNA leads to decreased bone resorption. Images PMID:8200964

Antisense antibiotics: a brief review of novel target discovery and delivery.

PubMed

Bai, Hui; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Meng, Jingru; Luo, Xiaoxing

2010-06-01

The nightmare of multi-drug resistant bacteria will still haunt if no panacea is ever found. Efforts on seeking desirable natural products with bactericidal property and screening chemically modified derivatives of traditional antibiotics have lagged behind the emergence of new multi-drug resistant bacteria. The concept of using antisense antibiotics, now as revolutionary as is on threshold has experienced ups and downs in the past decade. In the past five years, however, significant technology advances in the fields of microbial genomics, structural modification of oligonucleotides and efficient delivery system have led to fundamental progress in the research and in vivo application of this paradigm. The wealthy information provided in the microbial genomics era has allowed the identification and/or validation of a number of essential genes that may serve as possible targets for antisense inhibition; antisense oligodeoxynucleotides (ODNs) based on the 3rd generation of modified structures, e.g., peptide nucleic acids (PNAs) and phosphorodiamidate morpholino oligomers (PMOs) have shown great potency in gene expression inhibition in a sequence-specific and dosedependent manner at low micromolar concentrations; and cell penetrating peptide mediated delivery system has enabled the effective display of intracellular antisense inhibition of targeted genes both in vitro and in vivo. The new methods show promise in the discovery of novel gene-specific antisense antibiotics that will be useful in the future battle against drug-resistant bacterial infections. This review describes this promising paradigm, the targets that have been identified and the recent technologies on which it is delivered.

Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy.

PubMed

Krieg, A M; Tonkinson, J; Matson, S; Zhao, Q; Saxon, M; Zhang, L M; Bhanja, U; Yakubov, L; Stein, C A

1993-02-01

Phosphodiester oligodeoxynucleotides bearing a 5' cholesteryl (chol) modification bind to low density lipoprotein (LDL), apparently by partitioning the chol-modified oligonucleotides into the lipid layer. Both HL60 cells and primary mouse spleen T and B cells incubated with fluorescently labeled chol-modified oligonucleotide showed substantially increased cellular association by flow cytometry and increased internalization by confocal microscopy compared to an identical molecule not bearing the chol group. Cellular internalization of chol-modified oligonucleotide occurred at least partially through the LDL receptor; it was increased in mouse spleen cells by cell culture in lipoprotein-deficient medium and/or lovastatin, and it was decreased by culture in high serum medium. To determine whether chol-modified oligonucleotides are more potent antisense agents, we titered antisense unmodified phosphodiester and chol-modified oligonucleotides targeted against a mouse immunosuppressive protein. Murine spleen cells cultured with 20 microM phosphodiester antisense oligonucleotides had a 2-fold increase in RNA synthesis, indicating the expected lymphocyte activation. Antisense chol-modified oligonucleotides showed an 8-fold increase in relative potency: they caused a 2-fold increase in RNA synthesis at just 2.5 microM. The increased efficacy was blocked by heparin and was further increased by cell culture in 1% (vs. 10%) fetal bovine serum, suggesting that the effect may, at least in part, be mediated via the LDL receptor. Antisense chol-modified oligonucleotides are sequence specific and have increased potency as compared to unmodified oligonucleotides.

Novel Target for Ameliorating Pain and Other Problems after SCI: Spontaneous Activityin Nociceptors

DTIC Science & Technology

2015-10-01

Funding support (other than DoD) Mission Connect-TIRR Foundation, "Neuroprotective Effect of Targeting KCNQ/ Kv7 Channels in Spinal Cord Injury...the function of a sodium ion channel , Nav1.8, that is selectively expressed in primary afferent neurons (especially nociceptors) ameliorate reflex...our finding that antisense knockdown of TRPV1 channels or pharmacological blockade of TRPV1 channels -- which are expressed most abundantly in

Physicochemical and biological properties of self-assembled antisense/poly(amidoamine) dendrimer nanoparticles: the effect of dendrimer generation and charge ratio

PubMed Central

Nomani, Alireza; Haririan, Ismaeil; Rahimnia, Ramin; Fouladdel, Shamileh; Gazori, Tarane; Dinarvand, Rassoul; Omidi, Yadollah; Azizi, Ebrahim

2010-01-01

To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine) dendrimer (PAMAM) dendrimer and a short-stranded DNA (antisense oligonucleotide), multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS); zeta potential measurement; and atomic force microscopy (AFM). PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller molecules produce more heterodisperse and large nanoparticles when they are condensed with a cationic dendrimer. AFM images also showed that such nanoparticles were spherical. The stability of the antisense content of the nanoparticles was investigated over different charge ratios using polyacrylamide gel electrophoresis. It was clear from such analyses that much more than charge neutrality point was required to obtain stable nanoparticles. For cell uptake, self-assembled nanoparticles were prepared with PAMAM G5 and 5’-FITC labeled antisense and the uptake experiment was carried out in T47D cell culture. This investigation also shows that the cytotoxicity of the nanoparticles was dependent upon the generation and charge ratio of the PAMAM dendrimer, and the antisense concentration had no significant effect on the cytotoxicity. PMID:20517481

Antisense apolipoprotein B therapy: where do we stand?

PubMed

Akdim, Fatima; Stroes, Erik S G; Kastelein, John J P

2007-08-01

Antisense oligonucleotides are novel therapeutic agents that reduce the number of specific mRNAs available for translation of the encoded protein. ISIS 301012 is an antisense oligonucleotide developed to reduce the hepatic synthesis of apolipoprotein B-100. Apolipoprotein B-100 is made in the liver, and antisense oligonucleotides preferentially distribute to that organ, so antisense apolipoprotein B-100 may have potential as an efficacious lipid-lowering agent. Recently, in healthy volunteers and in mild dyslipidaemic patients, this strategy as monotherapy or in conjunction with statins has shown unparalleled efficacy in reducing apolipoprotein B-100 and LDL-cholesterol. Tolerance for this novel therapy is encouraging and safety concerns currently only relate to mild injection-site reactions and rare liver-function test abnormalities. It should be noted, however, that these safety results were obtained in relatively few individuals. ISIS 301012 has initially shown promising results in experimental animal models, and in clinical trials in humans. Besides the effect of reducing apolipoprotein B-100 and LDL-cholesterol, this compound also significantly lowers plasma triglycerides. Safety concerns related to the drug include increased liver-function tests. To date no evidence of hepatic steatosis has been reported. Nonetheless, clinical trials of longer duration are required to demonstrate further safety.

RNase III-Binding-mRNAs Revealed Novel Complementary Transcripts in Streptomyces

PubMed Central

Šetinová, Dita; Šmídová, Klára; Pohl, Pavel; Musić, Inesa; Bobek, Jan

2018-01-01

cis-Antisense RNAs (asRNAs) provide very simple and effective gene expression control due to the perfect complementarity between regulated and regulatory transcripts. In Streptomyces, the antibiotic-producing clade, the antisense control system is not yet understood, although it might direct the organism's complex development. Initial studies in Streptomyces have found a number of asRNAs. Apart from this, hundreds of mRNAs have been shown to bind RNase III, the double strand-specific endoribonuclease. In this study, we tested 17 mRNAs that have been previously co-precipitated with RNase III for antisense expression. Our RACE mapping showed that all of these mRNAs possess cognate asRNA. Additional tests for antisense expression uncovered as-adpA, as-rnc, as3983, as-sigB, as-sigH, and as-sigR RNAs. Northern blots detected the expression profiles of 18 novel transcripts. Noteworthy, we also found that only a minority of asRNAs respond to the absence of RNase III enzyme by increasing their cellular levels. Our findings suggest that antisense expression is widespread in Streptomyces, including genes of such important developmental regulators, as AdpA, RNase III, and sigma factors. PMID:29379487

RNase III-Binding-mRNAs Revealed Novel Complementary Transcripts in Streptomyces.

PubMed

Šetinová, Dita; Šmídová, Klára; Pohl, Pavel; Musić, Inesa; Bobek, Jan

2017-01-01

cis -Antisense RNAs (asRNAs) provide very simple and effective gene expression control due to the perfect complementarity between regulated and regulatory transcripts. In Streptomyces , the antibiotic-producing clade, the antisense control system is not yet understood, although it might direct the organism's complex development. Initial studies in Streptomyces have found a number of asRNAs. Apart from this, hundreds of mRNAs have been shown to bind RNase III, the double strand-specific endoribonuclease. In this study, we tested 17 mRNAs that have been previously co-precipitated with RNase III for antisense expression. Our RACE mapping showed that all of these mRNAs possess cognate asRNA. Additional tests for antisense expression uncovered as-adpA, as-rnc, as3983, as-sigB, as-sigH , and as-sigR RNAs. Northern blots detected the expression profiles of 18 novel transcripts. Noteworthy, we also found that only a minority of asRNAs respond to the absence of RNase III enzyme by increasing their cellular levels. Our findings suggest that antisense expression is widespread in Streptomyces , including genes of such important developmental regulators, as AdpA, RNase III, and sigma factors.

Upping the Antisense Ante.

ERIC Educational Resources Information Center

Weiss, Rick

1991-01-01

Discussed is a designer-drug technology called antisense which blocks messenger RNA's ability to carry information to protein producing sites in the cell. The applications of this drug to AIDS research, cancer therapy, and other diseases are discussed. (KR)

Impact of the excess sludge modification with selected chemical reagents on the increase of dissolved organic substances concentration compounds transformations in activated sludge.

PubMed

Zawieja, Iwona; Lidia, Wolny; Marta, Próba

2017-07-01

Submission of excess sludge initial disintegration process significantly affects the efficiency of anaerobic stabilization process. Expression of increasing the concentration of organic matter in dissolved form is to increase sludge disintegration. As a result of chemical modification is an increase of the chemical oxygen demand and the concentration of volatile fatty acids. The aim of this study was to determine the impact of the disintegration process with selected chemical reagents to increase the concentration of organic substances in dissolved form. The process of chemical disintegration of excess sludge was treated using the following reagents: Mg(OH) 2 , Ca(OH) 2 , HCl, H 2 SO 4 , H 2 O 2 . The modification was carried out at ambient temperature for 2, 6 and 24h. During sludge disintegration it was noticed the growth of indicators values that confirmed the susceptibility of prepared sludge to biodegradation. Copyright © 2017 Elsevier Inc. All rights reserved.

Substrate-Directed Catalytic Selective Chemical Reactions.

PubMed

Sawano, Takahiro; Yamamoto, Hisashi

2018-05-04

The development of highly efficient reactions at only the desired position is one of the most important subjects in organic chemistry. Most of the reactions in current organic chemistry are reagent- or catalyst-controlled reactions, and the regio- and stereoselectivity of the reactions are determined by the inherent nature of the reagent or catalyst. In sharp contrast, substrate-directed reaction determines the selectivity of the reactions by the functional group on the substrate and can strictly distinguish sterically and electronically similar multiple reaction sites in the substrate. In this Perspective, three topics of substrate-directed reaction are mainly reviewed: (1) directing group-assisted epoxidation of alkenes, (2) ring-opening reactions of epoxides by various nucleophiles, and (3) catalytic peptide synthesis. Our newly developed synthetic methods with new ligands including hydroxamic acid derived ligands realized not only highly efficient reactions but also pinpointed reactions at the expected position, demonstrating the substrate-directed reaction as a powerful method to achieve the desired regio- and stereoselective functionalization of molecules from different viewpoints of reagent- or catalyst-controlled reactions.

Reagent Selection Methodology for a Novel Explosives Detection Platform

ScienceCinema

Warner, Marvin

2018-02-14

This video describes research being conducted by Dr. Marvin Warner, a research scientist at Pacific Northwest National Laboratory, in the individual pieces of antibodies used to set up a chemical reaction that will give off light just by mixing reagents together with a sample that contains an explosive molecule. This technology would help detect if explosives are present with just the use of a handheld system or container.

Highly Stereoselective Gold-Catalyzed Coupling of Diazo Reagents and Fluorinated Enol Silyl Ethers to Tetrasubstituted Alkenes.

PubMed

Liao, Fu-Min; Cao, Zhong-Yan; Yu, Jin-Sheng; Zhou, Jian

2017-02-20

We report a highly stereoselective synthesis of all-carbon or fluorinated tetrasubstituted alkenes from diazo reagents and fluorinated enol silyl ethers, using C-F bond as a synthetic handle. Cationic Au I catalysis plays a key role in this reaction. Remarkable fluorine effects on the reactivity and selectivity was also observed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Removal of toxic metals from vanadium-contaminated soils using a washing method: Reagent selection and parameter optimization.

PubMed

Jiang, Jianguo; Yang, Meng; Gao, Yuchen; Wang, Jiaming; Li, Dean; Li, Tianran

2017-08-01

Vanadium (V) contamination in soils is an increasing worldwide concern facing human health and environmental conservation. The fractionation of a metal influences its mobility and biological toxicity. We analyzed the fractionations of V and several other metals using the BCR three-step sequential extraction procedure. Among methods for removing metal contamination, soil washing is an effective permanent treatment. We conducted experiments to select the proper reagents and to optimize extraction conditions. Citric acid, tartaric acid, oxalic acid, and Na 2 EDTA all exhibited high removal rates of the extractable state of V. With a liquid-to-solid ratio of 10, washing with 0.4 mol/L citric acid, 0.4 mol/L tartaric acid, 0.4 mol/L oxalic acid, and 0.12 mol/L Na 2 EDTA led to removal rates of 91%, 88%, 88%, and 61%, respectively. The effect of multiple washing on removal rate was also explored. According to the changes observed in metal fractionations, differences in removal rates among reagents is likely associated with their pK a value, pH in solution, and chemical structure. We concluded that treating with appropriate washing reagents under optimal conditions can greatly enhance the remediation of vanadium-contaminated soils. Copyright © 2017 Elsevier Ltd. All rights reserved.

Release profile and stability evaluation of optimized chitosan/alginate nanoparticles as EGFR antisense vector

PubMed Central

Azizi, Ebrahim; Namazi, Alireza; Haririan, Ismaeil; Fouladdel, Shamileh; Khoshayand, Mohammad R; Shotorbani, Parisa Y; Nomani, Alireza; Gazori, Taraneh

2010-01-01

Chitosan/alginate nanoparticles which had been optimized in our previous study using two different N/P ratios were chosen and their ability to release epidermal growth factor receptor (EGFR) antisense was investigated. In addition, the stability of these nanoparticles in aqueous medium and after freeze-drying was investigated. In the case of both N/P ratios (5, 25), nanoparticles started releasing EGFR antisense as soon as they were exposed to the medium and the release lasted for approximately 50 hours. Nanoparticle size, shape, zeta potential, and release profile did not show any significant change after the freeze-drying process (followed by reswelling). The nanoparticles were reswellable again after freeze-drying in phosphate buffer with a pH of 7.4 over a period of six hours. Agarose gel electrophoresis of the nanoparticles with the two different N/P ratios showed that these nanoparticles could protect EGFR antisense molecules for six hours. PMID:20957167

Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

NASA Astrophysics Data System (ADS)

Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

1996-11-01

In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

Genetic therapeutic approaches for Duchenne muscular dystrophy.

PubMed

Foster, Helen; Popplewell, Linda; Dickson, George

2012-07-01

Despite an expansive wealth of research following the discovery of the DMD gene 25 years ago, there is still no curative treatment for Duchenne muscular dystrophy. However, there are currently many promising lines of research, including cell-based therapies and pharmacological reagents to upregulate dystrophin via readthrough of nonsense mutations or by upregulation of the dystrophin homolog utrophin. Here we review genetic-based therapeutic strategies aimed at the amelioration of the DMD phenotype. These include the reintroduction of a copy of the DMD gene into an affected tissue by means of a viral vector; correction of the mutated DMD transcript by antisense oligonucleotide-induced exon skipping to restore the open reading frame; and direct modification of the DMD gene at a chromosomal level through genome editing. All these approaches are discussed in terms of the more recent advances, and the hurdles to be overcome if a comprehensive and effective treatment for DMD is to be found. These hurdles include the need to target all musculature of the body. Therefore any potential treatment would need to be administered systemically. In addition, any treatment needs to have a long-term effect, with the possibility of readministration, while avoiding any potentially detrimental immune response to the vector or transgene.

The Status of Exon Skipping as a Therapeutic Approach to Duchenne Muscular Dystrophy

PubMed Central

Lu, Qi-Long; Yokota, Toshifumi; Takeda, Shin'ichi; Garcia, Luis; Muntoni, Francesco; Partridge, Terence

2011-01-01

Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level. PMID:20978473

Selection of organic acid leaching reagent for recovery of zinc and manganese from zinc-carbon and alkaline spent batteries

NASA Astrophysics Data System (ADS)

Yuliusman; Amiliana, R. A.; Wulandari, P. T.; Ramadhan, I. T.; Kusumadewi, F. A.

2018-03-01

Zinc-carbon and alkaline batteries are often used in electronic equipment that requires small quantities of power. The waste from these batteries contains valuable metals, such as zinc and manganese, that are needed in many industries and can pollute the environment if not treated properly. This paper concerns the recovery of zinc and manganese metals from zinc-carbon and alkaline spent batteries with leaching method and using organic acid as the environmental friendly leaching reagent. Three different organic acids, namely citric acid, malic acid and aspartic acid, were used as leaching reagents and compared with sulfuric acid as non-organic acid reagents that often used for leaching. The presence of hydrogen peroxide as manganese reducers was investigated for both organic and non-organic leaching reagents. The result showed that citric acid can recover 64.37% Zinc and 51.32% Manganese, while malic acid and aspartic acid could recover less than these. Hydrogen peroxide gave the significant effect for leaching manganese with non-organic acid, but not with organic acid.

Synthetic single domain antibodies for the conformational trapping of membrane proteins

PubMed Central

Arnold, Fabian M; Stohler, Peter; Bocquet, Nicolas; Hug, Melanie N; Huber, Sylwia; Siegrist, Martin; Hetemann, Lisa; Gera, Jennifer; Gmür, Samira; Spies, Peter; Gygax, Daniel

2018-01-01

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins. PMID:29792401

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

NASA Astrophysics Data System (ADS)

Hari, Yvonne; Dugovič, Branislav; Istrate, Alena; Fignolé, Annabel; Leumann, Christian J.; Schürch, Stefan

2016-07-01

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

Apparatus and method for polymer synthesis using arrays

DOEpatents

Brennan, Thomas M.

1995-01-01

A polymer synthesis apparatus (20) for building a polymer chain including a head assembly (21) having an array of nozzles (22) with each nozzle coupled to a reservoir (23) of liquid reagent (24) , and a base assembly (25) having an array of reaction wells (26). A transport mechanism (27) aligns the reaction wells (26) and selected nozzles (22) for deposition of the liquid reagent (24) into selected reaction wells (26). A sliding seal (30) is positioned between the head assembly (21) and the base assembly (25) to form a common chamber (31) enclosing both the reaction well (26) and the nozzles (22) therein. A gas inlet (70) into the common chamber (31), upstream from the nozzles (22), and a gas outlet (71) out of the common chamber (31) , downstream from the nozzles (22) , sweeps the common chamber ( 31 ) of toxic fumes emitted by the reagents. Each reaction well (26) includes an orifice (74) extending into the well (26) which is of a size and dimension to form a capillary liquid seal to retain the reagent solution (76) in the well (26) for polymer chain growth therein. A pressure regulating device (82) is provided for controlling a pressure differential, between a first gas pressure exerted on the reaction well (26) and a second gas pressure exerted on an exit (80) of the orifice, such that upon the pressure differential exceeding a predetermined amount, the reagent solution (76) is expelled from the well (26) through the orifice (74). A method of synthesis of a polymer chain in a synthesis apparatus (20) is also included.

Apparatus and method for polymer synthesis using arrays

DOEpatents

Brennan, Thomas M.

1996-01-01

A polymer synthesis apparatus (20) for building a polymer chain including a head assembly (21) having an array of nozzles (22) with each nozzle coupled to a reservoir (23) of liquid reagent (24), and a base assembly (25) having an array of reaction wells (26). A transport mechanism (27) aligns the reaction wells (26) and selected nozzles (22) for deposition of the liquid reagent (24) into selected reaction wells (26). A sliding seal (30) is positioned between the head assembly (21) and the base assembly (25) to form a common chamber (31) enclosing both the reaction well (26) and the nozzles (22) therein. A gas inlet (70) into the common chamber (31), upstream from the nozzles (22), and a gas outlet (71) out of the common chamber (31), downstream from the nozzles (22), sweeps the common chamber (31) of toxic fumes emitted by the reagents. Each reaction well ( 26) includes an orifice (74) extending into the well (26) which is of a size and dimension to form a capillary liquid seal to retain the reagent solution (76) in the well (26) for polymer chain growth therein. A pressure regulating device (82 ) is provided for controlling a pressure differential, between a first gas pressure exerted on the reaction well (26) and a second gas pressure exerted on an exit (80) of the orifice, such that upon the pressure differential exceeding a predetermined amount, the reagent solution (76) is expelled from the well (26) through the orifice (74). A method of synthesis of a polymer chain in a synthesis apparatus (20) is also included.

C-fos down-regulation inhibits testosterone-dependent male sexual behavior and the associated learning

PubMed Central

Niessen, Neville-Andrew; Balthazart, Jacques; Ball, Gregory F.; Charlier, Thierry D.

2013-01-01

Environmental stimulation results in an increased expression of transcription factors called immediate early genes (IEG) in specific neuronal populations. In male Japanese quail, copulation with a female increases the expression of the IEGs zenk and c-fos in the medial preoptic nucleus (POM), a key nucleus controlling male sexual behavior. The functional significance of this increased IEG expression that follows performance of copulatory behavior is unknown. We addressed this question by repeatedly quantifying the performance of appetitive (learned social proximity response) and consummatory (actual copulation) sexual behavior in castrated, testosterone-treated males that received daily intracerebroventricular injection of an antisense oligodeoxynucleotide targeting c-fos or control vehicle. Daily antisense injections significantly inhibited expression of copulatory behavior as well as acquisition of the learned social proximity response. A strong reduction of the proximity response was still observed in antisense-treated birds that copulated with a female, ruling out the indirect effect of the absence of interactions with females on the learning process. After a two-day interruption of behavioral testing but not of antisense injections, birds were submitted to a final copulatory test that confirmed the behavioral inhibition in antisense-injected birds. Brains were collected 90 min after the behavioral testing for quantification of c-fos immunoreactive cells. A significant reduction of the number of c-fos-positive cells in POM but not in other brain regions was observed following antisense injection. Together, data suggest that c-fos expression in POM modulates copulatory behavior and sexual learning in male quail. PMID:23895306

Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia1

PubMed Central

Pennycooke, Joyce C.; Jones, Michelle L.; Stushnoff, Cecil

2003-01-01

α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants. PMID:14500789

Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense Transcription

PubMed Central

Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J.; van Hooff, Sander; van Leenen, Dik

2012-01-01

Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3′-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms. PMID:23028359

Facially Selective Cu-catalyzed Carbozincation of Cyclopropenes Using Arylzinc Reagents Formed by Sequential I/Mg/Zn exchange

PubMed Central

Tarwade, Vinod; Selvaraj, Ramajeyam; Fox, Joseph M.

2012-01-01

Described is a Cu-catalyzed directed carbozincation of cyclopropenes with organozinc reagents prepared by I/Mg/Zn exchange. This protocol broadens the scope with respect to functional group tolerance and enables use of aryl iodide precursors, rather than purified diorganozinc precursors. Critical to diastereoselectivity of the carbozincation step is the removal of magnesium halide salts after transmetallation with ZnCl2. PMID:23035947

Enantioselective and regiodivergent copper-catalyzed conjugate addition of trialkylaluminium reagents to extended nitro-Michael acceptors.

PubMed

Tissot, Matthieu; Müller, Daniel; Belot, Sébastien; Alexakis, Alexandre

2010-06-18

The first highly enantioselective and regiodivergent conjugate addition of trialkylaluminium reagents to nitrodienes and nitroenynes is described. By a design of the substrate and a fine-tuning of the reaction conditions, it is possible to selectively form the 1,4- or 1,6-adduct. The same combination of catalyst, copper source, and a ferrocene-based phosphine ligand afforded enantioselectivities up to 95% and 91%, respectively.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

PubMed

Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

2017-09-19

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

NASA Technical Reports Server (NTRS)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

Antisense Transcription Is Pervasive but Rarely Conserved in Enteric Bacteria

PubMed Central

Raghavan, Rahul; Sloan, Daniel B.; Ochman, Howard

2012-01-01

ABSTRACT Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell’s transcription machinery. PMID:22872780

Permissive Sense and Antisense Transcription from the 5′ and 3′ Long Terminal Repeats of Human T-Cell Leukemia Virus Type 1

PubMed Central

Polakowski, Nicholas; Hoang, Kimson

2016-01-01

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus, and, as such, its genome becomes chromosomally integrated following infection. The resulting provirus contains identical 5′ and 3′ peripheral long terminal repeats (LTRs) containing bidirectional promoters. Antisense transcription from the 3′ LTR regulates expression of a single gene, hbz, while sense transcription from the 5′ LTR controls expression of all other viral genes, including tax. Both the HBZ and Tax proteins are implicated in the development of adult T-cell leukemia (ATL), a T-cell malignancy caused by HTLV-1 infection. However, these proteins appear to harbor opposing molecular functions, indicating that they may act independently and at different time points prior to leukemogenesis. Here, we used bidirectional reporter constructs to test whether transcriptional interference serves as a mechanism that inhibits simultaneous expression of Tax and HBZ. We found that sense transcription did not interfere with antisense transcription from the 3′ LTR and vice versa, even with strong transcription emanating from the opposing direction. Therefore, bidirectional transcription across the provirus might not restrict hbz or tax expression. Single-cell analyses revealed that antisense transcription predominates in the absence of Tax, which transactivates viral sense transcription. Interestingly, a population of Tax-expressing cells exhibited antisense but not activated sense transcription. Consistent with the ability of Tax to induce cell cycle arrest, this population was arrested in G0/G1 phase. These results imply that cell cycle arrest inhibits Tax-mediated activation of sense transcription without affecting antisense transcription, which may be important for long-term viral latency. IMPORTANCE The chromosomally integrated form of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) contains identical DNA sequences, known as long terminal repeats (LTRs), at its 5′ and 3′ ends. The LTRs modulate transcription in both forward (sense) and reverse (antisense) directions. We found that sense transcription from the 5′ LTR does not interfere with antisense transcription from the 3′ LTR, allowing viral genes encoded on opposite DNA strands to be simultaneously transcribed. Two such genes are tax and hbz, and while they are thought to function at different times during the course of infection to promote leukemogenesis of infected T cells, our results indicate that they can be simultaneously transcribed. We also found that the ability of Tax to induce cell cycle arrest inhibits its fundamental function of activating viral sense transcription but does not affect antisense transcription. This regulatory mechanism may be important for long-term HTLV-1 infection. PMID:26792732

Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state.

PubMed

Shiokawa, D; Tanuma, S

2004-10-01

In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase gamma, in neuronal apoptosis. High expression of CAD, but not DNase gamma, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase gamma occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase gamma caused by its antisense RNA. The induction of DNase gamma is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase gamma. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase gamma depending on the differentiation state. Furthermore, DNase gamma is suggested to be involved in naturally occurring apoptosis in developing nervous systems.

An approach to enhance self-compensation capability in paper-based devices for chemical sensing.

PubMed

Lo, Shih-Jie; Chen, Kuan-Hung; Yao, Da-Jeng

2015-12-01

This paper describes a simple design for increasing the tolerance of reagent dislocation on a paper-based platform using a combination of wax-treated paper and a vortex mixer. To date, massive budgetary funds are required in the biotechnological industry to develop new applications; a large part of that cost is attributable to the screening of specific chemical compounds. Here, we propose using a liquid-handling robot to automatically deposit selected reagents on a paper-based platform. We also present a preliminary concept approach for developing a reagent placing device with simple and inexpensive features. A defect of inaccuracy was observed between droplet location and test well location after viewing the performance of the liquid-handling robot on our paper-based platform. Because of dislocation error resulting from robotic reagent placement, we decided to apply an external, rotational force following droplet placement in order to compensate for the distance of reagent dislocation. Note, the largest distance of reagent dislocation was determined by examining the results of altering applied reagent volume, but not concentration, in volumes from 5 µL to 30 µL in a series of experiments. As a result of these experiments, we observed that dislocation was positively affected by an increase in applied volume. A colorimetric assay for nitrite detection was also performed to confirm the feasibility of this method. This work, we believe, can minimize the cost of chemical compound screening for the biotechnological industry. Copyright © 2015 Elsevier B.V. All rights reserved.

Chiral reagents in glycosylation and modification of carbohydrates.

PubMed

Wang, Hao-Yuan; Blaszczyk, Stephanie A; Xiao, Guozhi; Tang, Weiping

2018-02-05

Carbohydrates play a significant role in numerous biological events, and the chemical synthesis of carbohydrates is vital for further studies to understand their various biological functions. Due to the structural complexity of carbohydrates, the stereoselective formation of glycosidic linkages and the site-selective modification of hydroxyl groups are very challenging and at the same time extremely important. In recent years, the rapid development of chiral reagents including both chiral auxiliaries and chiral catalysts has significantly improved the stereoselectivity for glycosylation reactions and the site-selectivity for the modification of carbohydrates. These new tools will greatly facilitate the efficient synthesis of oligosaccharides, polysaccharides, and glycoconjugates. In this tutorial review, we will summarize these advances and highlight the most recent examples.

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells

NASA Technical Reports Server (NTRS)

Duncan, R. L.; Kizer, N.; Barry, E. L.; Friedman, P. A.; Hruska, K. A.

1996-01-01

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts

PubMed Central

PETERS, NICK T.; ROHRBACH, JUSTIN A.; ZALEWSKI, BRIAN A.; BYRKETT, COLLEEN M.; VAUGHN, JACK C.

2003-01-01

We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression. PMID:12756328

Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

PubMed

Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam

2015-11-11

Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p < 0.05). At these PNA concentrations, 66 % of MRSA and 92 % of MRSP cells were killed by oxacillin (p < 0.01). Anti-ftsZ PNA at 7.5 and 2.5 μM reduced ftsZ mRNA in MRSA and MRSP, respectively (p ≤ 0.05). At these PNA concentrations, 86 % of MRSA cells and 95 % of MRSP cells were killed by oxacillin (p < 0.05). Anti-ftsZ PNAs resulted in swelling of bacterial cells. Scrambled PNA controls did not affect MRSA but sensitized MRSP moderately to oxacillin without affecting mRNA levels. The antisense PNAs effects observed provide in vitro proof of concept that this approach can be used to reverse β-lactam resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

The Seeds of Lotus japonicus Lines Transformed with Sense, Antisense, and Sense/Antisense Galactomannan Galactosyltransferase Constructs Have Structurally Altered Galactomannans in Their Endosperm Cell Walls1

PubMed Central

Edwards, Mary E.; Choo, Tze-Siang; Dickson, Cathryn A.; Scott, Catherine; Gidley, Michael J.; Reid, J.S. Grant

2004-01-01

Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1→6)-α-galactose (Gal) substitution of the (1→4)-β-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense (“hairpin loop”) constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T1 generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T1 generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T2 generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase. PMID:14988472

Simultaneous Down-Regulation of Caffeic/5-Hydroxy Ferulic Acid-O-Methyltransferase I and Cinnamoyl-Coenzyme A Reductase in the Progeny from a Cross between Tobacco Lines Homozygous for Each Transgene. Consequences for Plant Development and Lignin Synthesis1

PubMed Central

Pinçon, Gaelle; Chabannes, Matthieu; Lapierre, Catherine; Pollet, Brigitte; Ruel, Katia; Joseleau, Jean-Paul; Boudet, Alain M.; Legrand, Michel

2001-01-01

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants. PMID:11351078

Immunotechnology: Preparation of Immunotherapeutic Reagents and Development of Immunopharmacologic Vaccines.

DTIC Science & Technology

1981-10-01

Cryopreservation and HLA Typing of the Panel We have assembled a group of volunteer and paid blood donors, some of whom have been selected on the basis of...reagents used as controls. The cell donor panel is typed for human histocompatibility antigens. Dr. A.H. Johnson determines the HLA -A,B,C phenotypes and Dr...2 microglobulin and anti- HLA antibodies. Screening the fusions on glutaraldehyde fixed monolayers of B lymphoblastoid lines, T cell leukaemic lines

Scandium recovery from slags after oxidized nickel ore processing

NASA Astrophysics Data System (ADS)

Smyshlyaev, Denis; Botalov, Maxim; Bunkov, Grigory; Rychkov, Vladimir; Kirillov, Evgeny; Kirillov, Sergey; Semenishchev, Vladimir

2017-09-01

One of the possible sources of scandium production - waste (slags) from processing of oxidized nickel ores, has been considered in present research work. The hydrometallurgical method has been selected as the primary for scandium extraction. Different reagents for leaching of scandium, such as sulfuric acid, various carbonate salts and fluorides, have been tested. Sulfuric acid has been recognized as an optimal leaching reagent. Sulfuric acid concentration of 100 g L-1 allowed recovering up to 97 % of scandium.

The generation of concentration gradients using electroosmotic flow in micro reactors allowing stereoselective chemical synthesis.

PubMed

Skelton, V; Greenway, G M; Haswell, S J; Styring, P; Morgan, D O; Warrington, B H; Wong, S Y

2001-01-01

The stereoselective control of chemical reactions has been achieved by applying electrical fields in a micro reactor generating controlled concentration gradients of the reagent streams. The chemistry based upon well-established Wittig synthesis was carried out in a micro reactor device fabricated in borosilicate glass using photolithographic and wet etching techniques. The selectivity of the cis (Z) to trans (E) isomeric ratio in the product synthesised was controlled by varying the applied voltages to the reagent reservoirs within the micro reactor. This subsequently altered the relative reagent concentrations within the device resulting in Z/E ratios in the range 0.57-5.21. By comparison, a traditional batch method based on the same reaction length, concentration, solvent and stoichiometry (i.e., 1.0:1.5:1.0 reagent ratios) gave a Z/E in the range 2.8-3.0. However, when the stoichiometric ratios were varied up to ten times as much, the Z/E ratios varied in accordance to the micro reactor i.e., when the aldehyde is in excess, the Z isomer predominates whereas when the aldehyde is in low concentrations, the E isomer is the more favourable form. Thus indicating that localised concentration gradients generated by careful flow control due to the diffusion limited non-turbulent mixing regime within a micro reactor, leads to the observed stereo selectivity for the cis and trans isomers.

Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

PubMed

Alban, Silvana M; de Moura, Juliana Ferreira; Minozzo, João Carlos; Mira, Marcelo Távora; Soccol, Vanete Thomaz

2013-01-25

An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Method for producing size selected particles

DOEpatents

Krumdick, Gregory K.; Shin, Young Ho; Takeya, Kaname

2016-09-20

The invention provides a system for preparing specific sized particles, the system comprising a continuous stir tank reactor adapted to receive reactants; a centrifugal dispenser positioned downstream from the reactor and in fluid communication with the reactor; a particle separator positioned downstream of the dispenser; and a solution stream return conduit positioned between the separator and the reactor. Also provided is a method for preparing specific sized particles, the method comprising introducing reagent into a continuous stir reaction tank and allowing the reagents to react to produce product liquor containing particles; contacting the liquor particles with a centrifugal force for a time sufficient to generate particles of a predetermined size and morphology; and returning unused reagents and particles of a non-predetermined size to the tank.

Synthesis of enyne and aryl vinyl sulfoxides: functionalization via Pummerer rearrangement.

PubMed

Souza, Frederico B; Shamim, Anwar; Argomedo, Luiz M Z; Pimenta, Daniel C; Stefani, Hélio A

2015-11-01

An efficient methodology for the synthesis of aryl-substituted vinyl sulfoxides through direct substitution of aryl-substituted alkynyl grignard reagents on menthyl-p-toluenesulfinate followed by Suzuki-Miyaura cross-coupling reaction has been developed. It has also been described that the reaction of alkyl-substituted and cycloalkyl-substituted alkynyl grignard reagents with menthyl-p-toluenesulfinate led to two products, i.e., alkynyl sulfoxide derivatives, as a result of substitution, and enyne sulfoxide derivatives, which resulted from substitution followed by Michael type addition. It was possible to selectively synthesize the enyne sulfoxide derivatives by changing the concentration of the grignard reagent. These alkenyl sulfoxides were transformed into the corresponding [Formula: see text]-thio aldehydes in high yields via additive Pummerer rearrangement.

Strategies to introduce resistance to viroids (Book Chapter)

USDA-ARS?s Scientific Manuscript database

Little or no naturally occurring durable resistance to viroids has been found in most viroid host species; therefore efforts to engineer viroid resistance in these plant hosts have been made. These efforts include strategies that incorporate viroid-specific antisense RNAs, sense and antisense viroid...

Bcl-2 antisense therapy in B-cell malignant proliferative disorders.

PubMed

Chanan-Khan, Asher; Czuczman, Myron S

2004-08-01

Overexpression of Bcl-2 oncogene has been clinically associated with an aggressive clinical course, chemotherapy and radiotherapy resistance, and poor survival in patients with malignant B-cell disorders. Patients with relapsed or refractory chronic lymphocytic leukemia, multiple myeloma, or non-Hodgkin's lymphoma have limited therapeutic options. Preclinical and early clinical data have shown that Bcl-2 oncoprotein can be decreased by Bcl-2 antisense therapy. Also, downregulation of Bcl-2 protein can result in reversal of chemotherapy resistance and improved antitumor activity of biologic agents. Various clinical trials are evaluating the role of targeting Bcl-2 as a mechanism to enhance the antitumor potential of chemotherapy and immunotherapy. Early results from these clinical studies are encouraging and confirm the proof of principle for antisense therapy. As current data mature, these trials will hopefully validate preliminary results and establish Bcl-2 antisense as an important addition to the current armamentarium used in the treatment of patients with B-cell neoplasms.

A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons

PubMed Central

Zhao, Xiuli; Tang, Zongxiang; Zhang, Hongkang; Atianjoh, Fidelis E.; Zhao, Jian-Yuan; Liang, Lingli; Wang, Wei; Guan, Xiaowei; Kao, Sheng-Chin; Tiwari, Vinod; Gao, Yong-Jing; Hoffman, Paul N.; Cui, Hengmi; Li, Min; Dong, Xinzhong; Tao, Yuan-Xiang

2013-01-01

Neuropathic pain is a refractory disease characterized by maladaptive changes in gene transcription and translation within the sensory pathway. Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate in the development of neuropathic pain is unclear. Here we identify a conserved lncRNA for Kcna2 (named Kcna2 antisense RNA) in first-order sensory neurons of rat dorsal root ganglion (DRG). Peripheral nerve injury increases Kcna2 antisense RNA expression in injured DRG through activation of myeloid zinc finger protein 1, a transcription factor that binds to Kcna2 antisense RNA gene promoter. Mimicking this increase downregulates Kcna2, reduces total Kv current, increases excitability in DRG neurons, and produces neuropathic pain symptoms. Blocking this increase reverses nerve injury-induced downregulation of DRG Kcna2 and attenuates development and maintenance of neuropathic pain. These findings suggest native Kcna2 antisense RNA as a new therapeutic target for the treatment of neuropathic pain. PMID:23792947

Antisense and sense poly(A)-RNAs from the Xenopus laevis pyruvate dehydrogenase gene loci are regulated with message production during embryogenesis.

PubMed

Islam, N; Poitras, L; Gagnon, F; Moss, T

1996-10-17

The structure and temporal expression of two Xenopus cDNAs encoding the beta subunit of pyruvate dehydrogenase (XPdhE1 beta) have been determined. XPdhE1 beta was 88% homologous to mature human PdhE1 beta, but the putative N-terminal mitochondrial signal peptide was poorly conserved. Zygotic expression of XPdhE1 beta mRNA was detected at neural tube closure and increased until stage 40. RT-PCR cloning identified a short homology to a protein kinase open reading frame within the 3' non-coding sequence of the XPdhE1 beta cDNAs. This homology, which occurred on the antisense cDNA strand, was shown by strand specific RT-PCR to be transcribed in vivo as part of an antisense RNA. Northern analysis showed that this RNA formed part of an abundant and heterogeneous population of antisense and sense poly(A)-RNAs transcribed from the XPdhE1 beta loci and coordinately regulated with message production.

VRP09 Reduction of Corneal Scarring Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

DTIC Science & Technology

2012-10-01

selective of all gene-targeted, oligonucleotide-based drug approaches (better than ribozymes, antisense oligonucleotides ( ASO ), or microRNAs).(4) We will...respect to a scrambled siRNA control. For the migration assay, a circular region in the middle of the well was removed using a gel removal solution...oligonucleotides, ASOs ) into rabbit corneal cells and found that technique was very effective in delivering ASOs into the stroma and even into the endothelial cell

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

PubMed Central

Sherwood, Laura J.; Hayhurst, Andrew

2013-01-01

Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus. PMID:23577211

E-RNAi: a web application for the multi-species design of RNAi reagents—2010 update

PubMed Central

Horn, Thomas; Boutros, Michael

2010-01-01

The design of RNA interference (RNAi) reagents is an essential step for performing loss-of-function studies in many experimental systems. The availability of sequenced and annotated genomes greatly facilitates RNAi experiments in an increasing number of organisms that were previously not genetically tractable. The E-RNAi web-service, accessible at http://www.e-rnai.org/, provides a computational resource for the optimized design and evaluation of RNAi reagents. The 2010 update of E-RNAi now covers 12 genomes, including Drosophila, Caenorhabditis elegans, human, emerging model organisms such as Schmidtea mediterranea and Acyrthosiphon pisum, as well as the medically relevant vectors Anopheles gambiae and Aedes aegypti. The web service calculates RNAi reagents based on the input of target sequences, sequence identifiers or by visual selection of target regions through a genome browser interface. It identifies optimized RNAi target-sites by ranking sequences according to their predicted specificity, efficiency and complexity. E-RNAi also facilitates the design of secondary RNAi reagents for validation experiments, evaluation of pooled siRNA reagents and batch design. Results are presented online, as a downloadable HTML report and as tab-delimited files. PMID:20444868

Nickel-catalyzed coupling reaction of alkyl halides with aryl Grignard reagents in the presence of 1,3-butadiene: mechanistic studies of four-component coupling and competing cross-coupling reactions† †Electronic supplementary information (ESI) available: Detailed experimental and computational results, procedures, characterization data, copies of NMR charts, and crystallographic data. CCDC 1572238. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c7sc04675h

PubMed Central

Fukuoka, Asuka; Yokoyama, Wataru; Min, Xin; Hisaki, Ichiro; Kuniyasu, Hitoshi

2018-01-01

We describe the mechanism, substituent effects, and origins of the selectivity of the nickel-catalyzed four-component coupling reactions of alkyl fluorides, aryl Grignard reagents, and two molecules of 1,3-butadiene that affords a 1,6-octadiene carbon framework bearing alkyl and aryl groups at the 3- and 8-positions, respectively, and the competing cross-coupling reaction. Both the four-component coupling reaction and the cross-coupling reaction are triggered by the formation of anionic nickel complexes, which are generated by the oxidative dimerization of two molecules of 1,3-butadiene on Ni(0) and the subsequent complexation with the aryl Grignard reagents. The C–C bond formation of the alkyl fluorides with the γ-carbon of the anionic nickel complexes leads to the four-component coupling product, whereas the cross-coupling product is yielded via nucleophilic attack of the Ni center toward the alkyl fluorides. These steps are found to be the rate-determining and selectivity-determining steps of the whole catalytic cycle, in which the C–F bond of the alkyl fluorides is activated by the Mg cation rather than a Li or Zn cation. ortho-Substituents of the aryl Grignard reagents suppressed the cross-coupling reaction leading to the selective formation of the four-component products. Such steric effects of the ortho-substituents were clearly demonstrated by crystal structure characterizations of ate complexes and DFT calculations. The electronic effects of the para-substituent of the aryl Grignard reagents on both the selectivity and reaction rates are thoroughly discussed. The present mechanistic study offers new insight into anionic complexes, which are proposed as the key intermediates in catalytic transformations even though detailed mechanisms are not established in many cases, and demonstrates their synthetic utility as promising intermediates for C–C bond forming reactions, providing useful information for developing efficient and straightforward multicomponent reactions. PMID:29719693

Antisense oligonucleotide technologies in drug discovery.

PubMed

Aboul-Fadl, Tarek

2006-09-01

The principle of antisense oligonucleotide (AS-OD) technologies is based on the specific inhibition of unwanted gene expression by blocking mRNA activity. It has long appeared to be an ideal strategy to leverage new genomic knowledge for drug discovery and development. In recent years, AS-OD technologies have been widely used as potent and promising tools for this purpose. There is a rapid increase in the number of antisense molecules progressing in clinical trials. AS-OD technologies provide a simple and efficient approach for drug discovery and development and are expected to become a reality in the near future. This editorial describes the established and emerging AS-OD technologies in drug discovery.

Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity

PubMed Central

Matveeva, O. V.; Tsodikov, A. D.; Giddings, M.; Freier, S. M.; Wyatt, J. R.; Spiridonov, A. N.; Shabalina, S. A.; Gesteland, R. F.; Atkins, J. F.

2000-01-01

Design of antisense oligonucleotides targeting any mRNA can be much more efficient when several activity-enhancing motifs are included and activity-decreasing motifs are avoided. This conclusion was made after statistical analysis of data collected from >1000 experiments with phosphorothioate-modified oligonucleotides. Highly significant positive correlation between the presence of motifs CCAC, TCCC, ACTC, GCCA and CTCT in the oligonucleotide and its antisense efficiency was demonstrated. In addition, negative correlation was revealed for the motifs GGGG, ACTG, AAA and TAA. It was found that the likelihood of activity of an oligonucleotide against a desired mRNA target is sequence motif content dependent. PMID:10908347

2'-O-[2-[2-(N,N-Dimethylamino)ethoxy]ethyl] Modified Antisense Oligonucleotides: Symbiosis of Charge Interaction Factors and Stereoelectronic Effects

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

Prhavc, M.; Prakash, T.P.; Minasov, G.

Oligonucleotides with a novel, 2'-O-[2-[2-(N,N-dimethylamino)ethoxy]ethyl] (2'-O-DMAEOE) modification have been synthesized. This modification, a cationic analogue of the 2'-O-(2-methoxyethyl) (2'-O-MOE) modification, exhibits high binding affinity to target RNA (but not to DNA) and exceptional resistance to nuclease degradation. Analysis of the crystal structure of a self-complementary oligonucleotide containing a single 2'-O-DMAEOE modification explains the importance of charge factors and gauche effects on the observed antisense properties. 2'-O-DMAEOE modified oligonucleotides are ideal candidates for antisense drugs.

A unified model of Grignard reagent formation.

PubMed

Shao, Yunqi; Liu, Zhen; Huang, Pan; Liu, Boping

2018-04-25

Grignard reagents are among the most fundamental reagents in organic synthesis, yet studies have hitherto failed to fully explain the selectivity and kinetics of Grignard reagent formation (GRF). The present study provides new insights into the intermediates and pathways of GRF using density functional theory (DFT) calculations. Potential energy surfaces of RX dissociation along different directions reveal the origin of configuration retention of alkenyl and aromatic halides. Radical intermediates participate solely in the dissociation stage, and depend on the geometry of the reactant halide. Dissociation of organic halides yields stabilized surface anions, and the rest of the reaction is ionic in nature. MgX+/RMg+ were proposed as the key intermediates of Mg leaving from the surface in the self-activation of GRF, which explains the accelerated kinetics upon addition of RMgX or MgX2. The intermediacy of the cations was supported by a simple electrochemical experiment. To the best of our knowledge, this is the first unified ionic model (I-model) developed for resolving the controversial issues of GRF.

Lithium Enolates in the Enantioselective Construction of Tetrasubstituted Carbon Centers with Chiral Lithium Amides as Noncovalent Stereodirecting Auxiliaries.

PubMed

Yu, Kai; Lu, Ping; Jackson, Jeffrey J; Nguyen, Thuy-Ai D; Alvarado, Joseph; Stivala, Craig E; Ma, Yun; Mack, Kyle A; Hayton, Trevor W; Collum, David B; Zakarian, Armen

2017-01-11

Lithium enolates derived from carboxylic acids are ubiquitous intermediates in organic synthesis. Asymmetric transformations with these intermediates, a central goal of organic synthesis, are typically carried out with covalently attached chiral auxiliaries. An alternative approach is to utilize chiral reagents that form discrete, well-defined aggregates with lithium enolates, providing a chiral environment conducive of asymmetric bond formation. These reagents effectively act as noncovalent, or traceless, chiral auxiliaries. Lithium amides are an obvious choice for such reagents as they are known to form mixed aggregates with lithium enolates. We demonstrate here that mixed aggregates can effect highly enantioselective transformations of lithium enolates in several classes of reactions, most notably in transformations forming tetrasubstituted and quaternary carbon centers. Easy recovery of the chiral reagent by aqueous extraction is another practical advantage of this one-step protocol. Crystallographic, spectroscopic, and computational studies of the central reactive aggregate, which provide insight into the origins of selectivity, are also reported.

Natural antisense transcripts associated with salinity response in alfalfa

USDA-ARS?s Scientific Manuscript database

Natural antisense transcripts (NATs) are long non-coding RNAs (lncRNAs) complimentary to the messenger (sense) RNA (Wang et al. 2014). Many of them are involved in regulation of their own sense transcripts thus playing pivotal biological roles in all processes of organismal development and responses...

[Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNA and dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector].

PubMed

Sun, Dian Xing; Hu, Da Rong; Wu, Guang Hui; Hu, Xue Ling; Li, Juan; Fan, Gong Ren

2002-08-01

To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein. Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. The mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium. It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.

Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.

PubMed

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Moazzeni, Mohammad; Soheili, Zahra Soheila; Samiee, Shahram

2010-12-01

In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.

Antisense suppression of violaxanthin de-epoxidase in tobacco does not affect plant performance in controlled growth conditions.

PubMed

Chang, S H; Bugos, R C; Sun, W H; Yamamoto, H Y

2000-01-01

Violaxanthin de-epoxidase (VDE) catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 mumol m(-2) s(-1) for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 mumol m(-2) s(-1) under controlled growth conditions as compared to wild-type tobacco.

The Long Noncoding RNA Transcriptome of Dictyostelium discoideum Development.

PubMed

Rosengarten, Rafael D; Santhanam, Balaji; Kokosar, Janez; Shaulsky, Gad

2017-02-09

Dictyostelium discoideum live in the soil as single cells, engulfing bacteria and growing vegetatively. Upon starvation, tens of thousands of amoebae enter a developmental program that includes aggregation, multicellular differentiation, and sporulation. Major shifts across the protein-coding transcriptome accompany these developmental changes. However, no study has presented a global survey of long noncoding RNAs (ncRNAs) in D. discoideum To characterize the antisense and long intergenic noncoding RNA (lncRNA) transcriptome, we analyzed previously published developmental time course samples using an RNA-sequencing (RNA-seq) library preparation method that selectively depletes ribosomal RNAs (rRNAs). We detected the accumulation of transcripts for 9833 protein-coding messenger RNAs (mRNAs), 621 lncRNAs, and 162 putative antisense RNAs (asRNAs). The noncoding RNAs were interspersed throughout the genome, and were distinct in expression level, length, and nucleotide composition. The noncoding transcriptome displayed a temporal profile similar to the coding transcriptome, with stages of gradual change interspersed with larger leaps. The transcription profiles of some noncoding RNAs were strongly correlated with known differentially expressed coding RNAs, hinting at a functional role for these molecules during development. Examining the mitochondrial transcriptome, we modeled two novel antisense transcripts. We applied yet another ribosomal depletion method to a subset of the samples to better retain transfer RNA (tRNA) transcripts. We observed polymorphisms in tRNA anticodons that suggested a post-transcriptional means by which D. discoideum compensates for codons missing in the genomic complement of tRNAs. We concluded that the prevalence and characteristics of long ncRNAs indicate that these molecules are relevant to the progression of molecular and cellular phenotypes during development. Copyright © 2017 Rosengarten et al.

Effect of antisense oligonucleotides against cholesteryl ester transfer protein on the development of atherosclerosis in cholesterol-fed rabbits.

PubMed

Sugano, M; Makino, N; Sawada, S; Otsuka, S; Watanabe, M; Okamoto, H; Kamada, M; Mizushima, A

1998-02-27

Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl ester from high density lipoprotein (HDL) to apolipoprotein B (apoB)-containing lipoproteins. However, the exact role of CETP in the development of atherosclerosis has not been determined. In the present study, we examined the effect of the suppression of increased plasma CETP by intravenous injection with antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the development of atherosclerosis in rabbits fed a cholesterol diet. The ODNs against rabbit CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method to regulate liver gene expression. Twenty-two male Japanese White rabbits were used in the experiment. Eighteen animals were fed a standard rabbit chow supplemented with 0.3% cholesterol throughout the experiment for 16 weeks. At 8 weeks, they were divided into three groups (six animals in each group), among which the plasma total and HDL cholesterol concentrations did not significantly change. The control group received nothing, the sense group were injected with the sense ODNs complex, and the antisense group were injected with the antisense ODNs complex, respectively, for subsequent 8 weeks. ASOR. poly(L-lysine) ODNs complex were injected via the ear veins twice a week. Four animals were fed a standard rabbit diet for 16 weeks. The total cholesterol concentrations and the CETP mass in the animals injected with antisense ODNs were all significantly decreased in 12 and 16 weeks compared with those injected with sense ODNs and the control animals. The HDL cholesterol concentrations measured by the precipitation assay did not significantly change among the groups fed a cholesterol diet, and triglyceride concentrations did not significantly change in the four groups. However, at the end of the study, when the HDL cholesterol concentrations were measured after the isolation by ultracentrifugation and a column chromotography, they were significantly higher in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. A reduction of CETP mRNA and an increase of LDL receptor mRNA in the liver were observed in the animals injected with antisense ODNs compared with those injected with sense ODNs and the control animals. Aortic cholesterol contents and the aortic percentage lesion to total surface area were significantly lower in the animals injected with antisense ODNs than in the animals injected with sense ODNs and in the control animals. These findings showed for the first time that suppression of increased plasma CETP by the injection with antisense ODNs against CETP coupled to ASOR carrier molecules targeted to the liver could thus inhibit the atherosclerosis possibly by decreasing the plasma LDL + very low density lipoprotein (VLDL) cholesterol in cholesterol-fed rabbits.

Gallium metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides in complex mixtures

PubMed Central

Blacken, Grady R.; Sadílek, Martin; Tureček, František

2008-01-01

Metal affinity capture tandem mass spectrometry (MAC-MSMS) is evaluated in a comparative study of a lysine-derived nitrilotriacetic acid (Nα, Nα-bis-(carboxymethyl)lysine, LysNTA) and an aspartic-acid-related iminodiacetic acid (N-(4-aminobutyl)aspartic acid, AspIDA) as selective phosphopeptide detection reagents. Both LysNTA and AspIDA spontaneously form ternary complexes with GaIII and phosphorylated amino acids and phosphopeptides upon mixing in solution. Collision-induced dissociation of positive complex ions produced by electrospray produces common fragments (LysNTA + H)+ or (AspIDA + H)+ at m/z 263 and 205, respectively. MSMS precursor scans using these fragments as reporter ions allow one to selectively detect multiple charge states of phosphopeptides in mixtures. It follows from this comparative study that LysNTA is superior to AspIDA in detecting phosphopeptides, possibly because of the higher coordination number and greater stability constant for GaIII – phosphopeptide complexation of the former reagent. In a continuing development of MAC-MSMS for proteomics applications, we demonstrate its utility in a post-column reaction format. Using a simple post-column-reaction ‘T’ and syringe pump to deliver our chelating reagents, α-casein tryptic phosphopeptides can be selectively analyzed from a solution containing a twofold molar excess of bovine serum albumin. The MAC-MSMS method is shown to be superior to the commonly used neutral loss scan for the common loss of phosphoric acid. PMID:18265438

Selectivity in the Addition Reactions of Organometallic Reagents to Aziridine-2-carboxaldehydes: The Effects of Protecting Groups and Substitution Patterns

PubMed Central

Kulshrestha, Aman; Schomaker, Jennifer M.; Holmes, Daniel; Staples, Richard J.; Jackson, James E.; Borhan, Babak

2014-01-01

Good to excellent stereo-selectivity has been found in the addition reactions of Grignard and organo-zinc reagents to N-protected aziridine-2-carboxaldehydes. Specifically, high syn selectivity was obtained with benzyl-protected cis, tert-butyloxycar-bonyl-protected trans, and tosyl-pro-tected 2,3-disubstituted aziridine-2-car-boxaldehydes. Furthermore, rate and selectivity effects of ring substituents, temperature, solvent, and Lewis acid and base modifiers were studied. The diastereomeric preference of addition is dominated by the substrate aziri-dines’ substitution pattern and especially the electronic character and conformational preferences of the nitrogen protecting groups. To help rationalize the observed stereochemical outcomes, conformational and electronic structural analyses of a series of model systems representing the various substitution patterns have been explored by density functional calculations at the B3LYP/6–31G* level of theory with the SM8 solvation model to account for solvent effects. PMID:21928447

Headspace analysis of new psychoactive substances using a Selective Reagent Ionisation-Time of Flight-Mass Spectrometer

PubMed Central

Acton, W. Joe; Lanza, Matteo; Agarwal, Bishu; Jürschik, Simone; Sulzer, Philipp; Breiev, Kostiantyn; Jordan, Alfons; Hartungen, Eugen; Hanel, Gernot; Märk, Lukas; Mayhew, Chris A.; Märk, Tilmann D.

2014-01-01

The rapid expansion in the number and use of new psychoactive substances presents a significant analytical challenge because highly sensitive instrumentation capable of detecting a broad range of chemical compounds in real-time with a low rate of false positives is required. A Selective Reagent Ionisation-Time of Flight-Mass Spectrometry (SRI-ToF-MS) instrument is capable of meeting all of these requirements. With its high mass resolution (up to m/Δm of 8000), the application of variations in reduced electric field strength (E/N) and use of different reagent ions, the ambiguity of a nominal (monoisotopic) m/z is reduced and hence the identification of chemicals in a complex chemical environment with a high level of confidence is enabled. In this study we report the use of a SRI-ToF-MS instrument to investigate the reactions of H3O+, O2+, NO+ and Kr+ with 10 readily available (at the time of purchase) new psychoactive substances, namely 4-fluoroamphetamine, methiopropamine, ethcathinone, 4-methylethcathinone, N-ethylbuphedrone, ethylphenidate, 5-MeO-DALT, dimethocaine, 5-(2-aminopropyl)benzofuran and nitracaine. In particular, the dependence of product ion branching ratios on the reduced electric field strength for all reagent ions was investigated and is reported here. The results reported represent a significant amount of new data which will be of use for the development of drug detection techniques suitable for real world scenarios. PMID:25844048

Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

USDA-ARS?s Scientific Manuscript database

Natural antisense transcripts (NATs) are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded) or a different locus (trans-encoded). They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation....

Bcl-2 antisense therapy in B-cell malignancies.

PubMed

Chanan-Khan, Asher

2005-07-01

Bcl-2 is an apoptosis regulating protein, overexpression of which is associated with chemotherapy resistant disease, aggressive clinical course, and poor survival in patients with B-cell lymphoproliferative disorders. Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Downregulation of this oncoprotein, thus, represents a possible new way to target clinically aggressive disease. Preclinical studies have shown that this oncoprotein can be effectively decreased by Bcl-2 antisense in malignant lymphoid cells and can reverse chemotherapy resistance, as well as enhance the anti-apoptotic potential of both chemotherapeutic and biologic agents. Ongoing clinical trials are exploring the role of Bcl-2 downregulation with oblimersen (Bcl-2 antisense) in patients with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. Early results from these studies are promising and support the proof of the principle. As these studies are completed and mature data emerges, the role of Bcl-2 antisense therapy in the treatment of B-cell malignancies will become clearer.

Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

PubMed Central

Pierce, M L; Ruffner, D E

1998-01-01

Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

Review on investigations of antisense oligonucleotides with the use of mass spectrometry.

PubMed

Studzińska, Sylwia

2018-01-01

Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

PubMed Central

Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro

2014-01-01

We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination. PMID:24212579

TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells.

PubMed

Philipp, Katrin; Riedel, Frank; Germann, Günter; Hörmann, Karl; Sauerbier, Michael

2005-02-01

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

PubMed

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri.

PubMed

Tran, Chi Nhan; Giangrossi, Mara; Prosseda, Gianni; Brandi, Anna; Di Martino, Maria Letizia; Colonna, Bianca; Falconi, Maurizio

2011-10-01

The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5'-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense-antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem-loop motifs of the antisense region.

Tetrahedral DNA Nanoparticle Vector for Intracellular Delivery of Targeted Peptide Nucleic Acid Antisense Agents to Restore Antibiotic Sensitivity in Cefotaxime-Resistant Escherichia coli.

PubMed

Readman, John Benedict; Dickson, George; Coldham, Nick G

2017-06-01

The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-bla CTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 μM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying bla CTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 μM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.

Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

PubMed

Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

2001-01-01

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

Identification of targets of miRNA-221 and miRNA-222 in fulvestrant-resistant breast cancer

PubMed Central

Liu, Pengfei; Sun, Manna; Jiang, Wenhua; Zhao, Jinkun; Liang, Chunyong; Zhang, Huilai

2016-01-01

The present study aimed to identify the differentially expressed genes (DEGs) regulated by microRNA (miRNA)-221 and miRNA-222 that are associated with the resistance of breast cancer to fulvestrant. The GSE19777 transcription profile was downloaded from the Gene Expression Omnibus database, and includes data from three samples of antisense miRNA-221-transfected fulvestrant-resistant MCF7-FR breast cancer cells, three samples of antisense miRNA-222-transfected fulvestrant-resistant MCF7-FR cells and three samples of control inhibitor (green fluorescent protein)-treated fulvestrant-resistant MCF7-FR cells. The linear models for microarray data package in R/Bioconductor was employed to screen for DEGs in the miRNA-transfected cells, and the pheatmap package in R was used to perform two-way clustering. Pathway enrichment was conducted using the Gene Set Enrichment Analysis tool. Furthermore, a miRNA-messenger (m) RNA regulatory network depicting interactions between miRNA-targeted upregulated DEGs was constructed and visualized using Cytoscape. In total, 492 and 404 DEGs were identified for the antisense miRNA-221-transfected MCF7-FR cells and the antisense miRNA-222-transfected MCF7-FR cells, respectively. Genes of the pentose phosphate pathway (PPP) were significantly enriched in the antisense miRNA-221-transfected MCF7-FR cells. In addition, components of the Wnt signaling pathway and cell adhesion molecules (CAMs) were significantly enriched in the antisense miRNA-222-transfected MCF7-FR cells. In the miRNA-mRNA regulatory network, miRNA-222 was demonstrated to target protocadherin 10 (PCDH10). The results of the present study suggested that the PPP and Wnt signaling pathways, as well as CAMs and PCDH10, may be associated with the resistance of breast cancer to fulvestrant. PMID:27895744

[Inhibiting target gene expression and controlling growth of Epstein-Barr virus transformed cells by antisense RNA transcripts].

PubMed

Chen, Jian-jing; Raab-Traub, Nancy; Yao, Qing-yun; Zhang, Feng; Huang, Mei-ling; Kuang, Zhu-ji; Zhang, Xiao-shi; Ye, Yan-li; Gu, Li

2002-01-01

The latent membrane protein gene (LMP) of Epstein-Barr virus (EBV) was thought to play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). In this study, the authors investigated the effects of antisense RNA (AsRNA) on LMP for down regulating at the target gene over expression in EBV transformed lymphoid cells, and set up an antisense system to inhibit LMP expression. Constructing the single strand antisense transcription system in vitro, the authors have got large amount of AsRNA. Designing and setting up an antisense tracing system in situ (ATSIS), the authors could observe the living particles of AsRNA/sense RNA duplex dimer. With time lapse phase-contrast microscopy, the agglutination phenotype on living cells was easily detected by MTT test, the inhibition rate on EBV transformed cells was calculated. LMP 1.9 fragment ligated into pGEM vector in reverse orientation have been constructed and produced a plentiful amount of AsLMPmRNA which could incorporated into both B95-8 and C1936 cell lines by endophagocytosis and formed the duplex dimer of As/Sense RNA. This particles have been visualized in situ when labelling 35S isotope by ATSIS. When AsLMPmRNA acted as agents for specific inhibition to LMP over expression, the transform phenotype of cell agglutination have been suppressed and MTT particle formatin was apparently reduced both two EBV tansformed cell lines. AsLMPmRNA targets at sense strand have a high effectiveness of down-regulation on EBV-LMP overexpression. This down regulating function of LMP and growth inhibition on transformed cell is demonstrated by the antisenes tracing system in situ (ATSIS). The results provide a clue to overcome the latent EBV infection in human bodies all living long time and to prevent it inducing NPC in high incidence area by antisense strategies.

Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.

PubMed

Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin

2009-06-01

Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that transfected SK-Br-3 oncocytes by antisense probe had the lowest signal of all. The SPIO-labeled ASODN probe shows unique features including well-distributed spherical morphology, high conjugating rate and loading efficiency, and the signal intensity of SPIO-labeled ASODN-transfected SK-Br-3 oncocytes is reduced in MR imaging. These results indicate that the SPIO-labeled ASODN probe is potentially useful as a MR targeting contrast enhancing agent to specifically diagnose tumors which had over-expression of the c-erbB2 oncogene at an early stage.

Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells.

PubMed

Rakoczy, P E; Lai, M C; Baines, M G; Spilsbury, K; Constable, I J

1998-10-01

To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression. Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells. The biologic effect of CatS downregulation in ARPE19 cells was tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity. After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity. Recombinant adenoviruses were shown to be suitable for producing antisense CatS transcripts to modulate endogenous CatS expression in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments through the regulation of other lysosomal enzyme activity, such as the expression of CatD.

Evaluation of sensitivity and specificity of a standardized procedure using different reagents for the detection of lupus anticoagulants. The Working Group on Hemostasis of the Société Française de Biologie Clinique and for the Groupe d'Etudes sur I'Hémostase et la Thrombose.

PubMed

Goudemand, J; Caron, C; De Prost, D; Derlon, A; Borg, J Y; Sampol, J; Sié, P

1997-02-01

This study was designed to test the sensitivity and specificity of a combination of 3 phospholipid-dependent assays performed with various reagents, for the detection of lupus anticoagulant (LA). Plasmas containing an LA (n = 56) or displaying various confounding pathologies [58 intrinsic pathway factor deficiencies, 9 factor VIII inhibitors, 28 plasmas from patients treated with an oral anticoagulant (OAC)] were selected. In a first step, the efficiency of each assay and reagent was assessed using the Receiving Operating Characteristic (ROC) method. Optimal cut-offs providing both sensitivity and specificity > or = 80% were determined. The APTT assay and most of the phospholipid neutralization assays failed to discriminate factor VIII inhibitors from LA. In a second step, using the optimal cut-offs determined above, the results of all the possible combinations of the 3 assays performed with 4 different reagents were analyzed. Thirteen combinations of reagents allowed > or = 80% of plasmas of each category (LA, factor deficiency or OAC) to be correctly classified (3/3 positive test results in LA-containing plasmas and 0/3 positive results in LA-negative samples).

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

HBEGF, SRA1, and IK: Three cosegregating genes as determinants of cardiomyopathy.

PubMed

Friedrichs, Frauke; Zugck, Christian; Rauch, Gerd-Jörg; Ivandic, Boris; Weichenhan, Dieter; Müller-Bardorff, Margit; Meder, Benjamin; El Mokhtari, Nour Eddine; Regitz-Zagrosek, Vera; Hetzer, Roland; Schäfer, Arne; Schreiber, Stefan; Chen, Jian; Neuhaus, Isaac; Ji, Ruiru; Siemers, Nathan O; Frey, Norbert; Rottbauer, Wolfgang; Katus, Hugo A; Stoll, Monika

2009-03-01

Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary.

HBEGF, SRA1, and IK: Three cosegregating genes as determinants of cardiomyopathy

PubMed Central

Friedrichs, Frauke; Zugck, Christian; Rauch, Gerd-Jörg; Ivandic, Boris; Weichenhan, Dieter; Müller-Bardorff, Margit; Meder, Benjamin; El Mokhtari, Nour Eddine; Regitz-Zagrosek, Vera; Hetzer, Roland; Schäfer, Arne; Schreiber, Stefan; Chen, Jian; Neuhaus, Isaac; Ji, Ruiru; Siemers, Nathan O.; Frey, Norbert; Rottbauer, Wolfgang; Katus, Hugo A.; Stoll, Monika

2009-01-01

Human dilated cardiomyopathy (DCM), a disorder of the cardiac muscle, causes considerable morbidity and mortality and is one of the major causes of sudden cardiac death. Genetic factors play a role in the etiology and pathogenesis of DCM. Disease-associated genetic variations identified to date have been identified in single families or single sporadic patients and explain a minority of the etiology of DCM. We show that a 600-kb region of linkage disequilibrium (LD) on 5q31.2-3, harboring multiple genes, is associated with cardiomyopathy in three independent Caucasian populations (combined P-value = 0.00087). Functional assessment in zebrafish demonstrates that at least three genes, orthologous to loci in this LD block, HBEGF, IK, and SRA1, result independently in a phenotype of myocardial contractile dysfunction when their expression is reduced with morpholino antisense reagents. Evolutionary analysis across multiple vertebrate genomes suggests that this heart failure-associated LD block emerged by a series of genomic rearrangements across amphibian, avian, and mammalian genomes and is maintained as a cluster in mammals. Taken together, these observations challenge the simple notion that disease phenotypes can be traced to altered function of a single locus within a haplotype and suggest that a more detailed assessment of causality can be necessary. PMID:19064678

RNA interference-based therapeutics: new strategies to fight infectious disease.

PubMed

López-Fraga, M; Wright, N; Jiménez, A

2008-12-01

For many years, there has been an ongoing search for new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise "undruggable" with traditional pharmaceutical approaches involving small molecules or proteins. RNA interference (RNAi) strategies have raised a lot of attention and several compounds are currently being tested in clinical trials. Viruses are the obvious target for RNAi-therapy, as most are difficult to treat with conventional drugs, they become rapidly resistant to drug treatment and their genes differ substantially from human genes, minimizing side effects. Antisense strategy offers very high target specificity, i.e., any viral sequence could potentially be targeted using the complementary oligonucleotide sequence. Consequently, new antisense-based therapeutics have the potential to lead a revolution in the anti-infective drug development field. Additionally, the relatively short turnaround for efficacy testing of potential RNAi molecules and that any pathogen is theoretically amenable to rapid targeting, make them invaluable tools for treating a wide range of diseases. This review will focus on some of the current efforts to treat infectious disease with RNAi-based therapies and some of the obstacles that have appeared on the road to successful clinical intervention.

Antisense Inhibition of the 2-Oxoglutarate Dehydrogenase Complex in Tomato Demonstrates Its Importance for Plant Respiration and during Leaf Senescence and Fruit Maturation[W][OA

PubMed Central

Araújo, Wagner L.; Tohge, Takayuki; Osorio, Sonia; Lohse, Marc; Balbo, Ilse; Krahnert, Ina; Sienkiewicz-Porzucek, Agata; Usadel, Björn; Nunes-Nesi, Adriano; Fernie, Alisdair R.

2012-01-01

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of the gene encoding the E1 subunit of the 2-oxoglutarate dehydrogenase complex in the antisense orientation and exhibiting substantial reductions in the activity of this enzyme exhibit a considerably reduced rate of respiration. They were, however, characterized by largely unaltered photosynthetic rates and fruit yields but restricted leaf, stem, and root growth. These lines displayed markedly altered metabolic profiles, including changes in tricarboxylic acid cycle intermediates and in the majority of the amino acids but unaltered pyridine nucleotide content both in leaves and during the progression of fruit ripening. Moreover, they displayed a generally accelerated development exhibiting early flowering, accelerated fruit ripening, and a markedly earlier onset of leaf senescence. In addition, transcript and selective hormone profiling of gibberellins and abscisic acid revealed changes only in the former coupled to changes in transcripts encoding enzymes of gibberellin biosynthesis. The data obtained are discussed in the context of the importance of this enzyme in both photosynthetic and respiratory metabolism as well as in programs of plant development connected to carbon–nitrogen interactions. PMID:22751214

Antisense expression of an Arabidopsis ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress

NASA Technical Reports Server (NTRS)

Kim, S. H.; Arnold, D.; Lloyd, A.; Roux, S. J.

2001-01-01

We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

PubMed

Gritsun, T S; Gould, E A

1998-12-01

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

Anti-sense oligonucleotide therapies for the treatment of hyperlipidaemia.

PubMed

Wierzbicki, Anthony S; Viljoen, Adie

2016-09-01

Anti-sense oligonucleotide (ASO) therapies are a new development in clinical pharmacology offering greater specificity compared to small molecule inhibitors and the ability to target intracellular process' not susceptible to antibody-based therapies. This article reviews the chemical biology of ASOs and related RNA therapeutics. It then reviews the data on their use to treat hyperlipidaemia. Data on mipomersen - an ASO to apolipoprotein B-100(apoB) licensed for treatment of homozygous familial hypercholesterolaemia (FH) is presented. Few effective therapies are available to reduce atehrogenic lipoprotein (a) levels. An ASO therapy to apolipoprotein(a) (ISIS Apo(a)Rx) specifically reduced lipoprotein (a) levels by up to 78%. Treatment options for patients with familial chylomicronaemia syndrome (lipoprotein lipase deficiency; LPLD) or lipodystrophies are highly limited and often inadequate. Volanesorsen, an ASO to apolipoprotein C-3, shows promise in the treatment of LPLD and severe hypertriglyceridaemia as it increases clearance of triglyceride-rich lipoproteins and can normalise triglycerides in these patients. The uptake of the novel ASO therapies is likely to be limited to selected niche groups or orphan diseases. These will include homozygous FH, severe heterozygous FH for mipomersen; LPLD deficiency and lipodystrophy syndromes for volanesorsen and treatment of patients with high elevated Lp(a) levels.

Strategies for generating peptide radical cations via ion/ion reactions.

PubMed

Gilbert, Joshua D; Fisher, Christine M; Bu, Jiexun; Prentice, Boone M; Redwine, James G; McLuckey, Scott A

2015-02-01

Several approaches for the generation of peptide radical cations using ion/ion reactions coupled with either collision induced dissociation (CID) or ultraviolet photo dissociation (UVPD) are described here. Ion/ion reactions are used to generate electrostatic or covalent complexes comprised of a peptide and a radical reagent. The radical site of the reagent can be generated multiple ways. Reagents containing a carbon-iodine (C-I) bond are subjected to UVPD with 266-nm photons, which selectively cleaves the C-I bond homolytically. Alternatively, reagents containing azo functionalities are collisionally activated to yield radical sites on either side of the azo group. Both of these methods generate an initial radical site on the reagent, which then abstracts a hydrogen from the peptide while the peptide and reagent are held together by either electrostatic interactions or a covalent linkage. These methods are demonstrated via ion/ion reactions between the model peptide RARARAA (doubly protonated) and various distonic anionic radical reagents. The radical site abstracts a hydrogen atom from the peptide, while the charge site abstracts a proton. The net result is the conversion of a doubly protonated peptide to a peptide radical cation. The peptide radical cations have been fragmented via CID and the resulting product ion mass spectra are compared to the control CID spectrum of the singly protonated, even-electron species. This work is then extended to bradykinin, a more broadly studied peptide, for comparison with other radical peptide generation methods. The work presented here provides novel methods for generating peptide radical cations in the gas phase through ion/ion reaction complexes that do not require modification of the peptide in solution or generation of non-covalent complexes in the electrospray process. Copyright © 2015 John Wiley & Sons, Ltd.

Role of TAF12 in the Increased VDR Activity in Paget’s Disease of Bone

DTIC Science & Technology

2013-10-01

and 5’‐GCC AAA TGC AGT TTA AGC TCT GCT‐3’ (antisense). The gene‐specific primers for mouse b‐actin were 5’‐GGC CGT ACC ACT GGC ATC GTG ATG‐ 3...cycles. The gene‐specific primers for CYP24A1 mRNA were 5’‐CGG GTG GAC CAT TTA CAA CTC GG‐3’ (sense) and 5’‐CTC AAC AGG CTC ATT GTC TGT GG‐3’ (antisense...The gene specific designing primers for b‐actinwere 5’‐ GTG CGT GAC ATC AAA GAG‐3’ (sense) and 5’‐GCC ACA GGA TTC CAT ACC‐3’ (antisense). The

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry.

PubMed

Santa, Tomofumi

2011-01-01

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds. 2010 John Wiley & Sons, Ltd.

Chemoselective chromium(II)-catalyzed cross-coupling reactions of dichlorinated heteroaromatics with functionalized aryl grignard reagents.

PubMed

Steib, Andreas K; Kuzmina, Olesya M; Fernandez, Sarah; Malhotra, Sushant; Knochel, Paul

2015-01-26

Chromium(II) chloride catalyzes the chemoselective cross-coupling reaction of dichloropyridines with a range of functionalized (hetero)aromatic Grignard reagents at room temperature. Functional groups, such as esters and acetals, are well tolerated in this transformation. Previously challenging substrates, quinolines and isoquinolines, participate in the selective Cr-catalyzed cross-coupling in cyclopentyl methyl ether (CPME) as the solvent. The effective purging of Cr salts is demonstrated by using various solid supports. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

New Synthetic Approach for the Incorporation of 3,2-Hydroxypyridinone (HOPO) Ligands: Synthesis of Structurally Diverse Poly HOPO Chelators

PubMed Central

Arumugam, Jayanthi; Brown, Hayley A.; Jacobs, Hollie K.; Gopalan, Aravamudan S.

2011-01-01

The HOPO sulfonamide reagent, 3, was prepared from commercial 2,3-dihydroxypyridine in four steps in good yields. Sulfonamide 3 readily underwent selective alkylation with dibromides in the presence of base or could be coupled to alcohols using Mitsunobu conditions. The utility of this nucleophilic HOPO reagent was demonstrated by the synthesis some tris and tetraHOPO chelators. This approach for tethering HOPO ligands is unique and flexible as shown by the preparation of HOPO/iminocarboxylic acid chelator 17. PMID:21709749

Chemical sensing flow probe

DOEpatents

Laguna, George R.; Peter, Frank J.; Butler, Michael A.

1999-01-01

A new chemical probe determines the properties of an analyte using the light absorption of the products of a reagent/analyte reaction. The probe places a small reaction volume in contact with a large analyte volume. Analyte diffuses into the reaction volume. Reagent is selectively supplied to the reaction volume. The light absorption of the reaction in the reaction volume indicates properties of the original analyte. The probe is suitable for repeated use in remote or hostile environments. It does not require physical sampling of the analyte or result in significant regent contamination of the analyte reservoir.

Chemical sensing flow probe

DOEpatents

Laguna, G.R.; Peter, F.J.; Butler, M.A.

1999-02-16

A new chemical probe determines the properties of an analyte using the light absorption of the products of a reagent/analyte reaction. The probe places a small reaction volume in contact with a large analyte volume. Analyte diffuses into the reaction volume. Reagent is selectively supplied to the reaction volume. The light absorption of the reaction in the reaction volume indicates properties of the original analyte. The probe is suitable for repeated use in remote or hostile environments. It does not require physical sampling of the analyte or result in significant regent contamination of the analyte reservoir. 7 figs.

Stereoselective Synthesis of trans-Olefins by the Copper-Mediated SN2′ Reaction of Vinyl Oxazines with Grignard Reagents. Asymmetric Synthesis of D-threo-Sphingosines

PubMed Central

Singh, Om V.; Han, Hyunsoo

2007-01-01

The SN2′ reaction of 6-vinyl-5,6-dihydro-4H-[1,3]oxazines with Grignard reagents in the presence of CuCN was studied, and high trans selectivity for the formation of double bond was observed with a variety of RMgX. The SN2′ reaction, coupled with regioselective asymmetric aminohydroxylation reaction, provided a highly efficient route for the asymmetric synthesis of D-threo-N-acetylsphingosine. PMID:18958293

Peptide-based antibody alternatives for biological sensing in austere environments

NASA Astrophysics Data System (ADS)

Coppock, Matthew B.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

2017-02-01

The most critical component of a biosensor, the biorecognition element, must exhibit high selectivity and strong affinity for a target of interest in operational sensing. Monoclonal antibodies are the current standard reagents for such devices, but their adaptability, manufacturability, and stability greatly limit their effectiveness in fieldable sensors. Peptides have emerged as potential antibody replacements in such applications due to their similar binding performance, extreme chemical and thermal stabilities, and on-demand scalability. In conjunction with modeling capabilities, work at the Army Research Lab focuses on protein catalyzed capture (PCC) agent technology and bacterial display for the discovery of these novel peptide binding reagents. The synthetic, bottom-up PCC agent technology uses an iterative, in situ "click chemistry" approach to produce high performing peptides against specific epitopes translatable to the protein target. Bacterial display allows rapid reagent discovery due to the combination of fast bacterial growth and effective peptide sequence enrichment through multiple rounds of biopanning. Recent advances in both methods are highlighted in regards to the discovery of reagents against Army high priority protein targets for soldier safety, performance, and diagnostics.

Bridging disulfides for stable and defined antibody drug conjugates.

PubMed

Badescu, George; Bryant, Penny; Bird, Matthew; Henseleit, Korinna; Swierkosz, Julia; Parekh, Vimal; Tommasi, Rita; Pawlisz, Estera; Jurlewicz, Kosma; Farys, Monika; Camper, Nicolas; Sheng, XiaoBo; Fisher, Martin; Grygorash, Ruslan; Kyle, Andrew; Abhilash, Amrita; Frigerio, Mark; Edwards, Jeff; Godwin, Antony

2014-06-18

To improve both the homogeneity and the stability of ADCs, we have developed site-specific drug-conjugating reagents that covalently rebridge reduced disulfide bonds. The new reagents comprise a drug, a linker, and a bis-reactive conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulfide bond in antibodies and antibody fragments. A disulfide rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain disulfide of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in in vitro and in vivo cancer models. Disulfide rebridging conjugation is a general approach to prepare stable ADCs, which does not require the antibody to be recombinantly re-engineered for site-specific conjugation.

Method of preparing a high heating value fuel product

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

Somerville, R.; Fan, L.T.

1989-10-24

This patent describes a method of preparing a high heating value fuel product. The method comprising the steps of: blending a high heating value waste material with a cellulosic material; mixing an organic reagent to the blended mixture of the waste material and the cellulosic material, the organic reagent being a mixture having a 4-15 weight percent of a chemical selected from the group consisting of: triethylene, glycol, diethylene glycol, and glycerin propylene glycol; introducing a pozzolanic agent to the blended mixture for controlling the rate of solidification; and forming the blended mixture into a form suitable for handling. Alsomore » described is the same method with the mixture of the organic reagent further comprising: a 20-32 weight percent calcium chloride solution. Another method of preparing a fuel product is also described.« less

Divergent transcription is associated with promoters of transcriptional regulators

PubMed Central

2013-01-01

Background Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. Results We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. Conclusions We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. PMID:24365181

Optical wavelength selection for portable hemoglobin determination by near-infrared spectroscopy method

NASA Astrophysics Data System (ADS)

Tian, Han; Li, Ming; Wang, Yue; Sheng, Dinggao; Liu, Jun; Zhang, Linna

2017-11-01

Hemoglobin concentration is commonly used in clinical medicine to diagnose anemia, identify bleeding, and manage red blood cell transfusions. The golden standard method for determining hemoglobin concentration in blood requires reagent. Spectral methods were advantageous at fast and non-reagent measurement. However, model calibration with full spectrum is time-consuming. Moreover, it is necessary to use a few variables considering size and cost of instrumentation, especially for a portable biomedical instrument. This study presents different wavelength selection methods for optical wavelengths for total hemoglobin concentration determination in whole blood. The results showed that modelling using only two wavelengths combination (1143 nm, 1298 nm) can keep on the fine predictability with full spectrum. It appears that the proper selection of optical wavelengths can be more effective than using the whole spectra for determination hemoglobin in whole blood. We also discussed the influence of water absorptivity on the wavelength selection. This research provides valuable references for designing portable NIR instruments determining hemoglobin concentration, and may provide some experience for noninvasive hemoglobin measurement by NIR methods.

Antisense Oligonucleotide Therapy for Patients with Advanced Cancer | Center for Cancer Research

Cancer.gov

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the U.S. Improvements in therapy have increased the survival of patients with CRC from 10 months to two years, but for patients who stop responding to treatments, such as irinotecan, options for additional therapy are limited. Antisense oligonucleotides (ASOs) may offer advantages over traditional

Anti-Angiogenic Action of Neutral Endopeptidase

DTIC Science & Technology

2007-11-01

message levels of NEP in hypoxia treated PC cells. Messenger RNA levels of NEP decreased between 50-75% relative to normoxic controls with high...GAGCATC-3 (sense) and 5-ATATGAATTCTCAGCTCT- TAGCAGACATGGAAGAAAG-3 ( antisense ) for glutathione S-transferase (GST) fusion proteins and 5-ATGGCAGCCGG...GAGCATC-3 (sense) and 5-CCCCAAGCTTTTAGCTCT- TAGCAGACAT-3 ( antisense ) for maltose-binding protein fusion proteins, as previously described (13

Antisense Oligonucleotides Modulating Activation of a Nonsense-Mediated RNA Decay Switch Exon in the ATM Gene.

PubMed

Kralovicova, Jana; Moreno, Pedro M D; Cross, Nicholas C P; Pêgo, Ana Paula; Vorechovsky, Igor

2016-12-01

ATM (ataxia-telangiectasia, mutated) is an important cancer susceptibility gene that encodes a key apical kinase in the DNA damage response pathway. ATM mutations in the germ line result in ataxia-telangiectasia (A-T), a rare genetic syndrome associated with hypersensitivity to double-strand DNA breaks and predisposition to lymphoid malignancies. ATM expression is limited by a tightly regulated nonsense-mediated RNA decay (NMD) switch exon (termed NSE) located in intron 28. In this study, we identify antisense oligonucleotides that modulate NSE inclusion in mature transcripts by systematically targeting the entire 3.1-kb-long intron. Their identification was assisted by a segmental deletion analysis of transposed elements, revealing NSE repression upon removal of a distant antisense Alu and NSE activation upon elimination of a long terminal repeat transposon MER51A. Efficient NSE repression was achieved by delivering optimized splice-switching oligonucleotides to embryonic and lymphoblastoid cells using chitosan-based nanoparticles. Together, these results provide a basis for possible sequence-specific radiosensitization of cancer cells, highlight the power of intronic antisense oligonucleotides to modify gene expression, and demonstrate transposon-mediated regulation of NSEs.

Episome-generated N-myc antisense RNA restricts the differentiation potential of primitive neuroectodermal cell lines.

PubMed Central

Whitesell, L; Rosolen, A; Neckers, L M

1991-01-01

Neuroectodermal tumors of childhood provide a unique opportunity to examine the role of genes potentially regulating neuronal growth and differentiation because many cell lines derived from these tumors are composed of at least two distinct morphologic cell types. These types display variant phenotypic characteristics and spontaneously interconvert, or transdifferentiate, in vitro. The factors that regulate transdifferentiation are unknown. Application of antisense approaches to the transdifferentiation process has allowed us to explore the precise role that N-myc may play in regulating developing systems. We now report construction of an episomally replicating expression vector designed to generate RNA antisense to part of the human N-myc gene. Such a vector is able to specifically inhibit N-myc expression in cell lines carrying both normal and amplified N-myc alleles. Inhibition of N-myc expression blocks transdifferentiation in these lines, with accumulation of cells of an intermediate phenotype. A concomitant decrease in growth rate but not loss of tumorigenicity was observed in the N-myc nonamplified cell line CHP-100. Vector-generated antisense RNA should allow identification of genes specifically regulated by the proto-oncogene N-myc. Images PMID:1996098

Nanoparticle Delivery of Antisense Oligonucleotides and Their Application in the Exon Skipping Strategy for Duchenne Muscular Dystrophy

PubMed Central

Falzarano, Maria Sofia; Passarelli, Chiara

2014-01-01

Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy. PMID:24506782

Diversity of Antisense and Other Non-Coding RNAs in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species

PubMed Central

Bernick, David L.; Dennis, Patrick P.; Lui, Lauren M.; Lowe, Todd M.

2012-01-01

A great diversity of small, non-coding RNA (ncRNA) molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs (sRNA) in archaea is limited. We employed RNA-seq to identify novel sRNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense sRNAs encoded opposite to key regulatory (ferric uptake regulator), metabolic (triose-phosphate isomerase), and core transcriptional apparatus genes (transcription factor B). We also found a large increase in the number of conserved C/D box sRNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these sRNAs indicates they are relatively recent, stable adaptations. PMID:22783241

Biological and molecular characterization of cellular differentiation in Tetrahymena vorax: a potential biocontrol protozoan.

PubMed

Green, M M; LeBoeuf, R D; Churchill, P F

2000-01-01

Tetrahymena vorax (T. vorax) is an indigenous fresh water protozoan with the natural biological potential to maintain a specific aquatic microbial flora by ingesting and eliminating specific microorganism. To investigate the molecular mechanisms controlling Tetrahymena vorax (T. vorax) cellular differentiation from a small-mouth vegetative cell to a voracious large-mouth carnivore capable of ingesting prey ciliates and bacteria from aquatic environments, we use DNA subtraction and gene discovery techniques to identify and isolate T. vorax differentiation-specific genes. The physiological necessity for one newly discovered gene, SUBII-TG, was determined in vivo using an antisense oligonucleotide directed against the 5' SUBII-TG DNA sequence. The barriers to delivering antisense oligonucleotides to the cytoplasm of T. vorax were circumvented by employing a new but simple procedure of processing the oligonucleotide with the differentiation stimulus, stomatin. In these studies, the antisense oligonucleotide down-regulated SUBII-TG mRNA expression, and blocked differentiation and ingestion of prey ciliates. The ability to down-regulate SUBII-TG expression with the antisense oligonucleotide suggests that the molecular mechanisms controlling the natural biological activities of T. vorax can be manipulated to further study its cellular differentiation and potential as a biocontrol microorganism.

Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes

PubMed Central

Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.

2003-01-01

Transformation of Mucor circinelloides with self-replicative plasmids containing a wild-type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady-state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post-transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M.circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25-nucleotide RNA and a short 21-nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self-replicative nature of the triggering molecules, make M.circinelloides a suitable organism for investigating some unresolved questions in RNA silencing. PMID:12881432

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

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

Tian, Hongmei; Ma, Leyuan; Zhao, Cong

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leavesmore » and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.« less

Selective optical sensing of biothiols with Ellman's reagent: 5,5'-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles.

PubMed

Güçlü, Kubilay; Ozyürek, Mustafa; Güngör, Nilay; Baki, Sefa; Apak, Reşat

2013-09-10

Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB(2-)) anion as a result of reaction with biothiols was measured at 410nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57μM and acceptable linearity ranging from 0.4 to 29.0μM (r=0.998). Copyright © 2013 Elsevier B.V. All rights reserved.

Tuning a Protein-Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation.

PubMed

Pham, Grace H; Ou, Weijia; Bursulaya, Badry; DiDonato, Michael; Herath, Ananda; Jin, Yunho; Hao, Xueshi; Loren, Jon; Spraggon, Glen; Brock, Ansgar; Uno, Tetsuo; Geierstanger, Bernhard H; Cellitti, Susan E

2018-04-16

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Vitro Selection of pH-Activated DNA Nanostructures.

PubMed

Fong, Faye Yi; Oh, Seung Soo; Hawker, Craig J; Soh, H Tom

2016-12-05

We report the first in vitro selection of DNA nanostructures that switch their conformation when triggered by change in pH. Previously, most pH-active nanostructures were designed using known pH-active motifs, such as the i-motif or the triplex structure. In contrast, we performed de novo selections starting from a random library and generated nanostructures that can sequester and release Mipomersen, a clinically approved antisense DNA drug, in response to pH change. We demonstrate extraordinary pH-selectivity, releasing up to 714-fold more Mipomersen at pH 5.2 compared to pH 7.5. Interestingly, none of our nanostructures showed significant sequence similarity to known pH-sensitive motifs, suggesting that they may operate via novel structure-switching mechanisms. We believe our selection scheme is general and could be adopted for generating DNA nanostructures for many applications including drug delivery, sensors and pH-active surfaces. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) increases the expression of prostaglandin E2 receptor subtype EP4. The roles of phosphatidylinositol 3-kinase and CCAAT/enhancer-binding protein beta.

PubMed

Han, ShouWei; Ritzenthaler, Jeffrey D; Wingerd, Byron; Roman, Jesse

2005-09-30

The prostaglandin E2 receptor subtype EP4 has been implicated in the growth and progression of human non-small cell lung carcinoma (NSCLC). However, the factors that control its expression have not been entirely elucidated. Our studies show that NSCLC cells express peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) protein and that treatment with a selective PPARbeta/delta agonist (GW501516) increases EP4 mRNA and protein levels. GW501516 induced NSCLC cell proliferation, and this effect was prevented by PPARbeta/delta antisense or EP4 short interfering RNA (siRNA). GW501516 increased the phosphorylation of Akt and decreased PTEN expression. The selective inhibitor of phosphatidylinositol 3-kinase (PI3-K), wortmannin, and PPARbeta/delta antisense, abrogated the effect of GW501516 on EP4 expression, whereas that of the inhibitor of Erk did not. GW501516 also increased EP4 promoter activity through effects on the region between -1555 and -992 bp in the EP4 promoter, and mutation of the CCAAT/enhancer-binding protein (C/EBP) site in this region abrogated the effect of GW501516. GW501516 increased not only the binding activity of C/EBP to the NF-IL6 site in the EP4 promoter, which was prevented by the inhibitor of PI3-K, but also increased C/EBPbeta protein in a dose- and PPARbeta/delta-dependent manner. The effect of GW501516 on EP4 protein was eliminated in the presence of C/EBPbeta siRNA. Finally, we showed that pretreatment of NSCLC with GW501516 further increased NSCLC cell proliferation in response to exogenous dimethyl-prostaglandin E2 (PGE2) that was diminished in the presence of PPARbeta/delta antisense and EP4 siRNA. Taken together, these findings suggest that activation of PPARbeta/delta induces PGE2 receptor subtype EP4 expression through PI3-K signals and increases human lung carcinoma cell proliferation in response to PGE2. The increase in transcription of the EP4 gene by PPARbeta/delta agonist was associated with increased C/EBP binding activity in the NF-IL6 site of EP4 promoter region and C/EBPbeta protein expression that were mediated through both PI3-K/Akt and PPARbeta/delta signaling pathways.

Locating the Anion-selectivity Filter of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel

PubMed Central

Cheung, Min; Akabas, Myles H.

1997-01-01

The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel; the site and mechanism of charge selectivity is unknown. We previously reported that cysteines substituted, one at a time, for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353, in and flanking the sixth membrane-spanning segment (M6), reacted with charged, sulfhydryl-specific, methanethiosulfonate (MTS) reagents. We inferred that these residues are on the water-accessible surface of the protein and may line the ion channel. We have now measured the voltage-dependence of the reaction rates of the MTS reagents with the accessible, engineered cysteines. By comparing the reaction rates of negatively and positively charged MTS reagents with these cysteines, we measured the extent of anion selectivity from the extracellular end of the channel to eight of the accessible residues. We show that the major site determining anion vs. cation selectivity is near the cytoplasmic end of the channel; it favors anions by ∼25-fold and may involve the residues Arg347 and Arg352. From the voltage dependence of the reaction rates, we calculated the electrical distance to the accessible residues. For the residues from Leu333 to Ser341 the electrical distance is not significantly different than zero; it is significantly different than zero for the residues Thr351 to Gln353. The maximum electrical distance measured was 0.6 suggesting that the channel extends more cytoplasmically and may include residues flanking the cytoplasmic end of the M6 segment. Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter. PMID:9089437

Synthesis, physicochemical and biochemical studies of 1',2'-oxetane constrained adenosine and guanosine modified oligonucleotides, and their comparison with those of the corresponding cytidine and thymidine analogues.

PubMed

Pradeepkumar, Pushpangadan I; Cheruku, Pradeep; Plashkevych, Oleksandr; Acharya, Parag; Gohil, Suresh; Chattopadhyaya, Jyoti

2004-09-22

We have earlier reported the synthesis and antisense properties of the conformationally constrained oxetane-C and -T containing oligonucleotides, which have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells. Here we report on the straightforward syntheses of the oxetane-A and oxetane-G nucleosides as well as their incorporations into antisense oligonucleotides (AONs), and compare their structural and antisense properties with those of the T and C modified AONs (including the thermostability and RNase H recruitment capability of the AON/RNA hybrid duplex by Michaelis-Menten kinetic analyses, their resistance in the human serum, as well as in the presence of exo and endonucleases).

The role of antisense oligonucleotide therapy in patients with familial hypercholesterolemia: risks, benefits, and management recommendations.

PubMed

Agarwala, Anandita; Jones, Peter; Nambi, Vijay

2015-01-01

Antisense oligonucleotide therapy is a promising approach for the treatment of a broad variety of medical conditions. It functions at the cellular level by interfering with RNA function, often leading to degradation of specifically targeted abnormal gene products implicated in the disease process. Mipomersen is a novel antisense oligonucleotide directed at apolipoprotein (apoB)-100, the primary apolipoprotein associated with low-density lipoprotein cholesterol (LDL-C), which has recently been approved for the treatment of familial hypercholesterolemia. A number of clinical studies have demonstrated its efficacy in lowering LDL-C and apoB levels in patients with elevated LDL-C despite maximal medical therapy using conventional lipid-lowering agents. This review outlines the risks and benefits of therapy and provides recommendations on the use of mipomersen.

A Twist on Facial Selectivity of Hydride Reductions of Cyclic Ketones: Twist-Boat Conformers in Cyclohexanone, Piperidone, and Tropinone Reactions

PubMed Central

2015-01-01

The role of twist-boat conformers of cyclohexanones in hydride reductions was explored. The hydride reductions of a cis-2,6-disubstituted N-acylpiperidone, an N-acyltropinone, and tert-butylcyclohexanone by lithium aluminum hydride and by a bulky borohydride reagent were investigated computationally and compared to experiment. Our results indicate that in certain cases, factors such as substrate conformation, nucleophile bulkiness, and remote steric features can affect stereoselectivity in ways that are difficult to predict by the general Felkin–Anh model. In particular, we have calculated that a twist-boat conformation is relevant to the reactivity and facial selectivity of hydride reduction of cis-2,6-disubstituted N-acylpiperidones with a small hydride reagent (LiAlH4) but not with a bulky hydride (lithium triisopropylborohydride). PMID:25372509

Quinone-Catalyzed Selective Oxidation of Organic Molecules

PubMed Central

Wendlandt, Alison E.

2016-01-01

Lead In Quinones are common stoichiometric reagents in organic chemistry. High potential para-quinones, such as DDQ and chloranil, are widely used and typically promote hydride abstraction. In recent years, many catalytic applications of these methods have been achieved by using transition metals, electrochemistry or O2 to regenerate the oxidized quinone in situ. Complementary studies have led to the development of a different class of quinones that resemble the ortho-quinone cofactors in Copper Amine Oxidases and mediate efficient and selective aerobic and/or electrochemical dehydrogenation of amines. The latter reactions typically proceed via electrophilic transamination and/or addition-elimination reaction mechanisms, rather than hydride abstraction pathways. The collective observations show that the quinone structure has a significant influence on the reaction mechanism and have important implications for the development of new quinone reagents and quinone-catalyzed transformations. PMID:26530485

Collision-induced dissociative chemical cross-linking reagents and methodology: Applications to protein structural characterization using tandem mass spectrometry analysis.

PubMed

Soderblom, Erik J; Goshe, Michael B

2006-12-01

Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.

Adapter reagents for protein site specific dye labeling.

PubMed

Thompson, Darren A; Evans, Eric G B; Kasza, Tomas; Millhauser, Glenn L; Dawson, Philip E

2014-05-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. © 2014 Wiley Periodicals, Inc.

Adapter Reagents for Protein Site Specific Dye Labeling

PubMed Central

Thompson, Darren A.; Evans, Eric G. B.; Kasza, Tomas; Millhauser, Glenn L.; Dawson, Philip E.

2016-01-01

Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this aceto-phenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. PMID:24599728

Second-Generation Difluorinated Cyclooctynes for Copper-Free Click Chemistry

PubMed Central

2008-01-01

The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed “click chemistry”, is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists. PMID:18680289

Second-generation difluorinated cyclooctynes for copper-free click chemistry.

PubMed

Codelli, Julian A; Baskin, Jeremy M; Agard, Nicholas J; Bertozzi, Carolyn R

2008-08-27

The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed "click chemistry", is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists.

Chemical labelling for visualizing native AMPA receptors in live neurons

NASA Astrophysics Data System (ADS)

Wakayama, Sho; Kiyonaka, Shigeki; Arai, Itaru; Kakegawa, Wataru; Matsuda, Shinji; Ibata, Keiji; Nemoto, Yuri L.; Kusumi, Akihiro; Yuzaki, Michisuke; Hamachi, Itaru

2017-04-01

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

Intravesical NGF Antisense Therapy Using Lipid Nanoparticle for Interstitial Cystitis

DTIC Science & Technology

2016-12-01

bladder symptoms including urinary frequency and urgency. Previous studies have indicated that overexpression of nerve growth factor (NGF) is an... studies indicate overexpression of nerve growth factor (NGF) as a key factor in the symptom development of IC/BPS. NGF antisense oligonucleotides hold...Stability Testing  Ex -vivo stress testing II-2. Research Accomplishment Description AIM 1 Regulatory approval for animal research ; Obtain

Development of siRNA Technology to Prevent Scar Formation in Tendon Repair

DTIC Science & Technology

2013-12-01

Anti-sense RNA technologies: Under normal conditions cells produce small interfering (si) RNAs that inhibit protein synthesis and stimulate...stimulation of fibroblast proliferation and migration, collagen and fibronectin synthesis , and altered tissue remodeling through regulation of MMPs...expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nat Biotechnol 2000;18:862-7. 7. Guha M, Xu ZG, Tung D, Lanting L

Iodine(III)-Mediated Selective Intermolecular C-H Amination for the Chemical Diversification of Tryptamines.

PubMed

Bosnidou, Alexandra E; Millán, Alba; Ceballos, Javier; Martínez, Claudio; Muñiz, Kilian

2016-08-05

Defined hypervalent iodine reagents of the general structure PhI[N(SO2R)(SO2R')]2 promote the selective direct C-H-amination of the indole core of various tryptamines. Starting from a general C2-amination strategy, subsequent transformations enable a variety of site-selective functionalizations, which proceed with noteworthy high chemoselectivity and provide an overall access to structurally diversified products.

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage-display

PubMed Central

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K.

2012-01-01

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display. PMID:22985966

Quantification of selected volatile organic compounds in human urine by gas chromatography selective reagent ionization time of flight mass spectrometry (GC-SRI-TOF-MS) coupled with head-space solid-phase microextraction (HS-SPME).

PubMed

Mochalski, Paweł; Unterkofler, Karl

2016-08-07

Selective reagent ionization time of flight mass spectrometry with NO(+) as the reagent ion (SRI-TOF-MS(NO(+))) in conjunction with gas chromatography (GC) and head-space solid-phase microextraction (HS-SPME) was used to determine selected volatile organic compounds in human urine. A total of 16 volatiles exhibiting high incidence rates were quantified in the urine of 19 healthy volunteers. Amongst them there were ten ketones (acetone, 2-butanone, 3-methyl-2-butanone, 2-pentanone, 3-methyl-2-pentanone, 4-methyl-2-pentanone, 2-hexanone, 3-hexanone, 2-heptanone, and 4-heptanone), three volatile sulphur compounds (dimethyl sulfide, allyl methyl sulfide, and methyl propyl sulfide), and three heterocyclic compounds (furan, 2-methylfuran, 3-methylfuran). The concentrations of the species under study varied between 0.55 nmol L(-1) (0.05 nmol mmol(-1)creatinine) for allyl methyl sulfide and 11.6 μmol L(-1) (1.54 μmol mmol(-1)creatinine) for acetone considering medians. Limits of detection (LODs) ranged from 0.08 nmol L(-1) for allyl methyl sulfide to 1.0 nmol L(-1) for acetone and furan (with RSDs ranging from 5 to 9%). The presented experimental setup assists both real-time and GC analyses of volatile organic compounds, which can be performed consecutively using the same analytical system. Such an approach supports the novel concept of hybrid volatolomics, an approach which combines VOC profiles obtained from two or more body fluids to improve and complement the chemical information on the physiological status of an individual.

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.

PubMed

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K

2012-11-23

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious, and time-consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13 the N-terminal Forkhead-associated (FHA1) domain of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be nonfunctional due to misfolding in the bacterial periplasm. To overcome this limitation, we constructed a library of FHA1 variants by mutagenic PCR and isolated functional variants after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1 strand was discovered to be essential for phage display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermally stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20-25mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage display. Copyright © 2012 Elsevier Ltd. All rights reserved.

N-Methylpyrrolidone Hydroperoxide/Cs2 CO3 as an Excellent Reagent System for the Hydroxy-Directed Diastereoselective Epoxidation of Electron-Deficient Olefins.

PubMed

Victor, Napoleon John; Gana, Janardhanan; Muraleedharan, Kannoth Manheri

2015-10-12

This report introduces N-methylpyrrolidone hydroperoxide (NMPOOH)/base as an excellent reagent system for hydroxy-directed syn selective epoxidation of electron-deficient olefins, characterized by high diastereoselectivity, short reaction times and remarkable chemoselectivity, especially in presence of oxidatively labile nitrogen or sulfur atoms. NMPOOH also proves efficient in the oxidation of electron-deficient aromatic aldehydes, in the removal of oxazolidinone chiral auxiliary, and in the functionalization of alkenes and alkynes, showing wide application potential. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Complete regioselective addition of grignard reagents to pyrazine N-oxides, toward an efficient enantioselective synthesis of substituted piperazines.

PubMed

Andersson, Hans; Banchelin, Thomas Sainte-Luce; Das, Sajal; Gustafsson, Magnus; Olsson, Roger; Almqvist, Fredrik

2010-01-15

A conceptually new one-pot strategy for the synthesis of protected substituted piperazines via the addition of Grignard reagents to pyrazine N-oxides is presented. This strategy is high yielding (33-91% over three steps), step-efficient, and fast. The synthesized N,N-diprotected piperazines are convenient to handle and allow for orthogonal deprotection at either nitrogen for selective transformations. In addition, this is a synthetic route to enantiomerically enriched piperazines by using a combination of phenyl magnesium chloride and (-)-sparteine, which resulted in enantiomeric excesses up to 83%.

METHOD 530 DETERMINATION OF SELECT SEMIVOLATILE ORGANIC CHEMICALS IN DRINKING WATER BY SOLID PHASE EXTRACTION AND GAS CHROMATOGRAPHY/ MASS SPECTROMETRY (GC/MS)

EPA Science Inventory

1.1. This is a gas chromatography/mass spectrometry (GC/MS) method for the determination of selected semivolatile organic compounds in drinking waters. Accuracy and precision data have been generated in reagent water, and in finished ground and surface waters for the compounds li...

Regiospecific Epoxidation of Carvone: A Discovery-Oriented Experiment for Understanding the Selectivity and Mechanism of Epoxidation Reactions

ERIC Educational Resources Information Center

Mak, Kendrew K. W.; Lai, Y. M.; Siu, Yuk-Hong

2006-01-01

This article describes a discovery-oriented experiment for demonstrating the selectivity of two epoxidation reactions. Peroxy acids and alkaline H[subscript 2]O[subscript 2] are two commonly used reagents for alkene epoxidation. The former react preferentially with electron-rich alkenes while the latter works better with alpha,beta-unsaturated…

Marfan syndrome, magnesium status and medical prevention of cardiovascular complications by hemodynamic treatments and antisense gene therapy.

PubMed

Igondjo-Tchen, S; Pagès, N; Bac, P; Godeau, G; Durlach, J

2003-03-01

The medical management of Marfan Syndrome (MFS) mainly relies on early prevention of the aortic complications. Hemodynamic treatments try to diminish the forcefulness of cardiac contractions and to reduce blood pressure: for example long term administration of propranolol may significantly reduce the rate of increase in aortic ratio (aortic diameter/expected aortic diameter). Retardation of aortic dilatation may be most often observed by early treatment started when the baseline end-diastolic aortic root diameter is < 40 mm. It seems better to use beta-blockers without intrinsic sympathomimetic activity. Successful acceptance of beta-blockers may be limited by side-effects, but the efficiency of alternative hypotensive agents (calcium channel inhibitors, ACE inhibitors) is not yet validated. Gene therapy might constitute an etiologic specific treatment of MFS. FBN1-RZ1 hammerhead antisense ribozyme is able to suppress expression of the mutant FBN1 allele. The use of ribozymes as systemic therapeutic agents will depend on efficient delivery to its target, but the various proposed vectors raise yet unsolved problems. A hydrogel angioplasty balloon might be a possible vector for delivering an antisense ribozyme in the aortic wall specifically. Ribozymes--as deoxyribonucleotides--may be taken up by tissue upon local application. Further research should study ex vivo local application of antisense ribozyme on human aortic wall, before assessing in vivo efficiency and tolerance of this aortic local vectorisation. It is always necessary to maintain a balanced magnesium intake in patients with MFS. Firstly to prevent the multiple noxious effects of magnesium deficiency on cardiovascular targets. Secondly to ensure the best efficiency and the least toxicity of the hemodynamic drugs used as long term prophylactic treatment for cardiovascular complications and of the etiologic antisense magnesium-dependent gene therapy, in the future.

Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.

PubMed

Rutkauskaite, Edita; Volkmer, Dagmar; Shigeyama, Yukio; Schedel, Jörg; Pap, Geza; Müller-Ladner, Ulf; Meinecke, Ingmar; Alexander, Dorothea; Gay, Renate E; Drynda, Susanne; Neumann, Wolfram; Michel, Beat A; Aicher, Wilhelm K; Gay, Steffen; Pap, Thomas

2005-07-01

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

PubMed Central

Giannini, C D; Roth, W K; Piiper, A; Zeuzem, S

1999-01-01

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene. PMID:10373591

Small-interfering RNAs from natural antisense transcripts derived from a cellulose synthase gene modulate cell wall biosynthesis in barley

PubMed Central

Held, Michael A.; Penning, Bryan; Brandt, Amanda S.; Kessans, Sarah A.; Yong, Weidong; Scofield, Steven R.; Carpita, Nicholas C.

2008-01-01

Small-interfering RNAs (siRNAs) from natural cis-antisense pairs derived from the 3′-coding region of the barley (Hordeum vulgare) CesA6 cellulose synthase gene substantially increase in abundance during leaf elongation. Strand-specific RT-PCR confirmed the presence of an antisense transcript of HvCesA6 that extends ≥1230 bp from the 3′ end of the CesA-coding sequence. The increases in abundance of the CesA6 antisense transcript and the 21-nt and 24-nt siRNAs derived from the transcript are coincident with the down-regulation of primary wall CesAs, several Csl genes, and GT8 glycosyl transferase genes, and are correlated with the reduction in rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Virus induced gene silencing using unique target sequences derived from HvCesA genes attenuated expression not only of the HvCesA6 gene, but also of numerous nontarget Csls and the distantly related GT8 genes and reduced the incorporation of D-14C-Glc into cellulose and into mixed-linkage (1 → 3),(1 → 4)-β-D-glucans of the developing leaves. Unique target sequences for CslF and CslH conversely silenced the same genes and lowered rates of cellulose and (1 → 3),(1 → 4)-β-D-glucan synthesis. Our results indicate that the expression of individual members of the CesA/Csl superfamily and glycosyl transferases share common regulatory control points, and siRNAs from natural cis-antisense pairs derived from the CesA/Csl superfamily could function in this global regulation of cell-wall synthesis. PMID:19075248

Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

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

Murphy, Jane; Hall, William W.; Ratner, Lee

The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we foundmore » that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.« less

Targeted delivery of an antisense oligonucleotide in the retina: uptake, distribution, stability, and effect.

PubMed

Rakoczy, P E; Lai, M C; Watson, M; Seydel, U; Constable, I

1996-01-01

In this article, we describe the preliminary results of the development of an animal model that will enable us to study the effect of photoreceptor-derived debris accumulation on the normal function of the retina in vivo. An antisense oligonucleotide (Cat 5), saline, and two control oligonucleotides were injected into the vitreous of 7-week-old RCS-rdy+ rats. The uptake, distribution, and persistence of the antisense oligonucleotide in the retina was demonstrated by fluorescent confocal microscopy, and the stability of the oligonucleotide was shown by GeneScan analysis using a fluorescein-labeled derivative of Cat 5 (Cat 5F). The accumulation of photoreceptor-derived debris was monitored by the number of undigested phagosomes in the RPE layer by light microscopy. Following intravitreal injection of Cat 5F, penetration of the oligonucleotide was observed in the ganglion cell layer in 2 hours and in the photoreceptor and pigment epithelial layers 3 days later. However, at 7, 28, and 56 days postinjection, only the RPE layer had significant amounts of Cat 5F present. Using GeneScan analysis, it was demonstrated that the fluorescein-labeled oligonucleotide present in the RPE layer was not degraded and it retained its original 19-mer length. There was no statistically significant difference in the number of phagosomes found in the RPE layer of control uninjected, saline-injected, and two sense and two antisense oligonucleotides-injected animals at 7 and 28 days postinjection. In contrast, the number of phagosomes was significantly higher (p < 0.001) in the RPE layer of Cat 5 antisense oligonucleotide-injected animals at 7 and 28 days postinjection. This difference, however, disappeared by 56 days postinjection. The inner nuclear layers of the retina of control and experimental animals were not affected by the injections.

The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply*

PubMed Central

Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R.; Aro, Eva-Mari

2012-01-01

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. PMID:22854963

SPECTROPHOTOMETRIC DETERMINATION OF CALCIUM WITH GLYOXAL BIS (2-HYDROXY- ANIL)

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

Florence, T.M.; Morgan, J.

1961-03-01

A selective method is described for the spectrophotometric determination of calcium using glyoxal bis(2hydroxy-anil) as the chromogenic agent. A comprehensive study of interferences and reagent variables was made. (auth)

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging

PubMed Central

2015-01-01

Bioorthogonal reactions, including the strain-promoted azide–alkyne cycloaddition (SPAAC) and inverse electron demand Diels–Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines. PMID:26270632

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging.

PubMed

Murrey, Heather E; Judkins, Joshua C; Am Ende, Christopher W; Ballard, T Eric; Fang, Yinzhi; Riccardi, Keith; Di, Li; Guilmette, Edward R; Schwartz, Joel W; Fox, Joseph M; Johnson, Douglas S

2015-09-09

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

Fluorogen-Activating-Proteins as Universal Affinity Biosensors for Immunodetection

PubMed Central

Gallo, Eugenio; Vasilev, Kalin V.; Jarvik, Jonathan

2014-01-01

Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins –Protein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. PMID:24122476

Defining Global Gene Expression Changes of the Hypothalamic-Pituitary-Gonadal Axis in Female sGnRH-Antisense Transgenic Common Carp (Cyprinus carpio)

PubMed Central

Xu, Jing; Huang, Wei; Zhong, Chengrong; Luo, Daji; Li, Shuangfei; Zhu, Zuoyan; Hu, Wei

2011-01-01

Background The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. Methodology/Principal Findings In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. Conclusions/Significance This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish. PMID:21695218

Antisense Treatments for Biothreat Agents

DTIC Science & Technology

2006-08-01

2001) 19(4):360-364. 82. Nekhotiaeva N, Awasthi SK, Nielsen PE, Good L: Inhibition of Staphylococcus aureus gene expression and growth using...to PNA enhanced the entry of the antisense molecules and reduced expression of the bacterial target genes both in E coli [81] and Staphylococcus ... aureus [82]. Peptide-tagged PMOs can also efficiently inhibit bacterial growth in pure and infected cultures [75]. In a recent study, we observed that

Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents

Treesearch

Steven L. Voelker; Barbara Lachenbruch; Frederick C. Meinzer; Peter Kitin; Steven H. Strauss

2011-01-01

Reduced lignin content in perennial crops has been sought as a means to improve biomass processability for paper and biofuels production, but it is unclear how this could affect wood properties and tree form. Here, we studied a nontransgenic control and 14 transgenic events containing an antisense 4-coumarate:coenzyme A ligase (4CL) to discern the...

Antisense Oligonucleotide Therapy for Patients with Advanced Cancer | Center for Cancer Research

Cancer.gov

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the U.S. Improvements in therapy have increased the survival of patients with CRC from 10 months to two years, but for patients who stop responding to treatments, such as irinotecan, options for additional therapy are limited. Antisense oligonucleotides (ASOs) may offer advantages over traditional therapies if an appropriate target can be identified.

Annexin II-Dependent Mechanism of Breast Cancer Progression

DTIC Science & Technology

2008-06-01

and migratory capacities of the annexin II-suppressed cells. Methods: We used antisense RNA technology to silence the annexin II gene in MDA...gene in mDA-MB231 cells using polymerase chain reaction-based short hairpin RNA (1–7 months) b) Characterize the proliferative, invasive, and...MB231 cells according to methods described by Li et al. (24). Briefly, three different diothionated antisense nucleotides (ODN) were synthesized

ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy

PubMed Central

Ottesen, Eric W.

2017-01-01

Abstract Spinal muscular atrophy (SMA) is one of the leading genetic diseases of children and infants. SMA is caused by deletions or mutations of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, cannot compensate for the loss of SMN1 due to predominant skipping of exon 7. While various regulatory elements that modulate SMN2 exon 7 splicing have been proposed, intronic splicing silencer N1 (ISS-N1) has emerged as the most promising target thus far for antisense oligonucleotide-mediated splicing correction in SMA. Upon procuring exclusive license from the University of Massachussets Medical School in 2010, Ionis Pharmaceuticals (formerly ISIS Pharamaceuticals) began clinical development of Spinraza™ (synonyms: Nusinersen, IONIS-SMNRX, ISIS-SMNRX), an antisense drug based on ISS-N1 target. Spinraza™ showed very promising results at all steps of the clinical development and was approved by US Food and Drug Administration (FDA) on December 23, 2016. Spinraza™ is the first FDA-approved treatment for SMA and the first antisense drug to restore expression of a fully functional protein via splicing correction. The success of Spinraza™ underscores the potential of intronic sequences as promising therapeutic targets and sets the stage for further improvement of antisense drugs based on advanced oligonucleotide chemistries and delivery protocols. PMID:28400976

Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.

PubMed

Sartor, Gregory C; Powell, Samuel K; Velmeshev, Dmitry; Lin, David Y; Magistri, Marco; Wiedner, Hannah J; Malvezzi, Andrea M; Andrade, Nadja S; Faghihi, Mohammad A; Wahlestedt, Claes

2017-12-01

Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations. Copyright © 2017 Elsevier Inc. All rights reserved.

cis-antisense RNA, another level of gene regulation in bacteria.

PubMed

Georg, Jens; Hess, Wolfgang R

2011-06-01

A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology.

cis-Antisense RNA, Another Level of Gene Regulation in Bacteria

PubMed Central

Georg, Jens; Hess, Wolfgang R.

2011-01-01

Summary: A substantial amount of antisense transcription is a hallmark of gene expression in eukaryotes. However, antisense transcription was first demonstrated in bacteria almost 50 years ago. The transcriptomes of bacteria as different as Helicobacter pylori, Bacillus subtilis, Escherichia coli, Synechocystis sp. strain PCC6803, Mycoplasma pneumoniae, Sinorhizobium meliloti, Geobacter sulfurreducens, Vibrio cholerae, Chlamydia trachomatis, Pseudomonas syringae, and Staphylococcus aureus have now been reported to contain antisense RNA (asRNA) transcripts for a high percentage of genes. Bacterial asRNAs share functional similarities with trans-acting regulatory RNAs, but in addition, they use their own distinct mechanisms. Among their confirmed functional roles are transcription termination, codegradation, control of translation, transcriptional interference, and enhanced stability of their respective target transcripts. Here, we review recent publications indicating that asRNAs occur as frequently in simple unicellular bacteria as they do in higher organisms, and we provide a comprehensive overview of the experimentally confirmed characteristics of asRNA actions and intimately linked quantitative aspects. Emerging functional data suggest that asRNAs in bacteria mediate a plethora of effects and are involved in far more processes than were previously anticipated. Thus, the functional impact of asRNAs should be considered when developing new strategies against pathogenic bacteria and when optimizing bacterial strains for biotechnology. PMID:21646430

Integrated Safety Assessment of 2′-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers

PubMed Central

Crooke, Stanley T; Baker, Brenda F; Kwoh, T Jesse; Cheng, Wei; Schulz, Dan J; Xia, Shuting; Salgado, Nelson; Bui, Huynh-Hoa; Hart, Christopher E; Burel, Sebastien A; Younis, Husam S; Geary, Richard S; Henry, Scott P; Bhanot, Sanjay

2016-01-01

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2′-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2′-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied. PMID:27357629

QTLs Regulating the Contents of Antioxidants, Phenolics, and Flavonoids in Soybean Seeds Share a Common Genomic Region.

PubMed

Li, Man-Wah; Muñoz, Nacira B; Wong, Chi-Fai; Wong, Fuk-Ling; Wong, Kwong-Sen; Wong, Johanna Wing-Hang; Qi, Xinpeng; Li, Kwan-Pok; Ng, Ming-Sin; Lam, Hon-Ming

2016-01-01

Soybean seeds are a rich source of phenolic compounds, especially isoflavonoids, which are important nutraceuticals. Our study using 14 wild- and 16 cultivated-soybean accessions shows that seeds from cultivated soybeans generally contain lower total antioxidants compared to their wild counterparts, likely an unintended consequence of domestication or human selection. Using a recombinant inbred population resulting from a wild and a cultivated soybean parent and a bin map approach, we have identified an overlapping genomic region containing major quantitative trait loci (QTLs) that regulate the seed contents of total antioxidants, phenolics, and flavonoids. The QTL for seed antioxidant content contains 14 annotated genes based on the Williams 82 reference genome (Gmax1.01). None of these genes encodes functions that are related to the phenylpropanoid pathway of soybean. However, we found three putative Multidrug And Toxic Compound Extrusion (MATE) transporter genes within this QTL and one adjacent to it (GmMATE1-4). Moreover, we have identified non-synonymous changes between GmMATE1 and GmMATE2, and that GmMATE3 encodes an antisense transcript that expresses in pods. Whether the polymorphisms in GmMATE proteins are major determinants of the antioxidant contents, or whether the antisense transcripts of GmMATE3 play important regulatory roles, awaits further functional investigations.

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

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

Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.

2005-07-01

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased inmore » association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.« less

Will novel oral formulations change the management of inflammatory bowel disease?

PubMed

Nielsen, Ole Haagen; Seidelin, Jakob Benedict; Ainsworth, Mark; Coskun, Mehmet

2016-06-01

The traditional management of inflammatory bowel disease (IBD) with sulphasalazine/5-aminosalicylic acid, glucocorticoids and immunomodulators (i.e., thiopurines and methotrexate) was nearly two decades ago extended with intravenously or subcutaneously administered biologics (i.e., tumor necrosis factor inhibitors and later gut-selective integrin antagonists). However, recently, orally administered treatments with simple, well-characterized, and stable structures consisting of either small molecules or anti-sense therapy have been devised. This review discusses the current approaches with promising new oral drugs with distinct modes of action, including: the Janus kinase inhibitors (i.e., tofacitinib, filgotinib and peficitinib); the immunomodulatory drug (laquinimod); a small α4 antagonist (AJM300); agonists for sphingosine-phosphate receptors (i.e., ozanimod, APD334, and amiselimod), as well as anti-sense therapy (mongersen) targeting SMAD7, drugs which directly target intracellular pathways of relevance for intestinal inflammation. A new avenue using easily administered oral therapies for the management of IBD is being introduced. While their place in the clinical armamentarium remains to be proven, it is likely that many of these drugs will find their place in the treatment algorithm of IBD in the next few years. Thus, we will face times in which IBD therapy will be based on significantly more tablets than prescribed today.

Implementation of microfluidic sandwich ELISA for superior detection of plant pathogens.

PubMed

Thaitrong, Numrin; Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Karoonuthaisiri, Nitsara

2013-01-01

Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5-10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation.

Microfluidic device for robust generation of two-component liquid-in-air slugs with individually controlled composition

PubMed Central

Liu, Kan; Chen, Yi-Chun; Tseng, Hsian-Rong

2010-01-01

Using liquid slugs as microreactors and microvessels enable precise control over the conditions of their contents on short-time scales for a wide variety of applications. Particularly for screening applications, there is a need for control of slug parameters such as size and composition. We describe a new microfluidic approach for creating slugs in air, each comprising a size and composition that can be selected individually for each slug. Two-component slugs are formed by first metering the desired volume of each reagent, merging the two volumes into an end-to-end slug, and propelling the slug to induce mixing. Volume control is achieved by a novel mechanism: two closed chambers on the chip are initially filled with air, and a valve in each is briefly opened to admit one of the reagents. The pressure of each reagent can be individually selected and determines the amount of air compression, and thus the amount of liquid that is admitted into each chamber. We describe the theory of operation, characterize the slug generation chip, and demonstrate the creation of slugs of different compositions. The use of microvalves in this approach enables robust operation with different liquids, and also enables one to work with extremely small samples, even down to a few slug volumes. The latter is important for applications involving precious reagents such as optimizing the reaction conditions for radiolabeling biological molecules as tracers for positron emission tomography. Electronic supplementary material The online version of this article (doi:10.1007/s10404-010-0617-0) contains supplementary material, which is available to authorized users. PMID:20930933

Spectroscopic study of the reaction mechanism of buspirone interaction with iodine and tetracyanoethylene reagents and its applications.

PubMed

Zayed, M A; El-Habeeb, Abeer A

2009-06-01

The reactions between the drug buspirone (busp) in its base form and iodine amphoteric reagent (n-donor and/or sigma-acceptor) and with tetracyanoethylene as a pi-acceptor reagent (TCNE) have been studied spectrophotometrically at different reactant concentrations, time intervals, temperatures, and with different solvents and wavelengths, with the aim of selecting the conditions that give the most suitable molar extinction coefficients. This study aims chiefly to throw light on the nature of these reactions and to select the most proper conditions for spectrophotometric application of these reagents to determine this biologically active drug used in treating different diseases. The reaction mechanism involves the formation of busp-I(2) outer and inner sphere complexes. The separated busp-I(2) solid product obtained was investigated using elemental analyses, FT-IR, thermal analyses (TA) and electron ionization mass spectrometry (EI-MS) and was found to be biologically active. The reaction mechanism of busp-TCNE involves the formation of a charge transfer (CT) complex. The analytical parameters of the proposed spectrophotometric procedures were calculated. These procedures were applied in the analysis of busp in its formulations as a drug used to treat psychiatric illnesses. The values of the Sandell sensitivity, standard deviation (SD), relative standard deviation (RSD) and recovery percentage show the high sensitivity of these procedures. This study also presents a promising new busp-I(2) drug derivative that can be used more efficiently for the same purposes as its parent. It gives a clear idea about the possible metabolites and metabolic pathways of busp and its derivative that may occur in vivo. Copyright 2009 John Wiley & Sons, Ltd.

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

PubMed

Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

2012-01-01

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

Synthesis of Actinide Fluoride Complexes Using Trimethyltin Fluoride as a Mild and Selective Fluorinating Reagent

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

Kagan, Benjamin D.; Lichtscheidl, Alejandro G.; Erickson, Karla A.

Trimethyltin fluoride (Me₃SnF) is a mild and selective reagent for the installation of actinide fluoride bonds as demonstrated by the room temperature synthesis of a variety of organometallic and inorganic thorium(IV), uranium(IV), and uranium(V) fluoride complexes ((1,2,4-tBu₃C₅H₂)₂ThF₂, (C₅Me₅)₂U(F)(O-2,6-iPr₂C₆H₃), U(F)(O-2,6-tBu₂C₆H₃)₃, U(F)[N(SiMe₃)₂]₃ (C₅Me₅)₂UF₂(L) (L = O=PMe₃, O=PPh₃, O=PCy₃), and (C₅Me₅)₂U(F)(=N-2,6-iPr₂C₆H₃)) from their corresponding chloride, bromide, and iodide analogues. From these reactions, the new (C₅Me₅)₂UF₂(L) (L = O=PPh₃, O=PCy₃) uranium fluoride complexes were isolated and characterized by NMR spectroscopy and X-ray crystallography.

Extraction photometric determination of yperite by phthaleins.

PubMed

Halámek, E; Kobliha, Z

1999-01-01

Extraction spectrophotometric determination of sulfidic yperite, based on the reaction with four phthaleins, was developed. The method is technically simpler than the determination of yperites with reagent T-135 (alkaline-aqueous ethanolic thymolphthalein solution) because it does not require heating at 80 degrees C, cooling and acidification of the reaction mixture. Selection of the appropriate phthalein, and particularly optimization of the reagent composition and extraction of the coloured reaction product in chloroform, markedly increased the selectivity of the determination of yperites (HD, HN-3). The reaction is performed in a medium of increased polarity due to the low content of alcohol which enables the reaction to proceed at temperatures of 5-20 degrees C without any marked loss of sensitivity. Using (1)H and (13)C NMR spectroscopy, the reaction products of HD and o-cresolphthalein were identified and an ionic mechanism for the reaction of HD with phthaleins is suggested.

Photo-oxidation of PAHs with calcium peroxide as a source of the hydroxyl radicals

NASA Astrophysics Data System (ADS)

Kozak, Jolanta; Włodarczyk-Makuła, Maria

2018-02-01

The efficiency of the removal of selected PAHs from the pretreated coking wastewater with usage of CaO2, Fenton reagent (FeSO4) and UV rays are presented in this article. The investigations were carried out using coking wastewater originating from biological, industrial wastewater treatment plant. At the beginning of the experiment, the calcium peroxide (CaO2) powder as a source of hydroxyl radicals (OH•) and Fenton reagent were added to the samples of wastewater. Then, the samples were exposed to UV rays for 360 s. The process was carried out at pH 3.5-3.8. After photo-oxidation process a decrease in the PAHs concentration was observed. The removal efficiency of selected hydrocarbons was in the ranged of 89-98%. The effectiveness of PAHs degradation was directly proportional to the calcium peroxide dose.

A novel property of DNA - as a bioflotation reagent in mineral processing.

PubMed

Vasanthakumar, Balasubramanian; Ravishankar, Honnavar; Subramanian, Sankaran

2012-01-01

Environmental concerns regarding the use of certain chemicals in the froth flotation of minerals have led investigators to explore biological entities as potential substitutes for the reagents in vogue. Despite the fact that several microorganisms have been used for the separation of a variety of mineral systems, a detailed characterization of the biochemical molecules involved therein has not been reported so far. In this investigation, the selective flotation of sphalerite from a sphalerite-galena mineral mixture has been achieved using the cellular components of Bacillus species. The key constituent primarily responsible for the flotation of sphalerite has been identified as DNA, which functions as a bio-collector. Furthermore, using reconstitution studies, the obligatory need for the presence of non-DNA components as bio-depressants for galena has been demonstrated. A probable model involving these entities in the selective flotation of sphalerite from the mineral mixture has been discussed.

A Novel Property of DNA – As a Bioflotation Reagent in Mineral Processing

PubMed Central

Vasanthakumar, Balasubramanian; Ravishankar, Honnavar; Subramanian, Sankaran

2012-01-01

Environmental concerns regarding the use of certain chemicals in the froth flotation of minerals have led investigators to explore biological entities as potential substitutes for the reagents in vogue. Despite the fact that several microorganisms have been used for the separation of a variety of mineral systems, a detailed characterization of the biochemical molecules involved therein has not been reported so far. In this investigation, the selective flotation of sphalerite from a sphalerite-galena mineral mixture has been achieved using the cellular components of Bacillus species. The key constituent primarily responsible for the flotation of sphalerite has been identified as DNA, which functions as a bio-collector. Furthermore, using reconstitution studies, the obligatory need for the presence of non-DNA components as bio-depressants for galena has been demonstrated. A probable model involving these entities in the selective flotation of sphalerite from the mineral mixture has been discussed. PMID:22768298

Synthesis of Actinide Fluoride Complexes Using Trimethyltin Fluoride as a Mild and Selective Fluorinating Reagent

DOE PAGES

Kagan, Benjamin D.; Lichtscheidl, Alejandro G.; Erickson, Karla A.; ...

2017-11-07

Trimethyltin fluoride (Me₃SnF) is a mild and selective reagent for the installation of actinide fluoride bonds as demonstrated by the room temperature synthesis of a variety of organometallic and inorganic thorium(IV), uranium(IV), and uranium(V) fluoride complexes ((1,2,4-tBu₃C₅H₂)₂ThF₂, (C₅Me₅)₂U(F)(O-2,6-iPr₂C₆H₃), U(F)(O-2,6-tBu₂C₆H₃)₃, U(F)[N(SiMe₃)₂]₃ (C₅Me₅)₂UF₂(L) (L = O=PMe₃, O=PPh₃, O=PCy₃), and (C₅Me₅)₂U(F)(=N-2,6-iPr₂C₆H₃)) from their corresponding chloride, bromide, and iodide analogues. From these reactions, the new (C₅Me₅)₂UF₂(L) (L = O=PPh₃, O=PCy₃) uranium fluoride complexes were isolated and characterized by NMR spectroscopy and X-ray crystallography.

Phase I study of liposome-encapsulated c-raf antisense oligodeoxyribonucleotide infusion in combination with radiation therapy in patients with advanced malignancies.

PubMed

Dritschilo, Anatoly; Huang, Chao H; Rudin, Charles M; Marshall, John; Collins, Brian; Dul, Jeanne L; Zhang, Chuanbo; Kumar, Deepak; Gokhale, Prafulla C; Ahmad, Ateeq; Ahmad, Imran; Sherman, Jeffrey W; Kasid, Usha N

2006-02-15

Raf proteins are key elements of growth-related cellular signaling pathways and are a component of cancer cell resistance to radiation therapy. Antisense oligonucleotides to c-raf-1 permit highly selective inhibition of the gene product and offer a strategy for sensitizing cancer cells to radiation therapy. In this dose escalation study, we evaluated the safety of combined liposomal formulation of raf antisense oligonucleotide (LErafAON) and radiation therapy in patients with advanced malignancies. Patients with advanced solid tumors were treated with LErafAON in a phase I dose escalation study while receiving palliative radiation therapy. Drug-related and radiation-related toxicities were monitored. Pharmacokinetics and expression of c-raf-1 mRNA and Raf-1 protein were determined in peripheral blood mononuclear cells. Seventeen patients with palliative indications for radiation therapy were entered into this study. Thirteen patients received daily infusions of LErafAON and four received twice-weekly infusions. Radiation therapy was delivered in daily 300-cGy fractions over 2 weeks. Patients tolerated radiation, and no unexpected radiation-related side effects were observed. Drug-related reactions (grade > or =2), such as back pain, chills, dyspnea, fatigue, fever, flushing, and hypertension, were observed in most patients and were managed by premedication with corticosteroids and antihistamines. Serious adverse events occurred in five patients, including acute infusion-related symptoms, abnormal liver function tests, hypoxia, dehydration, diarrhea, esophagitis, fever, hypokalemia, pharyngitis, and tachypnea. Twelve of 17 patients were evaluable for tumor response at completion of treatment; four showed partial response, four showed stable disease, and four experienced progressive disease. The intact rafAON was detected in plasma for 30 minutes to several hours. Six patients with partial response or stable disease were evaluable for c-raf-1 mRNA and/or Raf-1 protein expression. Inhibition of c-raf-1 mRNA was observed in three of five patients. Raf-1 protein was inhibited in four of five patients. This is the first report of the combined modality treatment using antisense oligonucleotides with radiation therapy in patients with advanced cancer. A dose of 2.0 mg/kg of LErafAON administered twice weekly is tolerated with premedication and does not enhance radiation toxicity in patients. The observation of dose-dependent, infusion-related reactions has led to further modification of the liposomal composition for use in future clinical trials.

Antisense imaging of epidermal growth factor-induced p21(WAF-1/CIP-1) gene expression in MDA-MB-468 human breast cancer xenografts.

PubMed

Wang, Judy; Chen, Paul; Mrkobrada, Marko; Hu, Meiduo; Vallis, Katherine A; Reilly, Raymond M

2003-09-01

Molecular imaging of the expression of key genes which determine the response to DNA damage following cancer treatment may predict the effectiveness of a particular treatment strategy. A prominent early response gene for DNA damage is the gene encoding p21(WAF-1/CIP-1), a cyclin-dependent kinase inhibitor that regulates progression through the cell cycle. In this study, we explored the feasibility of imaging p21(WAF-1/CIP-1) gene expression at the mRNA level using an 18-mer phosphorothioated antisense oligodeoxynucleotide (ODN) labeled with (111)In. The known induction of the p21(WAF-1/CIP-1) gene in MDA-MB-468 human breast cancer cells following exposure to epidermal growth factor (EGF) was used as an experimental tool. Treatment of MDA-MB-468 cells in vitro with EGF (20 n M) increased the ratio of p21(WAF-1/CIP-1) mRNA/beta-actin mRNA threefold within 2 h as measured by the reverse transcription polymerase chain reaction (RT-PCR). A concentration-dependent inhibition of EGF-induced p21(WAF-1/CIP-1) protein expression was achieved in MDA-MB-468 cells by treatment with antisense ODNs with up to a tenfold decrease observed at 1 microM. There was a fourfold lower inhibition of p21(WAF-1/CIP-1) protein expression by control sense or random sequence ODNs. Intratumoral injections of EGF (15 microg/dayx3 days) were employed to induce p21(WAF-1/CIP-1) gene expression in MDA-MB-468 xenografts implanted subcutaneously into athymic mice. RT-PCR of explanted tumors showed a threefold increased level of p21(WAF-1/CIP-1) mRNA compared with normal saline-treated tumors. Successful imaging of EGF-induced p21(WAF-1/CIP-1) gene expression in MDA-MB-468 xenografts was achieved at 48 h post injection of (111)In-labeled antisense ODNs (3.7 MBq; 2 microg). Tumors displaying basal levels of p21(WAF-1/CIP-1) gene expression in the absence of EGF treatment could not be visualized. Biodistribution studies showed a significantly higher tumor accumulation of (111)In-labeled antisense ODNs in the presence of EGF induction of the p21(WAF-1/CIP-1) gene (0.32%+/-0.06% injected dose/g) compared with normal saline-treated control mice (0.11%+/-0.07% injected dose/g). The tumor/blood ratio for antisense ODNs in the presence of EGF induction of the p21(WAF-1/CIP-1) gene (4.87+/-0.87) was also significantly higher than for control random sequence ODNs (2.14+/-0.69) or for mice receiving antisense ODNs but not treated with EGF (2.07+/-0.37). We conclude that antisense imaging of upregulated p21(WAF-1/CIP-1) gene expression is feasible and could represent a promising new molecular imaging strategy for monitoring tumor response in cancer patients. To our knowledge, this study also describes the first report of molecular imaging of the upregulated expression of a downstream gene target of the EGFR, a transmembrane tyrosine kinase receptor.

Iron-catalysed fluoroaromatic coupling reactions under catalytic modulation with 1,2-bis(diphenylphosphino)benzene.

PubMed

Hatakeyama, Takuji; Kondo, Yoshiyuki; Fujiwara, Yu-Ichi; Takaya, Hikaru; Ito, Shingo; Nakamura, Eiichi; Nakamura, Masaharu

2009-03-14

A catalytic amount of 1,2-bis(diphenylphosphino)benzene (DPPBz) achieves selective cleavage of sp(3)-carbon-halogen bond in the iron-catalysed cross-coupling between polyfluorinated arylzinc reagents and alkyl halides, which was unachievable with a stoichiometric modifier such as TMEDA; the selective iron-catalysed fluoroaromatic coupling provides easy and practical access to polyfluorinated aromatic compounds.

A novel surface-enhanced Raman scattering nanosensor for detecting multiple heavy metal ions based on 2-mercaptoisonicotinic acid functionalized gold nanoparticles.

PubMed

Tan, Enzhong; Yin, Penggang; Lang, Xiufeng; Zhang, Hongyan; Guo, Lin

2012-11-01

A novel, effective and simple surface-enhanced Raman scattering (SERS) nanosensor for selectively and sensitively detecting heavy metal ions in aqueous solution has been developed in the form of 2-mercaptoisonicotinic acid (2 MNA)-modified gold nanoparticles (AuNPs). Multiple heavy metal ions can be identified and quantified by using relative peak intensity ratios of selected vibrational bands in the SERS spectra of 2 MNA. Especially, concentration of Hg(2+) and Pb(2+) ions are determined by comparing the intensity ratios of the bands 1160/1230 cm(-1) for Hg(2+) and 861/815 cm(-1) (or 815/1392 cm(-1)) for Pb(2+), with detection limits of 3.4×10(-8) and 1.0×10(-7)M, respectively. 2 MNA-AuNPs sensors show a high selectivity for Hg(2+) without masking reagent, and they can also be highly selective for Pb(2+) when using sodium thiosulphate and l-cysteine as masking reagents. These results demonstrate that these 2 MNA-AuNPs nanosensors are promising candidates for in situ heavy metal ions detection and quantification, maybe even inside living cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Lack of anti-tumor activity with the β-catenin expression inhibitor EZN-3892 in the C57BL/6J Min/+ model of intestinal carcinogenesis

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

Hasson, Rian M.; Briggs, Alexandra; Rizvi, Hira

2014-02-14

Highlights: • Wnt/β-catenin signaling is aberrantly activated in most colorectal cancers. • Locked nucleic acid (LNA)-based antisense is a novel tool for cancer therapy. • β-Catenin inhibition was observed in mature intestinal tissue of LNA-treated mice. • Further investigation of Wnt/β-catenin targeted therapies is warranted. - Abstract: Background: Previously, we showed that short-term inhibition of β-catenin expression and reversal of aberrant β-catenin subcellular localization by the selective COX-2 inhibitor celecoxib is associated with adenoma regression in the C57BL/6J Min/+ mouse. Conversly, long-term administration resulted in tumor resistance, leading us to investigate alternative methods for selective β-catenin chemoprevention. In this study,more » we hypothesized that disruption of β-catenin expression by EZN-3892, a selective locked nucleic acid (LNA)-based β-catenin inhibitor, would counteract the tumorigenic effect of Apc loss in Min/+ adenomas while preserving normal intestinal function. Materials and methods: C57BL/6J Apc{sup +/+} wild-type (WT) and Min/+ mice were treated with the maximum tolerated dose (MTD) of EZN-3892 (30 mg/kg). Drug effect on tumor numbers, β-catenin protein expression, and nuclear β-catenin localization were determined. Results: Although the tumor phenotype and β-catenin nuclear localization in Min/+ mice did not change following drug administration, we observed a decrease in β-catenin expression levels in the mature intestinal tissue of treated Min/+ and WT mice, providing proof of principle regarding successful delivery of the LNA-based antisense vehicle. Higher doses of EZN-3892 resulted in fatal outcomes in Min/+ mice, likely due to β-catenin ablation in the intestinal tissue and loss of function. Conclusions: Our data support the critical role of Wnt/β-catenin signaling in maintaining intestinal homeostasis and highlight the challenges of effective drug delivery to target disease without permanent toxicity to normal cellular function.« less

Qualitative and quantitative evaluation of derivatization reagents for different types of protein-bound carbonyl groups.

PubMed

Bollineni, Ravi Chand; Fedorova, Maria; Hoffmann, Ralf

2013-09-07

Mass spectrometry (MS) of 'carbonylated proteins' often involves derivatization of reactive carbonyl groups to facilitate their enrichment, identification and quantification. Among the many reported reagents, 2,4-dinitrophenylhydrazine (DNPH), biotin hydrazide (BHZ) and O-(biotinylcarbazoylmethyl) hydroxylamine (ARP) are the most frequently used. Despite their common use in carbonylation research, their reactivity towards protein-bound carbonyls has not been quantitatively evaluated in detail, to the best of our knowledge. Thus we studied the reactivity and specificity of these reagents towards different classes of reactive carbonyl groups (e.g. aldehydes, ketones and lactams), each being represented by a synthetic peptide carrying an accordingly modified residue. All three tagging reagents were selective for aliphatic aldehydes and ketones. Lactams and carbonyl-containing tryptophan oxidation products, however, were labelled only at low levels or not at all. Whereas DNPH derivatization was efficient under the published standard conditions, the derivatization conditions for BHZ and ARP had to be altered. Acidic conditions provided quantitative labelling yields for ARP. Peptides derivatized with DNPH, BHZ and ARP fragmented efficiently in tandem mass spectrometry, when the experimental conditions were chosen carefully for each reagent. Importantly, the tested carbonylated peptides did not cross-react with amino groups in other proteins present during sample preparations or enzymatic digestion. Thus, it appears favourable to digest proteins first and then derivatise the reactive carbonyl groups more efficiently at the peptide level under acidic conditions. The carbonylated model peptides used in this study might be valid internal standards for carbonylation proteomics.

Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization.

PubMed

Welch, Leslie; Dong, Xiao; Hewitt, Daniel; Irwin, Michelle; McCarty, Luke; Tsai, Christina; Baginski, Tomasz

2018-06-02

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies. Copyright © 2018 Elsevier B.V. All rights reserved.

Static micromixer-coaxial electrospray synthesis of theranostic lipoplexes.

PubMed

Wu, Yun; Li, Lei; Mao, Yicheng; Lee, Ly James

2012-03-27

Theranostic lipoplexes are an integrated nanotherapeutic system with diagnostic imaging capability and therapeutic functions. They hold great promise to improve current cancer treatments; however, producing uniform theranostic lipoplexes with multiple components in a reproducible manner is a highly challenging task. Conventional methods, such as bulk mixing, are not able to achieve this goal because of their macroscale and random nature. Here we report a novel technique, called the static micromixer-coaxial electrospray (MCE), to synthesize theranostic lipoplexes in a single step with high reproducibility. In this work, quantum dots (QD605) and Cy5-labeled antisense oligodeoxynucleotides (Cy5-G3139) were chosen as the model imaging reagent and therapeutic drug, respectively. Compared with bulk mixing, QD605/Cy5-G3139-loaded lipoplexes produced by MCE were highly uniform with polydispersity of 0.024 ± 0.006 and mean diameter by volume of 194 ± 15 nm. MCE also showed higher encapsulation efficiency of QD605 and Cy5-G3139. QD605 and Cy5 also formed the Förster resonance energy transfer pair, and thus the cellular uptake and intracellular fate of theranostic lipoplexes could be visualized by flow cytometry and confocal microscopy. The lipoplexes were efficiently delivered to A549 cells (non-small cell lung cancer cell line) and down-regulated the Bcl-2 gene expression by 48 ± 6%. © 2012 American Chemical Society

Anti-NGF Local Therapy for Autonomic Dysreflexia in Spinal Cord Injury

DTIC Science & Technology

2013-10-01

growth factor in the urothelium of sham treated rats, which was decreased 5 by antisense treatment (Fig. 4A) and (5) increased nerve growth...sensitization. B Figure 4. A: Antisense OND mediated suppression of acetic acid (AA) induced NGF protein expression in urothelium . AA exposure...upregulation in the bladder urothelium of SCI rats  Detection of hyperexcitability of bladder afferent neurons due to the reduction of A-type K+ channel

Growth Suppression and Therapy Sensitization of Breast Cancer

DTIC Science & Technology

2000-07-01

determined by performed on two independent occasions. PCR amplification of a given housekeeping gene have been shown to correspond to determinations of...h incubation in the presence or absence of 1 mM cisplatin expressed housekeeping gene, dihydrofolate reductase (DHFR). (Platinol, aqueous solution at... G3PDH :j G3PDH Figure 9. A549 cells were treated with 3 different antisense oligonucleotides complementary to JNKI mRNA (including the active antisense

Generation of Soluble Receptor Activator of NF-kappa B Ligand is Critical for Osteolytic Bone Metastasis

DTIC Science & Technology

2009-10-01

differentiation and activation of osteoclast precursors. Targeting RANKL expression with antisense oligonucleotides (RANKL- ASO ) decreased RANKL expression and...1,175 Ci/mmol at 10 mCi/mL). Two microliters of the reaction mixture were separated on a 12% SDS-polyacrylamide gel and subsequently visualized...OPG), a decoy receptor for RANKL, at the TB-interface was also increased. Targeting RANKL expression with antisense oligonucleotides (RANKL- ASO

Imaging Oncogene Expression

PubMed Central

Mukherjee, Archana; Wickstrom, Eric

2009-01-01

This review briefly outlines the importance of molecular imaging, particularly imaging of endogenous gene expression for noninvasive genetic analysis of radiographic masses. The concept of antisense imaging agents and the advantages and challenges in the development of hybridization probes for in vivo imaging are described. An overview of the investigations on oncogene expression imaging is given. Finally, the need for further improvement in antisense-based imaging agents and directions to improve oncogene mRNA targeting is stated. PMID:19264436

Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

NASA Astrophysics Data System (ADS)

Lau, Jeffrey M. C.; Muslin, Anthony J.

The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

Versatile Method for the Site-Specific Modification of DNA with Boron Clusters: Anti-Epidermal Growth Factor Receptor (EGFR) Antisense Oligonucleotide Case.

PubMed

Ebenryter-Olbińska, Katarzyna; Kaniowski, Damian; Sobczak, Milena; Wojtczak, Błażej A; Janczak, Sławomir; Wielgus, Ewelina; Nawrot, Barbara; Leśnikowski, Zbigniew J

2017-11-21

A general and convenient approach for the incorporation of different types of boron clusters into specific locations of the DNA-oligonucleotide chain based on the automated phosphoramidite method of oligonucleotide synthesis and post-synthetic "click chemistry" modification has been developed. Pronounced effects of boron-cluster modification on the physico- and biochemical properties of the antisense oligonucleotides were observed. The silencing activity of antisense oligonucleotides bearing a single boron cluster modification in the middle of the oligonucleotide chain was substantially higher than that of unmodified oligonucleotides. This finding may be of importance for the design of therapeutic nucleic acids with improved properties. The proposed synthetic methodology broadens the availability of nucleic acid-boron cluster conjugates and opens up new avenues for their potential practical use. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Inhibition of monocytes adhesion to the intima of arterial wall by local expression of antisense monocyte chemotactic protein-1].

PubMed

Wu, Q; Qiao, H; Wang, Z; Zhang, H; Liu, P; Xu, M; Ren, G; Zhao, S; She, M

2000-04-01

To study the mechanism of monocyte recruitment in atherogenesis and to clarify the effect of monocyte chemotactic protein-1 (MCP-1) in this process. Femoral arteries isolated from the rabbits which had been fed with a high cholesterol diet and locally perfused with MM-LDL within the artery beforehand, were used as the models. Antisense MCP-1cDNA was transferred into the arterial wall by injecting recombinant LNCX-anti-MCP-1/liposomal complex in the femoral sheath and the periarterial tissue. Expression of antisense MCP-1 mediated by recombinant LNCX plasmid/lipsomal complex gene transfer enabled to inhibit MCP-1 gene expression and adhesion of monocyte to the intima. MCP-1 plays an important role on the recruitment of monocytes in the arterial wall, which provides a potential clue in developing a gene therapy project for the prevention and treatment of atherogenesis.

Antisense oligonucleotide inhibition of cholesteryl ester transfer protein enhances RCT in hyperlipidemic, CETP transgenic, LDLr-/- mice.

PubMed

Bell, Thomas A; Graham, Mark J; Lee, Richard G; Mullick, Adam E; Fu, Wuxia; Norris, Dan; Crooke, Rosanne M

2013-10-01

Due to their ability to promote positive effects across all of the lipoprotein classes, cholesteryl ester transfer protein (CETP) inhibitors are currently being developed as therapeutic agents for cardiovascular disease. In these studies, we compared an antisense oligonucleotide (ASO) inhibitor of CETP to the CETP small molecule inhibitor anacetrapib. In hyperlipidemic CETP transgenic (tg) mice, both drugs provided comparable reductions in total plasma cholesterol, decreases in CETP activity, and increases in HDL cholesterol. However, only mice treated with the antisense inhibitor showed an enhanced effect on macrophage reverse cholesterol transport, presumably due to differences in HDL apolipoprotein composition and decreases in plasma triglyceride. Additionally, the ASO-mediated reductions in CETP mRNA were associated with less accumulation of aortic cholesterol. These preliminary findings suggest that CETP ASOs may represent an alternative means to inhibit that target and to support their continued development as a treatment for cardiovascular disease in man.

Inhibition of adenovirus 5 replication in COS-1 cells by antisense RNAs against the viral E1a region.

PubMed

Miroshnichenko, O I; Ponomareva, T I; Tikchonenko, T I

1989-12-07

To study the effect of antisense E1a RNA (asRNA) on adenovirus development, two types of adenovirus 5 E1a antisense constructs have been engineered. One was complementary to the viral DNA region [nucleotide (nt) positions 500-720] regulated by the metallothionein-I promoter, and the other was complementary to the DNA regions (nt positions 630-1570) under control of the long terminal repeat Moloney mouse leukosis virus promoter. Both asRNA constructs were cloned into a plasmid containing the simian virus 40 origin of replication, the gene controlling geneticin (G418) resistance (G418R), and other regulatory elements. The COS-1 cells, which contained up to 100 copies of the engineered plasmids, synthesized antiviral asRNAs, which provided 71 to over 95% inhibition of adenoviral replication, in comparison to the control cells not synthesizing asRNAs.

Bacterial antisense RNAs are mainly the product of transcriptional noise.

PubMed

Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria

2016-03-01

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

Bacterial antisense RNAs are mainly the product of transcriptional noise

PubMed Central

Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

2016-01-01

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.

PubMed

Froim, D; Hopkins, C E; Belenky, A; Cohen, A S

1997-11-01

The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation.

Method for phosphorothioate antisense DNA sequencing by capillary electrophoresis with UV detection.

PubMed Central

Froim, D; Hopkins, C E; Belenky, A; Cohen, A S

1997-01-01

The progress of antisense DNA therapy demands development of reliable and convenient methods for sequencing short single-stranded oligonucleotides. A method of phosphorothioate antisense DNA sequencing analysis using UV detection coupled to capillary electrophoresis (CE) has been developed based on a modified chain termination sequencing method. The proposed method reduces the sequencing cost since it uses affordable CE-UV instrumentation and requires no labeling with minimal sample processing before analysis. Cycle sequencing with ThermoSequenase generates quantities of sequencing products that are readily detectable by UV. Discrimination of undesired components from sequencing products in the reaction mixture, previously accomplished by fluorescent or radioactive labeling, is now achieved by bringing concentrations of undesired components below the UV detection range which yields a 'clean', well defined sequence. UV detection coupled with CE offers additional conveniences for sequencing since it can be accomplished with commercially available CE-UV equipment and is readily amenable to automation. PMID:9336449

Microchemical Pen: An Open Microreactor for Region-Selective Surface Modification.

PubMed

Mao, Sifeng; Sato, Chiho; Suzuki, Yuma; Yang, Jianmin; Zeng, Hulie; Nakajima, Hizuru; Yang, Ming; Lin, Jin-Ming; Uchiyama, Katsumi

2016-10-18

Various micro surface-modification approaches including photolithography, dip-pen lithography and ink-jet systems have been developed and used to extend the functionalities of solid surfaces. While those approaches work in the "open space", push-pull systems which work in solutions have recently drawn considerable attention. However, the confining flows performed by push-pull systems have realized only the dispense process, while microscale, region-selective chemical reactions have remained unattainable. This study reports a microchemical pen that enables region-selective chemical reactions for the micro surface modification/patterning. The chemical pen is based on the principle of microfluidic laminar flows and the resulting mixing of reagents by the mutual diffusion. The tiny diffusion layer performs as the working region. This report represents the first demonstration of an open microreactor in which two different reagents react on a real solid sample. The multifunctional characteristics of the microchemical pen are confirmed by different types of reactions in many research areas, including inorganic chemistry, polymer science, electrochemistry and biological sample treatment. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of γ-hydroxypropyl P-chirogenic (±)-phosphorus oxide derivatives by regioselective ring-opening of oxaphospholane 2-oxide precursors

PubMed Central

Binyamin, Iris; Meidan-Shani, Shoval

2015-01-01

Summary The synthesis of P-chirogenic (±)-phosphine oxides and phosphinates via selective nucleophilic ring opening of the corresponding oxaphospholanes is described. Two representative substrates: the phosphonate 2-ethoxy-1,2-oxaphospholane 2-oxide and the phosphinate 2-phenyl-1,2-oxaphospholane 2-oxide were reacted with various Grignard reagents to produce a single alkyl/aryl product. These products may possess further functionalities in addition to the phosphorus center such as the γ-hydroxypropyl group which results from the ring opening and π-donor moieties such as aryl, allyl, propargyl and allene which originates from the Grignard reagent. PMID:26425187

Arylation, alkenylation, and alkylation of 2-halopyridine N-oxides with grignard reagents: a solution to the problem of C2/C6 regioselective functionalization of pyridine derivatives.

PubMed

Zhang, Song; Liao, Lian-Yan; Zhang, Fang; Duan, Xin-Fang

2013-03-15

A facile arylation, alkenylation, and alkylation of functionalized 2-halopyridine N-oxides with various Grignard reagents was developed. It represented a highly efficient and selective C-H bond functionalization of pyridine derivatives in the presence of reactive C-Cl or C-Br bonds. Using Cl or Br as a blocking group, C2/C6 site-controllable functionalization of pyridine derivatives has been achieved. Various pyridine compounds can be prepared as illustrated in the total syntheses of Onychine, dielsine, and PARP-1 inhibitor GPI 16539.

Preparation System and Method

NASA Technical Reports Server (NTRS)

Zhang, Ye (Inventor); Wu, Honglu (Inventor)

2015-01-01

Systems and methods for preparing a sample for further analysis are provided. The system can include an enclosure. A membrane can be disposed within the enclosure. First and second reservoirs can be disposed within the enclosure, and at least one of the first and second reservoirs can be adapted to have a reagent disposed therein. A valve can be disposed within the enclosure and in fluid communication with the first or second reservoirs or both. The valve can also be in fluid communication with the membrane. The valve can be adapted to selectively regulate the flow of the reagent from the first reservoir, through the membrane, and into the second reservoir.

Separation of V(V)-4-(2-pyridylazo)resorcinolato complex from a large excess reagent using an ODS cartridge for high-performance liquid chromatography.

PubMed

Takahashi, Toru; Kaneko, Emiko; Yotsuyanagi, Takao

2006-12-01

A selective off-line preconcentration technique for the V(V) complex with 4-(2-pyridylazo)resorcinol has been developed and successfully applied to the determination of V(V) in an air-borne sample. The target complex was separated from excess reagent using an ODS cartridge and water as the eluent. The complex was then concentrated on another ODS cartridge using tetrabutylammonium bromide and eluted with methanol; the eluate was applied to a one-drop concentration/HPLC. A detection limit as low as (6.05 +/- 0.82)x 10(-11) M (5 ppt) was achieved.

Routine low-level monitoring of polar pesticides and pesticide degradates by HPLC/ESI-MS: Evaluating long-term performance

USGS Publications Warehouse

Furlong, E.T.; Martin, Jeffrey D.; Werner, S.L.; Gates, Paul M.

2002-01-01

The sensitivity and selective determination of polar pesticides were analyzed using high-performance liquid chromatography/electrospray ionization-mass spectrometry (HPLC/ESI-MS). The effects of multiple operators and instruments on method performance were evaluated using 440 pairs of fortified reagent-water and blank reagent-water samples. The influence of varying environmental matrices on recovery and precision were also analyzed using 200 fortified ambient water samples and duplicate ambient water samples. The results show that compound stability in filtered water was matrix-, chemical class- and compound-dependent which ranged from 1 day to 2 weeks.

Cell specific aptamer-photosensitizer conjugates as a molecular tool in photodynamic therapy

PubMed Central

Mallikaratchy, Prabodhika; Tang, Zhiwen

2010-01-01

This paper describes the application of a molecular construct of a photosensitizer and an aptamer for photo-therapeutically targeting tumor cells. The key step in increasing selectivity in chemotherapeutic drugs is to create effective molecular platforms that could target cancer cells but not normal cells. Recently, we have developed a strategy via cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to obtain cell specific aptamers using intact viable cells as targets to select aptamers that can recognize cell membrane proteins with high selectivity and excellent affinity. We have identified an aptamer TD05 that only recognizes Ramos cells, a Burkitt’s lymphoma cell line. Here, the high specificity of aptamers in target cell binding and an efficient phototherapy reagent, Ce6, are molecularly engineered to construct a highly selective Aptamer-photosensitizer conjugates (APS) to effectively destroy target cancer cells. Introduction of the APS conjugates followed by irradiation of light selectively destroyed target Ramos cells but not acute lymphoblastic leukemia and myeloid leukemia cell lines. This study demonstrates that the use of cancer specific aptamers conjugated to a photosensitizer will enhance the selectivity of photodynamic therapy. Coupled with the advantages of the cell-SELEX in generating multiple effective aptamers for diseased cell recognition, we will be able to develop highly efficient photosensitizer based therapeutical reagents for clinical applications. PMID:18058891

Antisense Down-Regulation of the FaPG1 Gene Reveals an Unexpected Central Role for Polygalacturonase in Strawberry Fruit Softening1[W

PubMed Central

Quesada, Miguel A.; Blanco-Portales, Rosario; Posé, Sara; García-Gago, Juan A.; Jiménez-Bermúdez, Silvia; Muñoz-Serrano, Andrés; Caballero, José L.; Pliego-Alfaro, Fernando; Mercado, José A.; Muñoz-Blanco, Juan

2009-01-01

The strawberry (Fragaria × ananassa ‘Chandler’) fruit undergoes a fast softening during ripening. Polygalacturonase (PG) activity is low during this process, but two ripening-related PG genes, FaPG1 and FaPG2, have been cloned. Both genes were up-regulated during fruit ripening and were also negatively regulated by auxin. To further assess the role of FaPG1 on strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the 35S promoter (APG lines) were obtained. Sixteen out of 30 independent transgenic lines showed fruit yields similar to those of the control. Several quality parameters were measured in ripe fruits from these 16 lines. Fruit weight was slightly reduced in four lines, and most of them showed an increase in soluble solid content. Half of these lines yielded fruits significantly firmer than did the control. Four APG lines were selected, their ripened fruits being on average 163% firmer than the control. The postharvest softening of APG fruits was also diminished. Ripened fruits from the four selected lines showed a 90% to 95% decrease in FaPG1 transcript abundance, whereas the level of FaPG2 was not significantly altered. Total PG activity was reduced in three of these lines when compared with control fruits. Cell wall extracts from APG fruits showed a reduction in pectin solubilization and an increase in pectins covalently bound to the cell wall. A comparative transcriptomic analysis of gene expression between the ripened receptacle of the control and those of the APG fruits (comprising 1,250 receptacle expressed sequence tags) did not show any statistically significant change. These results indicate that FaPG1 plays a central role in strawberry softening. PMID:19395408

Physical and Functional Interaction of NCX1 and EAAC1 Transporters Leading to Glutamate-Enhanced ATP Production in Brain Mitochondria

PubMed Central

Arcangeli, Sara; Nasti, Annamaria Assunta; Giordano, Antonio; Amoroso, Salvatore

2012-01-01

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na+-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production. PMID:22479505

Decoy Oligonucleotide Rescues IGF1R Expression from MicroRNA-223 Suppression

PubMed Central

Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3’ untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5’, central or 3’ region of mature miR-223 suppressed miR-223 targeting the 3’UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3’UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3’UTRs have similar binding sites for miR-223 with IGF1R 3’UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting. PMID:24324762

Decoy oligonucleotide rescues IGF1R expression from MicroRNA-223 suppression.

PubMed

Wu, Li Hui; Cai, Qian Qian; Dong, Yi Wei; Wang, Rong; He, Bao Mei; Qi, Bing; Xu, Chang Jun; Wu, Xing Zhong

2013-01-01

A mature miRNA generally suppresses hundreds of mRNA targets. To evaluate the selective effect of synthetic oligonucleotide decoys on hsa-miR-223 activity, reporters containing 3' untranslated regions (UTR) of IGF1R, FOXO1, POLR3G, FOXO3, CDC27, FBXW7 and PAXIP1 mRNAs were constructed for the luciferase assay. The oligonucleotide decoys were designed and synthesized according to mature miR-223 sequence and its target mRNA sequence. Quantitative RT-PCR & western analysis were used to measure miR-223-targeted mRNA expression, Interestingly, apart from the antisense oligonucleotide, decoy nucleotides which were complementary to the 5', central or 3' region of mature miR-223 suppressed miR-223 targeting the 3'UTR of IGF1R, FOXO1, FOXO3, CDC27, POLR3G, and FBXW7 mRNAs and rescued the expression of these genes to varying degrees from miR-223 suppression at both mRNA and protein levels. All decoys had no effect on PAXIP1 which was not targeted by miR-223. The decoy 1 that was based on the sequence of IGF1R 3'UTR rescued the expression of IGF1R more significantly than other decoy nucleotides except the antisense decoy 4. Decoy 1 also rescued the expression of FOXO3 and POLR3G of which their 3'UTRs have similar binding sites for miR-223 with IGF1R 3'UTR. However decoy 1 failed to recover Sp1, CDC27 and FBXW7 expression. These data support that the sequence-specific decoy oligonucleotides might represent exogenous competing RNA which selectively inhibits microRNA targeting.

Selective inhibition of alpha1B-adrenergic receptor expression and function using a phosphorothioate antisense oligodeoxynucleotide.

PubMed

Gonzalez-Cabrera, P J; Iversen, P L; Liu, M F; Scofield, M A; Jeffries, W B

1998-06-01

To investigate alpha1B-adrenoceptor function, we developed a phosphorothioate antisense oligodeoxynucleotide (AO) to inhibit the expression of the alpha1B-adrenoceptor subtype in DDT1 MF2 cells. We measured the cellular uptake of the AO and its effect on alpha1B-adrenoceptor mRNA expression, protein density, and coupling to phospholipase C. Cells treated with either a control oligodeoxynucleotide (CO) or medium alone served as control groups. Confocal microscopy demonstrated that DDT1 MF2 cells internalized carboxyfluorescein-labeled (FAM) AO within 30 min. Analysis of cellular lysates showed that approximately 50% of the intracellular FAM-AO was present as an intact 18-mer for up to 48 hr. Incubation of cells with AO for 48 hr decreased alpha1B-adrenoceptor density ([3H]prazosin Bmax) versus control groups by 12% (1 microM AO) and 72% (10 microM AO). In time course experiments, AO (10 microM) reduced alpha1B-adrenoceptor density by 28, 64, and 68% versus controls after 24, 48, and 72 hr of exposure, respectively. alpha1B-Adrenoceptor mRNA concentration (measured by RT-PCR) was reduced by 25% in cells treated for 48 hr with 10 microM AO versus controls. AO pretreatment (10 microM, 48 hr) reduced the maximum response to agonist-stimulated [3H]inositol phosphate accumulation. The maximal response of the full agonist norepinephrine was reduced by 30% after AO treatment, and by 73% for the partial agonist naphazoline. In contrast, AO did not affect histamine-stimulated total [3H]inositol phosphate accumulation. Thus, AO effectively reduced alpha1B-adrenoceptor subtype expression and function in vitro, suggesting a potential to selectively inhibit alpha1B-adrenoceptor function in vivo.

A review of the fundamental studies of the copper activation mechanisms for selective flotation of the sulfide minerals, sphalerite and pyrite.

PubMed

Chandra, A P; Gerson, A R

2009-01-30

A review of the considerable, but often contradictory, literature examining the specific surface reactions associated with copper adsorption onto the common metal sulfide minerals sphalerite, (Zn,Fe)S, and pyrite (FeS(2)), and the effect of the co-location of the two minerals is presented. Copper "activation", involving the surface adsorption of copper species from solution onto mineral surfaces to activate the surface for hydrophobic collector attachment, is an important step in the flotation and separation of minerals in an ore. Due to the complexity of metal sulfide mineral containing systems this activation process and the emergence of activation products on the mineral surfaces are not fully understood for most sulfide minerals even after decades of research. Factors such as copper concentration, activation time, pH, surface charge, extent of pre-oxidation, water and surface contaminants, pulp potential and galvanic interactions are important factors affecting copper activation of sphalerite and pyrite. A high pH, the correct reagent concentration and activation time and a short time delay between reagent additions is favourable for separation of sphalerite from pyrite. Sufficient oxidation potential is also needed (through O(2) conditioning) to maintain effective galvanic interactions between sphalerite and pyrite. This ensures pyrite is sufficiently depressed while sphalerite floats. Good water quality with low concentrations of contaminant ions, such as Pb(2+)and Fe(2+), is also needed to limit inadvertent activation and flotation of pyrite into zinc concentrates. Selectivity can further be increased and reagent use minimised by opting for inert grinding and by carefully choosing selective pyrite depressants such as sulfoxy or cyanide reagents. Studies that approximate plant conditions are essential for the development of better separation techniques and methodologies. Improved experimental approaches and surface sensitive techniques with high spatial resolution are needed to precisely verify surface structures formed after copper activation. Sphalerite and pyrite surfaces are characterised by varying amounts of steps and defects, and this heterogeneity suggests co-existence of more than one copper-sulfide structure after activation.

Visual Servoing for Optimization of Anticancer Drug Uptake in Human Breast Cancer Cells

DTIC Science & Technology

2000-09-01

successfully obtained new DOE Medical Applications Program funding for this research (included in Appendix G: Automated Imaging System for Guiding Antisense ...Guiding Antisense Compounds to Specific mRNVA targets in Living Cells ) that will support this integration and development work with Dr. Parvin and Deep...a DNA and RNA binding fluorescence probe with a very different emission wavelengths, depending on whether it is bound to DNA or RNA ). Cells were then

Chemical labelling for visualizing native AMPA receptors in live neurons

PubMed Central

Wakayama, Sho; Kiyonaka, Shigeki; Arai, Itaru; Kakegawa, Wataru; Matsuda, Shinji; Ibata, Keiji; Nemoto, Yuri L.; Kusumi, Akihiro; Yuzaki, Michisuke; Hamachi, Itaru

2017-01-01

The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders. PMID:28387242

ISS-N1 makes the First FDA-approved Drug for Spinal Muscular Atrophy.

PubMed

Ottesen, Eric W

2017-01-01

Spinal muscular atrophy (SMA) is one of the leading genetic diseases of children and infants. SMA is caused by deletions or mutations of Survival Motor Neuron 1 ( SMN1 ) gene. SMN2 , a nearly identical copy of SMN1 , cannot compensate for the loss of SMN1 due to predominant skipping of exon 7. While various regulatory elements that modulate SMN2 exon 7 splicing have been proposed, intronic splicing silencer N1 (ISS-N1) has emerged as the most promising target thus far for antisense oligonucleotide-mediated splicing correction in SMA. Upon procuring exclusive license from the University of Massachussets Medical School in 2010, Ionis Pharmaceuticals (formerly ISIS Pharamaceuticals) began clinical development of Spinraza ™ (synonyms: Nusinersen, IONIS-SMN RX , ISIS-SMN RX ), an antisense drug based on ISS-N1 target. Spinraza ™ showed very promising results at all steps of the clinical development and was approved by US Food and Drug Administration (FDA) on December 23, 2016. Spinraza ™ is the first FDA-approved treatment for SMA and the first antisense drug to restore expression of a fully functional protein via splicing correction. The success of Spinraza ™ underscores the potential of intronic sequences as promising therapeutic targets and sets the stage for further improvement of antisense drugs based on advanced oligonucleotide chemistries and delivery protocols.

Spleen-specific suppression of TNF-alpha by cationic hydrogel-delivered antisense nucleotides for the prevention of arthritis in animal models.

PubMed

Dong, Lei; Xia, Suhua; Chen, Huan; Chen, Jiangning; Zhang, Junfeng

2009-09-01

This study developed a transplantable platform based on cationic hydrogels to deliver antisense oligodeoxynucleotides (ASOs) targeting the mRNA of TNF-alpha. Cationic agarose (c-agarose) was obtained by conjugating ethylenediamine to agarose via an N,N'-carbonyldiimidazole (CDI)-activation method. ASO-c-agarose system was constructed by mixing ASO in cationic agarose gel of proper concentration and gelation temperature. In vivo assessment of ASO distribution suggested that the system specifically target to spleen, wherein the c-agarose-delivered ASO had a concentration remarkably 50-fold higher than that of the naked ASO. The distribution of c-agarose-delivered ASO was scarcely detectable in liver and kidney. Next, three types of animal models were setup to evaluate the therapeutic efficacies of ASO-Gel, including the adjuvant-induced arthritis (AA), carrageen/lipopolysaccharide (LPS)-induced arthritis (CLA) and collagen-induced arthritis (CIA) models. The effects of ASO-c-agarose in alleviating inflammation and tissue destruction were evidenced in more than 90% of the testing animals, with decrease of main inflammatory cytokines, lightening of joint swelling and tissue damage, as well as increase in their body weights. All these findings suggest that this highly operable devise for the conveyance of antisense nucleotides together with its spleen-targeting property, could become a useful means of antisense-based therapeutics against rheumatoid arthritis and other diseases.

Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development.

PubMed

Foerst-Potts, L; Sadler, T W

1997-05-01

In mouse embryos, the muscle segment homeobox genes, Msx-1 and Msx-2 are expressed during critical stages of neural tube, neural crest, and craniofacial development, suggesting that these genes play important roles in organogenesis and cell differentiation. Although the patterns of expression are intriguing, little is known about the function of these genes in vertebrate embryonic development. Therefore, the expression of both genes, separately and together, was disrupted using antisense oligodeoxynucleotides and whole embryo culture techniques. Antisense attenuation of Msx-1 during early stages of neurulation produced hypoplasia of the maxillary, mandibular, and frontonasal prominences, eye anomalies, and somite and neural tube abnormalities. Eye defects consisted of enlarged optic vesicles, which may ultimately result in micropthalmia similar to that observed in Small eye mice homozygous for mutations in the Pax-6 gene. Histological sections and SEM analysis revealed a thinning of the neuroepithelium in the diencephalon and optic vesicle and mesenchymal deficiencies in the craniofacial region. Injections of Msx-2 antisense oligodeoxynucleotides produced similar malformations as those targeting Msx-1, with the exception that there was an increase in number and severity of neural tube and somite defects. Embryos injected with the combination of Msx-1 + Msx-2 antisense oligodeoxynucleotides showed no novel abnormalities, suggesting that the genes do not operate in a redundant manner.

[Inhibitory effect of VEGF antisense phosphorothioate oligodeoxynucleotides on the growth of human salivary adenoid cystic carcinoma xenografts in nude mice].

PubMed

Li, Xiao-guang; Wang, Xu-xia; Li, Teng-yu; Wang, Yan-xiu; Gao, Jing; Ni, Chun-xiao

2012-12-01

To investigate the inhibitory effect of VEGF antisense phosphorothioate oligodeoxynucleoiides on the growth of human salivary adenoid cystic carcinoma (SACC) xenografts in nude mice. The VEGF-ASODN was synthesised artificially. After the model of human SACC xenografts in nude mice was established, they were random1y divided into three groups: antisense group, scrambled group and normal saline group. A control group without cancer was also established. Antisense(66 μg), scrambled sequence(66 μg) and normal saline(once every 3 days and 7 times in all) were injected in three experimental groups, respectively. Two days after therapy, the mice were sacrificed. Serums were used for detection of VEGF protein. All tumors were measured and weighted. The quantity of VEGF mRNA and protein and PLI, MVD was detected by hybridization in situ and immunohistochemistry. SPSS13.0 software package was used for statistical analysis. The VEGF-ASODN could suppress the expression of VEGF in human SACC xenografts in nude mice and reduce VEGF protein in serum of nude mice significantly. It cou1d also reduce the volume and weight of xenografts and could reduce the expression of VEGF mRNA and its protein, PCNA and CD34. By inhibiting the expression of VEGF, VEGF-ASODN can inhabit proliferation of human SACC xenografts in nude mice.

Characterization of fructose-bisphosphate aldolase regulated by gibberellin in roots of rice seedling.

PubMed

Konishi, Hirosato; Yamane, Hisakazu; Maeshima, Masayoshi; Komatsu, Setsuko

2004-12-01

Fructose-bisphosphate aldolase is a glycolytic enzyme whose activity increases in rice roots treated with gibberellin (GA). To investigate the relationship between aldolase and root growth, GA-induced root aldolase was characterized. GA3 promoted an increase in aldolase accumulation when 0.1 microM GA3 was added exogenously to rice roots. Aldolase accumulated abundantly in roots, especially in the apical region. To examine the effect of aldolase function on root growth, transgenic rice plants expressing antisense aldolase were constructed. Root growth of aldolase-antisense transgenic rice was repressed compared with that of the vector control transgenic rice. Although aldolase activity increased by 25% in vector control rice roots treated with 0.1 microM GA3, FBPA activity increased very little by 0.1 microM GA3 treatment in the root of aldolase-antisense transgenic rice. Furthermore, aldolase co-immunoprecipitated with antibodies against vacuolar H+ -ATPase in rice roots. In the root of OsCDPK13-antisense transgenic rice, aldolase did not accumulate even after treatment with GA3. These results suggest that the activation of glycolytic pathway function accelerates root growth and that GA3-induced root aldolase may be modulated through OsCDPK13. Aldolase physically associates with vacuolar H-ATPase in roots and may regulate the vacuolar H-ATPase mediated control of cell elongation that determines root length.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase.

PubMed

Ralph, J; Hatfield, R D; Piquemal, J; Yahiaoui, N; Pean, M; Lapierre, C; Boudet, A M

1998-10-27

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl-SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl-SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Antisense expression of the peptide transport gene AtPTR2-B delays flowering and arrests seed development in transgenic Arabidopsis plants.

PubMed Central

Song, W; Koh, S; Czako, M; Marton, L; Drenkard, E; Becker, J M; Stacey, G

1997-01-01

Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246. PMID:9232875

PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

PubMed

Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik F; Persson, Robert; Møller, Marianne R; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen M; Koch, Troels

2012-02-01

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

Shine-Dalgarno sequence enhances the efficiency of lacZ repression by artificial anti-lac antisense RNAs in Escherichia coli.

PubMed

Stefan, Alessandra; Schwarz, Flavio; Bressanin, Daniela; Hochkoeppler, Alejandro

2010-11-01

Silencing of the lacZ gene in Escherichia coli was attempted by means of the expression of antisense RNAs (asRNAs) in vivo. A short fragment of lacZ was cloned into the pBAD expression vector, in reverse orientation, using the EcoRI and PstI restriction sites. This construct (pBAD-Zcal1) was used to transform E. coli cells, and the antisense transcription was induced simply by adding arabinose to the culture medium. We demonstrated that the Zcal1 asRNA effectively silenced lacZ using β-galactosidase activity determinations, SDS-PAGE, and Western blotting. Because the concentration of the lac mRNA was always high in cells that expressed Zcal1, we hypothesize that this antisense acts by inhibiting messenger translation. Similar analyses, performed with a series of site-specific Zcal1 mutants, showed that the Shine-Dalgarno sequence, which is conferred by the pBAD vector, is an essential requisite for silencing competence. Indeed, the presence of the intact Shine-Dalgarno sequence positively affects asRNA stability and, hence, silencing effectiveness. Our observations will contribute to the understanding of the main determinants of silencing as exerted by asRNAs as well as provide useful support for the design of robust and efficient prokaryotic gene silencers. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Fate and Transport of Select Hydraulic Fracturing Compounds of Potential Concern

EPA Pesticide Factsheets

Use of proprietary mixtures of reagents in fracing fluids injected in deep zones, has led to controversy over potential contamination of drinking water aquifers. This presentation focuses on the different classes of compounds identified in fracing fluids.

Modulation of Sodium Iodide Symporter in Thyroid Cancer

PubMed Central

Lakshmanan, Aparna; Scarberry, Daniel

2015-01-01

Radioactive iodine (RAI) is a key therapeutic modality for thyroid cancer. Loss of RAI uptake in thyroid cancer inversely correlates with patient’s survival. In this review, we focus on the challenges encountered in delivering sufficient doses of I-131 to eradicate metastatic lesions without increasing the risk of unwanted side effects. Sodium iodide symporter (NIS) mediates iodide influx, and NIS expression and function can be selectively enhanced in thyroid cells by thyroid-stimulating hormone. We summarize our current knowledge of NIS modulation in normal and cancer thyroid cells, and we propose that several reagents evaluated in clinical trials for other diseases can be used to restore or further increase RAI accumulation in thyroid cancer. Once validated in preclinical mouse models and clinical trials, these reagents, mostly small-molecule inhibitors, can be readily translated into clinical practice. We review available genetically engineered mouse models of thyroid cancer in terms of their tumor development and progression as well as their thyroid function. These mice will not only provide important insights into the mechanisms underlying the loss of RAI uptake in thyroid tumors but will also serve as preclinical animal models to evaluate the efficacy of candidate reagents to selectively increase RAI uptake in thyroid cancers. Taken together, we anticipate that the optimal use of RAI in the clinical management of thyroid cancer is yet to come in the near future. PMID:25234361

Winery wastewater treatment by combination of Cryptococcus laurentii and Fenton's reagent.

PubMed

Santos, Cátia; Lucas, Marco S; Dias, Albino A; Bezerra, Rui M F; Peres, José A; Sampaio, Ana

2014-12-01

Winery wastewaters (WW) have high levels of organic matter, resulting in high COD and BOD and suspended solids. This paper studies the combination of biological and chemical processes in WW treatment. Among 10 yeast isolates, Filobasidium sp. (AGG 577) and Cryptococcus laurentii (AGG 726) were selected due to their superior performance in COD removal. During WW degradation, COD and total polyphenols (TPP) content removal of 89-90% for Filobasidium sp. and 90-93% for C. laurentii were obtained. However, despite similar degradation efficiency for both yeasts, COD kinetics and pH evolution during treatment reveals that C. laurentii presents a faster response than Filobasidium sp. The toxicity (inhibition of Vibrio fischeri luminescence) of C. laurentii treated WW decreases to an inhibition value below 2.5%. However, treated WW exceeds the legal limits, making necessary an additional treatment. In this case, the selection of Fenton's reagent as a chemical final polish step process is a good compromise between efficiency and lower practical complexity. The best results for both COD and TPP removal were obtained with H2O2 initial concentration of 39.2mM and a H2O2:Fe(2+) molar ratio of 15:1. The combined C. laurentii - Fenton's reagent treatment of WW achieved a total reduction of 98% and 96%, for COD and TPP, respectively. Copyright © 2014 Elsevier Ltd. All rights reserved.

Photochemical reactions of aromatic compounds and the concept of the photon as a traceless reagent.

PubMed

Hoffmann, Norbert

2012-11-01

Electronic excitation significantly changes the reactivity of chemical compounds. Compared to ground state reactions, photochemical reactions considerably enlarge the application spectrum of a particular functional group in organic synthesis. Multistep syntheses may be simplified and perspectives for target oriented synthesis (TOS) and diversity oriented synthesis (DOS) are developed. New compound families become available or may be obtained more easily. In contrast to common chemical reagents, photons don't generate side products resulting from the transformation of a chemical reagent. Therefore, they are considered as a traceless reagent. Consequently, photochemical reactions play a central role in the methodology of sustainable chemistry. This aspect has been recognized since the beginning of the 20th century. As with many other photochemical transformations, photochemical reactions of aromatic, benzene-like compounds illustrate well the advantages in this context. Photochemical cycloadditions of aromatic compounds have been investigated for a long time. Currently, they are applied in various fields of organic synthesis. They are also studied in supramolecular structures. The phenomena of reactivity and stereoselectivity are investigated. During recent years, photochemical electron transfer mediated reactions are particularly focused. Such transformations have likewise been performed with aromatic compounds. Reactivity and selectivity as well as application to organic synthesis are studied.

Reverse flow injection spectrophotometric determination of ciprofloxacin in pharmaceuticals using iron from soil as a green reagent

NASA Astrophysics Data System (ADS)

Palamy, Sysay; Ruengsitagoon, Wirat

2018-02-01

A novel reverse flow injection spectrophotometric method for the determination of ciprofloxacin was successfully combined with the on-line introduction of an iron solution extracted from soil as green reagent. The assay was optimized by a univariate method to select the optimum conditions for the highest absorbance and highest stability of the complex. Beer-Lambert's law (λmax = 440 nm) is obeyed in the range 0.5-50 μg mL- 1 with a correlation coefficient (r2) of 0.9976 and 0.9996 using soil as green reagent from Khon Kaen, Thailand and Vientiane, Laos, respectively. The average percentage recoveries were in the range of 98.55-102.14% and the precision was in the range of 0.80-1.73%. The limit of detection and the limit of quantitation were 0.20 and 0.69 μg mL- 1, respectively, with a sampling rate of over 46 samples h- 1. The method was successfully applied to the determination of ciprofloxacin in commercial pharmaceutical formulations. The results were in good agreement with those obtained by the reference HPLC method using a t-test at 95% of confidence level for comparison. This method is suitable for laboratories looking for alternative analytical methods using green reagents.

Determination of total antioxidant capacity of humic acids using CUPRAC, Folin-Ciocalteu, noble metal nanoparticle- and solid-liquid extraction-based methods.

PubMed

Karadirek, Şeyda; Kanmaz, Nergis; Balta, Zeynep; Demirçivi, Pelin; Üzer, Ayşem; Hızal, Jülide; Apak, Reşat

2016-06-01

Total antioxidant capacity (TAC) of humic acid (HA) samples was determined using CUPRAC (CUPric Reducing Antioxidant Capacity), FC (Folin-Ciocalteu), QUENCHER-CUPRAC, QUENCHER-FC, Ag-NP (Silver nanoparticle)‒ and Au-NP (Gold nanoparticle)‒based methods. Conventional FC and modified FC (MFC) methods were applied to solid samples. Because of decreased solubility of Folin-Ciocalteu's phenol reagent in organic solvents, solvent effect on TAC measurement was investigated using QUENCHER-CUPRAC assay by using ethanol:distilled water and dimethyl sulfoxide:distilled water with varying ratios. To see the combined effect of solubilization (leaching) and TAC measurement of humic acids simultaneously, QUENCHER experiments were performed at 25°C and 50°C; QUENCHER-CUPRAC and QUENCHER-FC methods agreed well and had similar precision in F-statistics. Although the Gibbs free energy change (ΔG°) of the oxidation of HA dihydroxy phenols with the test reagents were negative, the ΔG° was positive only for the reaction of CUPRAC reagent with isolated monohydric phenols, showing CUPRAC selectivity toward polyphenolic antioxidants. This is the first work on the antioxidant capacity measurement of HA having a sparingly soluble matrix where enhanced solubilization of bound phenolics is achieved with coupled oxidation by TAC reagents. Copyright © 2016 Elsevier B.V. All rights reserved.

ISIS 301012 gene therapy for hypercholesterolemia: sense, antisense, or nonsense?

PubMed

Ito, Matthew K

2007-10-01

To present an overview of antisense technology and to review and assess available literature on the chemistry, pharmacology, pharmacokinetics, drug interactions, preclinical and clinical studies, dosing, and adverse events of ISIS 301012 in the treatment of hyperlipidemia. PubMed database searches were conducted from 1966 to May 2007 using the search terms ISIS 301012, antisense, oligonucleotide, hypercholesterolemia, hyperlipidemia, and apolipoprotein B. Bibliographies of relevant review articles and information from the manufacturer were reviewed for additional references. Available English-language literature, including abstracts, preclinical, and clinical trials, review articles, and scientific presentations were examined. Apolipoprotein B is an important structural protein on the surface of atherogenic lipoproteins such as remnant very-low-density lipoprotein and low-density lipoprotein and facilitates the clearance of these particles from the circulation by binding to the low-density lipoprotein receptor. Overproduction of apolipoprotein B or reduced receptor-mediated clearance of lipoproteins leads to elevated serum cholesterol levels and premature atherosclerosis. ISIS 301012 is an antisense oligonucleotide that inhibits apolipoprotein B production by binding directly to and reducing the expression of apolipoprotein B messenger RNA. In a clinical trial, ISIS 301012 50-400 mg administered weekly via subcutaneous injection for 4 weeks reduced apolipoprotein B by 14.3-47.4% and low-density lipoprotein cholesterol by 5.9-40% at 55 days. The most frequent adverse event was injection-site erythema that resolved spontaneously. Studies are ongoing to further define the safety, efficacy, and pharmacokinetics of ISIS 301012 as add-on therapy in patients with heterozygous and homozygous familial hypercholesterolemia. No pharmacokinetic interactions have been demonstrated with ezetimibe and simvastatin. ISIS 301012 is the first agent to enter clinical trials utilizing an antisense mechanism for reducing the production of apolipoprotein B. Further studies are needed to verify its safety, efficacy, and position of therapy in the dyslipidemic patient.

The 5′-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB

PubMed Central

López-Aguilar, Celeste; Romero-López, Cristina; Espinosa, Manuel; Berzal-Herranz, Alfredo; del Solar, Gloria

2015-01-01

Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5′-tail and an 8-nt-long 3′-terminal U-rich stretch. Here, the 3′ and 5′ regions of the 5′-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5′-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a “kissing” interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex. PMID:26175752

Upregulation of endothelial receptor for oxidized LDL (LOX-1) by oxidized LDL and implications in apoptosis of human coronary artery endothelial cells: evidence from use of antisense LOX-1 mRNA and chemical inhibitors.

PubMed

Li, D; Mehta, J L

2000-04-01

A specific lectin-like endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL-induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 microg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL-mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL-mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL-mediated apoptosis of HCAECs. Nuclear factor (NF)-kappaB was markedly activated in ox-LDL-treated HCAECs. The critical role of NF-kappaB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL-mediated NF-kappaB activation. In this process, an NF-kappaB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL-mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL-induced apoptosis is mediated by the action of LOX-1. In this process, NF-kappaB activation may play an important role as a signal transduction mechanism.

Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

PubMed

Rutten, Julie W; Dauwerse, Hans G; Peters, Dorien J M; Goldfarb, Andrew; Venselaar, Hanka; Haffner, Christof; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M; Lesnik Oberstein, Saskia A J

2016-04-01

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations. Transfection of these antisense oligonucleotides into CADASIL patient-derived cerebral vascular smooth muscle cells resulted in successful exon skipping, without abrogating NOTCH3 signalling. Combined, these data provide proof of concept for this novel application of exon skipping, and are a first step towards the development of a rational therapeutic approach applicable to up to 94% of CADASIL-causing mutations. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Selective-Reagent-Ionization Mass Spectrometry: New Prospects for Atmospheric Research

NASA Astrophysics Data System (ADS)

Sulzer, Philipp; Jordan, Alfons; Hartungen, Eugen; Hanel, Gernot; Jürschik, Simone; Herbig, Jens; Märk, Lukas; Märk, Tilmann D.

2014-05-01

Proton-Transfer-Reaction Mass Spectrometry (PTR-MS), which was introduced to the scientific community in the 1990's, has quickly evolved into a well-established technology for atmospheric research and environmental chemistry [1]. Advantages of PTR-MS are i) high sensitivities of several hundred cps/ppbv, ii) detection limits at or below the pptv level, iii) direct injection sampling (i.e. no sample preparation), iv) response times in the 100 ms regime and v) online quantification. However, one drawback is a somehow limited selectivity, as in case of quadrupole mass filter based instruments only information about nominal m/z are available. In Time-Of-Flight (TOF) mass analyzer based instruments selectivity is drastically increased by a high mass resolution of up to 8000 m/Δm, but e.g. isomers still cannot be separated. In 2009 we introduced an advanced version of PTR-MS, which permits switching the reagent ions from H3O+ to NO+ and O2+, respectively [2]. This novel type of instrumentation was called Selective-Reagent-Ionization Mass Spectrometry (SRI-MS) and has been successfully used to separate isomers, e.g. the biogenic compounds isoprene and 2-methyl-3-buten-2-ol as shown by Karl et al. [3]. Switching the reagent ions dramatically increases selectivity and thus applicability of SRI-MS in atmospheric research. Here we report on the latest results utilizing an even more advanced embodiment of SRI-MS enabling the use of the additional reagent ions Kr+ and Xe+ [4]. With this technology important atmospheric compounds, such as CO2, CO, CH4, O2, etc. can be quantified and selectivity is increased even further. We present comparison data between diesel and gasoline car exhaust gases and quantitative data on indoor air for these compounds, which are not detectable with classical PTR-MS. Additionally, we show very recent examples of isomers which cannot be separated with PTR-MS but can clearly be distinguished with SRI-MS. Finally, we give an overview of ongoing SRI-MS developments, which include TOF based instruments with increased sensitivity of one order of magnitude (i.e. in the 103 cps/ppbv regime) by means of using a quadrupole ion guide between the drift tube and the TOF analyzer. It is expected that these developments will have a serious impact in atmospheric research, because increased sensitivity implies reduced measurement times and thus, e.g. even more accurate flux measurements. References [1] J. de Gouw, C. Warneke, T. Karl, G. Eerdekens, C. van der Veen, R. Fall, Mass Spectrometry Reviews, 26 (2007), 223-257. [2] A. Jordan, S. Haidacher, G. Hanel, E. Hartungen, J. Herbig, L. Märk, R. Schottkowsky, H. Seehauser, P. Sulzer, T.D. Märk, International Journal of Mass Spectrometry, 286 (2009), 32 - 38. [3] T. Karl, A. Hansel, L. Cappellin, L. Kaser, I. Herdlinger-Blatt, W. Jud, Atmospheric Chemistry and Physics, 12/24 (2012), 11877-11884. [4] P. Sulzer, A. Edtbauer, E. Hartungen, S. Juerschik, A. Jordan, G. Hanel, S. Feil, S. Jaksch, L. Märk, T.D. Märk, International Journal of Mass Spectrometry, 321 (2012), 66-70. Acknowledgement We acknowledge financial support by the Austrian Research Promotion Agency (FFG), Wien.

Whole Blood Cell Staining Device

NASA Technical Reports Server (NTRS)

Sams, Clarence F.; Clift, Vaughan L.; McDonald, Kelly E.

2000-01-01

An apparatus and method for staining particular cell markers is disclosed. The apparatus includes a flexible tube that is reversibly pinched into compartments with one or more clamps. Each compartment of the tube contains a separate reagent and is in selective fluid communication with adjoining compartments.

Organic Chemical Attribution Signatures for the Sourcing of a Mustard Agent and Its Starting Materials

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

Fraga, Carlos G.; Bronk, Krys; Dockendorff, Brian P.

Chemical attribution signatures (CAS) are being investigated for the sourcing of chemical warfare (CW) agents and their starting materials that may be implicated in chemical attacks or CW proliferation. The work reported here demonstrates for the first time trace impurities produced during the synthesis of tris(2-chloroethyl)amine (HN3) that point to specific reagent stocks used in the synthesis of this CW agent. Thirty batches of HN3 were synthesized using different combinations of commercial stocks of triethanolamine (TEA), thionyl chloride, chloroform, and acetone. The HN3 batches and reagent stocks were then analyzed for impurities by gas chromatography/mass spectrometry. Reaction-produced impurities indicative ofmore » specific TEA and chloroform stocks were exclusively discovered in HN3 batches made with those reagent stocks. In addition, some reagent impurities were found in the HN3 batches that were presumably not altered during synthesis and believed to be indicative of reagent type regardless of stock. Supervised classification using partial least squares discriminant analysis (PLSDA) on the impurity profiles of chloroform samples from seven stocks resulted in an average classification error by cross-validation of 2.4%. A classification error of zero was obtained using the seven-stock PLSDA model on a validation set of samples from an arbitrarily selected chloroform stock. In a separate analysis, all samples from two of seven chloroform stocks that were purposely not modeled had their samples matched to a chloroform stock rather than assigned a “no class” classification.« less

Sensible use of antisense: how to use oligonucleotides as research tools.

PubMed

Myers, K J; Dean, N M

2000-01-01

In the past decade, there has been a vast increase in the amount of gene sequence information that has the potential to revolutionize the way diseases are both categorized and treated. Old diagnoses, largely anatomical or descriptive in nature, are likely to be superceded by the molecular characterization of the disease. The recognition that certain genes drive key disease processes will also enable the rational design of gene-specific therapeutics. Antisense oligonucleotides represent a technology that should play multiple roles in this process.

VRP09 Reduction of Corneal Scarring Following Blast and Burn Injuries to Cornea Using siRNAs Targeting TGFb and CTGF

DTIC Science & Technology

2013-03-01

oligonucleotide-based drug approaches (better than ribozymes, antisense oligonucleotides ( ASO ), or microRNAs). (4) To accomplish these objectives, we...negative control scrambled ASO (designated NC). The combination of siRNAs T1 and R1 produced a knockdown of ~80% of TGFb1 protein in the conditioned...sequences (antisense oligonucleotides, ASOs ) into rabbit corneal cells and found that technique was very effective in delivering ASOs into the stroma and

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

PubMed

Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W; Clark, Chad R; Bharucha, Adil; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong-Heon

2017-12-21

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

The sensitivity of benzene cluster cation chemical ionization mass spectrometry to select biogenic terpenes

NASA Astrophysics Data System (ADS)

Lavi, Avi; Vermeuel, Michael P.; Novak, Gordon A.; Bertram, Timothy H.

2018-06-01

Benzene cluster cations are a sensitive and selective reagent ion for chemical ionization of select biogenic volatile organic compounds. We have previously reported the sensitivity of a field deployable chemical ionization time-of-flight mass spectrometer (CI-ToFMS), using benzene cluster cation ion chemistry, for detection of dimethyl sulfide, isoprene and α-pinene. Here, we present laboratory measurements of the sensitivity of the same instrument to a series of terpenes, including isoprene, α-pinene, β-pinene, D-limonene, ocimene, β-myrcene, farnesene, α-humulene, β-caryophyllene, and isolongifolene at atmospherically relevant mixing ratios (< 100 pptv). In addition, we determine the dependence of CI-ToFMS sensitivity on the reagent ion neutral delivery concentration and water vapor concentration. We show that isoprene is primarily detected as an adduct (C5H8 ṡ C6H6+) with a sensitivity ranging between 4 and 10 ncps ppt-1, which depends strongly on the reagent ion precursor concentration, de-clustering voltages, and specific humidity (SH). Monoterpenes are detected primarily as the molecular ion (C10H16+) with an average sensitivity, across the five measured compounds, of 14 ± 3 ncps ppt-1 for SH between 7 and 14 g kg-1, typical of the boreal forest during summer. Sesquiterpenes are detected primarily as the molecular ion (C15H24+) with an average sensitivity, across the four measured compounds, of 9.6 ± 2.3 ncps ppt-1, that is also independent of specific humidity. Comparable sensitivities across broad classes of terpenes (e.g., monoterpenes and sesquiterpenes), coupled to the limited dependence on specific humidity, suggest that benzene cluster cation CI-ToFMS is suitable for field studies of biosphere-atmosphere interactions.

Strategies for In Vivo Screening and Mitigation of Hepatotoxicity Associated with Antisense Drugs.

PubMed

Kamola, Piotr J; Maratou, Klio; Wilson, Paul A; Rush, Kay; Mullaney, Tanya; McKevitt, Tom; Evans, Paula; Ridings, Jim; Chowdhury, Probash; Roulois, Aude; Fairchild, Ann; McCawley, Sean; Cartwright, Karen; Gooderham, Nigel J; Gant, Timothy W; Moores, Kitty; Hughes, Stephen A; Edbrooke, Mark R; Clark, Kenneth; Parry, Joel D

2017-09-15

Antisense oligonucleotide (ASO) gapmers downregulate gene expression by inducing enzyme-dependent degradation of targeted RNA and represent a promising therapeutic platform for addressing previously undruggable genes. Unfortunately, their therapeutic application, particularly that of the more potent chemistries (e.g., locked-nucleic-acid-containing gapmers), has been hampered by their frequent hepatoxicity, which could be driven by hybridization-mediated interactions. An early de-risking of this liability is a crucial component of developing safe, ASO-based drugs. To rank ASOs based on their effect on the liver, we have developed an acute screen in the mouse that can be applied early in the drug development cycle. A single-dose (3-day) screen with streamlined endpoints (i.e., plasma transaminase levels and liver weights) was observed to be predictive of ASO hepatotoxicity ranking established based on a repeat-dose (15 day) study. Furthermore, to study the underlying mechanisms of liver toxicity, we applied transcriptome profiling and pathway analyses and show that adverse in vivo liver phenotypes correlate with the number of potent, hybridization-mediated off-target effects (OTEs). We propose that a combination of in silico OTE predictions, streamlined in vivo hepatotoxicity screening, and a transcriptome-wide selectivity screen is a valid approach to identifying and progressing safer compounds. Copyright © 2017 GSK R&D. Published by Elsevier Inc. All rights reserved.

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

PubMed Central

Burel, Sebastien A.; Hart, Christopher E.; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M.; Hung, Gene; Dan, Amy; Prakash, T.P.; Seth, Punit P.; Swayze, Eric E.; Bennett, C. Frank; Crooke, Stanley T.; Henry, Scott P.

2016-01-01

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

PubMed

Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

1990-03-01

The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

Multidimensional data analysis in immunophenotyping.

PubMed

Loken, M R

2001-05-01

The complexity of cell populations requires careful selection of reagents to detect cells of interest and distinguish them from other types. Additional reagents are frequently used to provide independent criteria for cell identification. Two or three monoclonal antibodies in combination with forward and right-angle light scatter generate a data set that is difficult to visualize because the data must be represented in four- or five-dimensional space. The separation between cell populations provided by the multiple characteristics is best visualized by multidimensional analysis using all parameters simultaneously to identify populations within the resulting hyperspace. Groups of cells are distinguished based on a combination of characteristics not apparent in any usual two-dimensional representation of the data.

Progress and developments in the turbo Grignard reagent i-PrMgCl·LiCl: a ten-year journey.

PubMed

Bao, Robert Li-Yuan; Zhao, Rong; Shi, Lei

2015-04-25

Over the past decade, the effectiveness of i-PrMgCl·LiCl has been constantly highlighted by a number of research groups. Its enhanced nucleophilicity brings prosperity to highly functionalized Grignard reagents, other useful bimetallic (alkali-metal) agents and nucleophilic alkylation products under mild reaction conditions. In this feature article, a comprehensive, systematical and in-depth overview of i-PrMgCl·LiCl is provided in a multidisciplinary idea. It involves the structural and kinetic perspectives of i-PrMgCl·LiCl as well as its unique reactivity and selectivity, with knowledge of the former helping to rationalize trends of the later.

System and Method for Isolation of Samples

NASA Technical Reports Server (NTRS)

Zhang, Ye (Inventor); Wu, Honglu (Inventor)

2014-01-01

Systems and methods for isolating samples are provided. The system comprises a first membrane and a second membrane disposed within an enclosure. First and second reservoirs can also be disposed within the enclosure and adapted to contain one or more reagents therein. A first valve can be disposed within the enclosure and in fluid communication with the first reservoir, the second reservoir, or both. The first valve can also be in fluid communication with the first or second membranes or both. The first valve can be adapted to selectively regulate the flow of the reagents from the first reservoir, through at least one of the first and second membranes, and into the second reservoir.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry.

PubMed

Ornatsky, Olga I; Kinach, Robert; Bandura, Dmitry R; Lou, Xudong; Tanner, Scott D; Baranov, Vladimir I; Nitz, Mark; Winnik, Mitchell A

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.

Using microRNA as an alternative treatment for hyperlipidemia and cardiovascular disease: cardio-miRs in the pipeline.

PubMed

Hennessy, Elizabeth J; Moore, Kathryn J

2013-09-01

It is now appreciated that over 90% of the human genome is comprised of noncoding RNAs that have the ability to affect other components of the genome and regulate gene expression. This has galvanized the development of RNA-based therapeutics for a myriad of diseases, including cancer, inflammatory conditions, and cardiovascular disease. Several classes of RNA therapeutics are currently under clinical development, including antisense oligonucleotides, small interfering RNA, and microRNA mimetics and inhibitors. The field of antisense technology saw a huge leap forward with the recent Food and Drug Administration approval of the first antisense therapy, directed against apolipoprotein B, for the treatment of familial hypercholesterolemia. In addition, recent progress in the development of approaches to inhibit microRNAs has helped to illuminate their roles in repressing gene networks and also revealed their potential as therapeutic targets. In this review, these exciting opportunities in the field of drug discovery, with a focus on emerging therapeutics in the field of cardiovascular disease, are summarized.

Orlistat and antisense-miRNA-loaded PLGA-PEG nanoparticles for enhanced triple negative breast cancer therapy

PubMed Central

Bhargava-Shah, Aarohi; Foygel, Kira; Devulapally, Rammohan; Paulmurugan, Ramasamy

2016-01-01

Background: This study explores the use of hydrophilic poly(ethylene glycol)-conjugated poly(lactic-co-glycolic acid) nanoparticles (PLGA-PEG-NPs) as delivery system to improve the antitumor effect of antiobesity drug orlistat for triple-negative breast cancer (TNBC) therapy by improving its bioavailability. Materials & methods: PLGA-PEG-NPs were synthesized by emulsion-diffusion-evaporation method, and the experiments were conducted in vitro in MDA-MB-231 and SKBr3 TNBC and normal breast fibroblast cells. Results: Delivery of orlistat via PLGA-PEG-NPs reduced its IC50 compared with free orlistat. Combined treatment of orlistat-loaded NPs and doxorubicin or antisense-miR-21-loaded NPs significantly enhanced apoptotic effect compared with independent doxorubicin, anti-miR-21-loaded NPs, orlistat-loaded NPs or free orlistat treatments. Conclusion: We demonstrate that orlistat in combination with antisense-miR-21 or current chemotherapy holds great promise as a novel and versatile treatment agent for TNBC. PMID:26787319

Targeting of Repeated Sequences Unique to a Gene Results in Significant Increases in Antisense Oligonucleotide Potency

PubMed Central

Vickers, Timothy A.; Freier, Susan M.; Bui, Huynh-Hoa; Watt, Andrew; Crooke, Stanley T.

2014-01-01

A new strategy for identifying potent RNase H-dependent antisense oligonucleotides (ASOs) is presented. Our analysis of the human transcriptome revealed that a significant proportion of genes contain unique repeated sequences of 16 or more nucleotides in length. Activities of ASOs targeting these repeated sites in several representative genes were compared to those of ASOs targeting unique single sites in the same transcript. Antisense activity at repeated sites was also evaluated in a highly controlled minigene system. Targeting both native and minigene repeat sites resulted in significant increases in potency as compared to targeting of non-repeated sites. The increased potency at these sites is a result of increased frequency of ASO/RNA interactions which, in turn, increases the probability of a productive interaction between the ASO/RNA heteroduplex and human RNase H1 in the cell. These results suggest a new, highly efficient strategy for rapid identification of highly potent ASOs. PMID:25334092

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules

PubMed Central

Gifford, Lida K.; Opalinska, Joanna B.; Jordan, David; Pattanayak, Vikram; Greenham, Paul; Kalota, Anna; Robbins, Michelle; Vernovsky, Kathy; Rodriguez, Lesbeth C.; Do, Bao T.; Lu, Ponzy; Gewirtz, Alan M.

2005-01-01

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20–30 base oligodeoxynucleotides with 5–6 bp complementary ends to which a 5′ fluorophore and 3′ quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem–loop of the SQRM suggests that SQRM be made to target natural stem–loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells. PMID:15718294

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

DOE PAGES

Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.; ...

2016-09-20

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less

Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

PubMed

Kawano, Mitsuoki

2012-12-01

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

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

Hawkes, Emily J.; Hennelly, Scott P.; Novikova, Irina V.

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. Here, we investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probingmore » and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.« less

Fluorescence Characterization of Gold Modified Liposomes with Antisense N-myc DNA Bound to the Magnetisable Particles with Encapsulated Anticancer Drugs (Doxorubicin, Ellipticine and Etoposide).

PubMed

Skalickova, Sylvie; Nejdl, Lukas; Kudr, Jiri; Ruttkay-Nedecky, Branislav; Jimenez, Ana Maria Jimenez; Kopel, Pavel; Kremplova, Monika; Masarik, Michal; Stiborova, Marie; Eckschlager, Tomas; Adam, Vojtech; Kizek, Rene

2016-02-25

Liposome-based drug delivery systems hold great potential for cancer therapy. The aim of this study was to design a nanodevice for targeted anchoring of liposomes (with and without cholesterol) with encapsulated anticancer drugs and antisense N-myc gene oligonucleotide attached to its surface. To meet this main aim, liposomes with encapsulated doxorubicin, ellipticine and etoposide were prepared. They were further characterized by measuring their fluorescence intensity, whereas the encapsulation efficiency was estimated to be 16%. The hybridization process of individual oligonucleotides forming the nanoconstruct was investigated spectrophotometrically and electrochemically. The concentrations of ellipticine, doxorubicin and etoposide attached to the nanoconstruct in gold nanoparticle-modified liposomes were found to be 14, 5 and 2 µg·mL(-1), respectively. The study succeeded in demonstrating that liposomes are suitable for the transport of anticancer drugs and the antisense oligonucleotide, which can block the expression of the N-myc gene.

The Role of Mesopontine NGF in Sleep and Wakefulness

PubMed Central

Ramos, Oscar V.; Torterolo, Pablo; Lim, Vincent; Chase, Michael H.; Sampogna, Sharon; Yamuy, Jack

2011-01-01

The microinjection of nerve growth factor (NGF) into the cat pontine tegmentum rapidly induces rapid eye movement (REM) sleep. To determine if NGF is involved in naturally-occurring REM sleep, we examined whether it is present in mesopontine cholinergic structures that promote the initiation of REM sleep, and whether the blockade of NGF production in these structures suppresses REM sleep. We found that cholinergic neurons in the cat dorsolateral mesopontine tegmentum exhibited NGF-like immunoreactivity. In addition, the microinjection of an oligodeoxyribonucleotide (OD) directed against cat NGF mRNA into this region resulted in a reduction in the time spent in REM sleep in conjunction with an increase in the time spent in wakefulness. Sleep and wakefulness returned to baseline conditions 2 to 5 days after antisense OD administration. The preceding antisense OD-induced effects occurred in conjunction with the suppression of NGF-like immunoreactivity within the site of antisense OD injection. These data support the hypothesis that NGF is involved in the modulation of naturally-occurring sleep and wakefulness. PMID:21840513

Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

NASA Astrophysics Data System (ADS)

Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

2014-02-01

Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice.

PubMed

Graham, Mark J; Lemonidis, Kristina M; Whipple, Charles P; Subramaniam, Amuthakannan; Monia, Brett P; Crooke, Stanley T; Crooke, Rosanne M

2007-04-01

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.

Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B

PubMed Central

Crooke, Stanley T; Geary, Richard S

2013-01-01

Mipomersen is a second generation antisense oligonucleotide that targets apolipoprotein B. It has been studied thoroughly in clinical trials (more than 800 subjects), including four randomized double-blind placebo controlled phase 3 studies involving 391 patients, and is in registration for the treatment of severe hypercholesterolaemia. The pharmacokinetic and pharmacodynamic properties of mipomersen are well characterized. Mipomersen is rapidly and extensively absorbed after subcutaneous administration and has an elimination half-life of approximately 30 days across species. It is cleared by nuclease metabolism and renal excretion of the metabolites. Mipomersen reduces all apolipoprotein B containing atherogenic particles and displays dose dependent reductions between 50–400 mg week−1, both as a single agent and in the presence of maximal lipid lowering therapy. No drug–drug interactions have been identified. Mipomersen is a representative of second generation antisense drugs, all of which have similar properties, and is thus representative of the behaviour of the class of drugs. PMID:23013161

Investigation of Reagent Gases for the Positive Chemical Ionization of Select Polybrominated Diphenyl Ethers

EPA Science Inventory

Polybrominated diphenyl ethers (PBDEs) fall into the class of compounds known as brominated flame retardants and their incorporation in a multitude of products is responsible for saving numerous lives. However, toxicology studies have alerted researchers to the potential adverse...

Late-stage chemoselective functional-group manipulation of bioactive natural products with super-electrophilic silylium ions

NASA Astrophysics Data System (ADS)

Bender, Trandon A.; Payne, Philippa R.; Gagné, Michel R.

2018-01-01

The selective (and controllable) modification of complex molecules with disparate functional groups (for example, natural products) is a long-standing challenge that has been addressed using catalysts tuned to perform singular transformations (for example, C-H hydroxylation). A method whereby reactions with diverse functional groups within a single natural product are feasible depending on which catalyst or reagent is chosen would widen the possible structures one could obtain. Fluoroarylborane catalysts can heterolytically split Si-H bonds to yield an oxophilic silylium (R3Si+) equivalent along with a reducing (H-) equivalent. Together, these reactive intermediates enable the reduction of multiple functional groups. Exogenous phosphine Lewis bases further modify the catalyst speciation and attenuate aggressive silylium ions for the selective modification of complex natural products. Manipulation of the catalyst, silane reagent and the reaction conditions provides experimental control over which site is modified (and how). Applying this catalytic method to complex bioactive compounds (natural products or drugs) provides a powerful tool for studying structure-activity relationships.

Nonsymmetrical 3,4-dithienylmaleimides by cross-coupling reactions with indium organometallics: synthesis and photochemical studies.

PubMed

Mosquera, Angeles; Férnandez, M Isabel; Canle Lopez, Moisés; Pérez Sestelo, José; Sarandeses, Luis A

2014-10-27

The synthesis and photochemical study of novel nonsymmetrical 1,2-dithienylethenes (DTEs) with a maleimide bridge have been carried out. The synthetic approach to the DTEs was based on successive selective palladium-catalyzed cross-coupling reactions of 5-susbtituted-2-methyl-3-thiophenyl indium reagents with 3,4-dichloromaleimides. The required organoindium reagents were prepared from 2-methyl-3,5-dibromothiophene by a selective (C-5) coupling reaction with triorganoindium compounds (R3 In) and subsequent metal-halogen exchange. The coupling reactions usually gave good yields and have a high atom economy with substoichiometric amounts of R3 In. The results of photochemical studies show that these novel dithienylmaleimides undergo a photocyclization reaction upon irradiation in the UV region and a photocycloreversion after excitation in the visible region, thus they can be used as photochemical switches. ON-OFF operations can be repeated in successive cycles without appreciable loss of effectiveness in the process. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions

PubMed Central

Depke, Maren; Pané-Farré, Jan; Debarbouille, Michel; van der Kooi-Pol, Magdalena M.; Guérin, Cyprien; Dérozier, Sandra; Hiron, Aurelia; Jarmer, Hanne; Leduc, Aurélie; Michalik, Stephan; Reilman, Ewoud; Schaffer, Marc; Schmidt, Frank; Bessières, Philippe; Noirot, Philippe; Hecker, Michael; Msadek, Tarek; Völker, Uwe; van Dijl, Jan Maarten

2016-01-01

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria. PMID:27035918

Engineering the expression level of cytosolic nucleoside diphosphate kinase in transgenic Solanum tuberosum roots alters growth, respiration and carbon metabolism.

PubMed

Dorion, Sonia; Clendenning, Audrey; Rivoal, Jean

2017-03-01

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphate from a donor nucleoside triphosphate to an acceptor nucleoside diphosphate. In this study we used a targeted metabolomic approach and measurement of physiological parameters to report the effects of the genetic manipulation of cytosolic NDPK (NDPK1) expression on physiology and carbon metabolism in potato (Solanum tuberosum) roots. Sense and antisense NDPK1 constructs were introduced in potato using Agrobacterium rhizogenes to generate a population of root clones displaying a 40-fold difference in NDPK activity. Root growth, O 2 uptake, flux of carbon between sucrose and CO 2 , levels of reactive oxygen species and some tricarboxylic acid cycle intermediates were positively correlated with levels of NDPK1 expression. In addition, NDPK1 levels positively affected UDP-glucose and cellulose contents. The activation state of ADP-glucose pyrophosphorylase, a key enzyme in starch synthesis, was higher in antisense roots than in roots overexpressing NDPK1. Further analyses demonstrated that ADP-glucose pyrophosphorylase was more oxidized, and therefore less active, in sense clones than antisense clones. Consequently, antisense NDPK1 roots accumulated more starch and the starch to cellulose ratio was negatively affected by the level of NDPK1. These data support the idea that modulation of NDPK1 affects the distribution of carbon between starch and cellulose biosynthetic pathways. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

BcMF11, a novel non-coding RNA gene from Brassica campestris, is required for pollen development and male fertility.

PubMed

Song, Jiang-Hua; Cao, Jia-Shu; Wang, Cheng-Gang

2013-01-01

KEY MESSAGE : BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology. We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes' extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility.

Adenovirus-mediated transfer of HPV 16 E6/E7 antisense RNA combined with cisplatin inhibits cellular growth and induces apoptosis in HPV-positive head and neck cancer cells.

PubMed

Kojima, Yasutaka; Otsuki, Naoki; Kubo, Mie; Kitamoto, Junko; Takata, Eri; Saito, Hiroki; Kosaka, Kyoko; Morishita, Naoya; Uehara, Natsumi; Shirakawa, Toshiro; Nibu, Ken-Ich

2018-05-24

Human papillomavirus (HPV) infection has been identified as an etiologic factor of head and neck cancers (HNCs). We explored the potential use of antisense HPV RNA transcripts for gene therapy and its effect in combination with cisplatin (CDDP) for HPV-positive HNCs. We introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into UM-SCC-47 cells harboring HPV 16 and YCU-T892 cells that were HPV-negative using a recombinant adenoviral vector, Ad-E6/E7-AS. We then analyzed the effects of the introduction of Ad-E7-AS on cell and tumor growth and the synergistic effect with CDDP in vitro and in vivo. After infection of Ad-E6/E7-AS, the cellular growth of UM-SCC-47 cells were suppressed, but not that of YCU-T892 cells. E7 protein expression was suppressed, and p53 and pRb protein expression increased after infection of Ad-E7-AS. Cell growth and tumorigenicity were greatly suppressed in combination with CDDP compared with Ad-E7-AS or CDDP treatment alone in vitro. Ad-E7-AS combined with CDDP treatment significantly reduced the volumes of established subcutaneous tumors. Transfection with HPV 16 E7 antisense RNA combined with CDDP treatment might be a potentially useful approach to the therapy of HPV 16-positive HNC.

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy

PubMed Central

Singh, Natalia N.; Howell, Matthew D.; Androphy, Elliot J.; Singh, Ravindra N.

2017-01-01

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 due to predominant skipping of exon 7 during pre-mRNA splicing. With the recent FDA approval of nusinersen (Spinraza™), the potential for correction of SMN2 exon 7 splicing as a SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions. PMID:28485722

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy.

PubMed

Singh, N N; Howell, M D; Androphy, E J; Singh, R N

2017-09-01

Spinal muscular atrophy (SMA), a prominent genetic disease of infant mortality, is caused by low levels of survival motor neuron (SMN) protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1 present in humans, cannot compensate for the loss of SMN1 because of predominant skipping of exon 7 during pre-mRNA splicing. With the recent US Food and Drug Administration approval of nusinersen (Spinraza), the potential for correction of SMN2 exon 7 splicing as an SMA therapy has been affirmed. Nusinersen is an antisense oligonucleotide that targets intronic splicing silencer N1 (ISS-N1) discovered in 2004 at the University of Massachusetts Medical School. ISS-N1 has emerged as the model target for testing the therapeutic efficacy of antisense oligonucleotides using different chemistries as well as different mouse models of SMA. Here, we provide a historical account of events that led to the discovery of ISS-N1 and describe the impact of independent validations that raised the profile of ISS-N1 as one of the most potent antisense targets for the treatment of a genetic disease. Recent approval of nusinersen provides a much-needed boost for antisense technology that is just beginning to realize its potential. Beyond treating SMA, the ISS-N1 target offers myriad potentials for perfecting various aspects of the nucleic-acid-based technology for the amelioration of the countless number of pathological conditions.

Dissecting the hybridization of oligonucleotides to structured complementary sequences.

PubMed

Peracchi, Alessio

2016-06-01

When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.

Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana.

PubMed

Nanjo, T; Kobayashi, M; Yoshiba, Y; Sanada, Y; Wada, K; Tsukaya, H; Kakubari, Y; Yamaguchi-Shinozaki, K; Shinozaki, K

1999-04-01

Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), in plants. To elucidate essential roles of Pro, we generated antisense transgenic Arabidopsis plants with a P5CS cDNA. Several transgenics accumulated Pro at a significantly lower level than wild-type plants, providing direct evidence for a key role of P5CS in Pro production in Arabidopsis. These antisense transgenics showed morphological alterations in leaves and a defect in elongation of inflorescences. Furthermore, transgenic leaves were hypersensitive to osmotic stress. Microscopic analysis of transgenic leaves, in which the mutated phenotype clearly occurred, showed morphological abnormalities of epidermal and parenchymatous cells and retardation of differentiation of vascular systems. These phenotypes were suppressed by exogenous L-Pro but not by D-Pro or other Pro analogues. In addition, Pro deficiency did not broadly affect all proteins but specifically affected structural proteins of cell walls in the antisense transgenic plants. These results indicate that Pro is not just an osmoregulator in stressed plants but has a unique function involved in osmotolerance as well as in morphogenesis as a major constituent of cell wall structural proteins in plants.

Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

PubMed

Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

2014-07-01

Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

PubMed Central

Ralph, John; Hatfield, Ronald D.; Piquemal, Joël; Yahiaoui, Nabila; Pean, Michel; Lapierre, Catherine; Boudet, Alain M.

1998-01-01

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies. PMID:9788995

Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells.

PubMed

Yasui, Takahiro; Fujita, Keiji; Asai, Kiyofumi; Kohri, Kenjiro

2002-02-01

The association of calcium crystals with renal tubular cells is an important factor during the formation of urinary stones. We previously reported the strong expression of osteopontin (OPN) on renal tubular cells in the stone-forming kidney, suggesting that OPN plays a role in the crystal-cell interaction. In the present study, we examined the biological consequences of inhibiting OPN expression at the translational level on the formation and adhesion of crystals. We synthesized antisense OPN expression vector (pTet-OPNas) using the tetracycline-regulated expression system. The pTet-OPNas was constructed using a mouse OPN cDNA sequence in an inverted (antisense) orientation. Two clones (NRK-52E/ASs) were identified by transfection of pTet-OPNas into NRK-52E cells and they showed a marked reduction of OPN synthesis in the absence of tetracycline. Calcium oxalate (CaOx) crystal suspension was spread homogeneously on top of the NRK-52E cells. After incubation, the association of CaOx crystals and cells was visualized by scanning electron microscopy. Intact NRK-52E cells, NRK-52E cells transfected with empty vector and tetracycline-treated antisense clones (NRK-52E/ASs), under identical conditions, were associated with CaOx crystals. In contrast, the expression of antisense OPN prevented the association of CaOx crystals with NRK-52E cells. Osteopontin plays a crucial role in the adhesion process of CaOx crystals to renal tubular cells in stone formation.

Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice.

PubMed

Yamamoto, Tsuyoshi; Harada-Shiba, Mariko; Nakatani, Moeka; Wada, Shunsuke; Yasuhara, Hidenori; Narukawa, Keisuke; Sasaki, Kiyomi; Shibata, Masa-Aki; Torigoe, Hidetaka; Yamaoka, Tetsuji; Imanishi, Takeshi; Obika, Satoshi

2012-05-15

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2',4'-BNA (also called as locked nucleic acid (LNA)) and 2',4'-BNA(NC) chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2',4'-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2',4'-BNA-AON, 2',4'-BNA(NC)-AON showed an earlier LDL-C-lowering effect and was more tolerable in mice. Our results validate the optimization of 2',4'-BNA(NC)-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes

PubMed Central

Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

2015-01-01

Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3′ maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. PMID:26209135

The MORPHEUS II protein crystallization screen

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

Gorrec, Fabrice, E-mail: fgorrec@mrc-lmb.cam.ac.uk

2015-06-27

MORPHEUS II is a 96-condition initial crystallization screen formulated de novo. The screen incorporates reagents selected from the Protein Data Bank to yield crystals that are not observed in traditional conditions. In addition, the formulation facilitates the optimization and cryoprotection of crystals. High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected frommore » the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.« less

Chemiluminescence evidence supporting the selective role of ligands in the permanganate oxidation of micropollutants.

PubMed

Roderick, Mark S; Adcock, Jacqui L; Terry, Jessica M; Smith, Zoe M; Parry, Samuel; Linton, Stuart M; Thornton, Megan T; Barrow, Colin J; Francis, Paul S

2013-10-10

The selective increase in the oxidation rate of certain organic compounds with permanganate in the presence of environmental "ligands" and reduced species has been ascribed to the different reactivity of the target compounds toward Mn(III), which bears striking similarities to recent independent investigations into the use of permanganate as a chemiluminescence reagent. In spite of the importance of Mn(III) in the light-producing pathway, the dependence of the oxidation mechanism for any given compound on this intermediate could not be determined solely through the emission intensity. However, target compounds susceptible to single-electron oxidation by Mn(III) (such as bisphenol A and triclosan) can be easily distinguished by the dramatic increase in chemiluminescence intensity when a permanganate reagent containing high, stable concentrations of Mn(III) is used. The differences are accentuated under the low pH conditions that favor the chemiluminescence emission due to the greater reactivity of Mn(III) and the greater influence of complexing agents. This study supports the previously postulated selective role of ligands and reducing agents in permanganate oxidations and demonstrates a new approach to explore the chemistry of environmental manganese redox processes.

The chemistry of TALSPEAK: A review of the science

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

Nash, Kenneth L.

Here, the TALSPEAK Process (Trivalent Actinide Lanthanide Separation with Phosphorus-reagent Extraction from Aqueous Komplexes) was originally developed at Oak Ridge National Laboratory by B. Weaver and F.A. Kappelmann in the 1960s. It was envisioned initially as an alternative to the TRAMEX process (selective extraction of trivalent actinides by tertiary or quaternary amines over fission product lanthanides from concentrated LiCl solutions). TALSPEAK proposed the selective extraction of trivalent lanthanides away from the actinides, which are retained in the aqueous phase as aminopolycarboxylate complexes. After several decades of research and development, the conventional TALSPEAK process (based on di-(2-ethylhexyl) phosphoric acid (extractant) inmore » 1,4-di-isopropylbenzene (diluent) and a concentrated lactate buffer containing diethylenetriamine-N,N,N',N",N"-pentaacetic acid (actinide-selective holdback reagent)) has become a widely recognized benchmark for advanced aqueous partitioning of the trivalent 4f/5f elements. TALSPEAK chemistry has also been utilized as an actinide-selective stripping agent (Reverse TALSPEAK) with some notable success. Under ideal conditions, conventional TALSPEAK separates Am 3+ from Nd 3+ (the usual limiting pair) with a single-stage separation factor of about 100; both lighter and heavier lanthanides are more completely separated from Am 3+. Despite this apparent efficiency, TALSPEAK has not seen enthusiastic adoption for advanced partitioning of nuclear fuels at process scale for two principle reasons: 1) all adaptations of TALSPEAK chemistry to process scale applications require rigid pH control within a narrow range of pH, and 2) phase transfer kinetics are often slower than ideal. To compensate for these effects, high concentrations of the buffer (0.5-2 M H/Na lactate) are required. Acknowledgement of these complications in TALSPEAK process development has inspired significant research activities dedicated to improving understanding of the basic chemistry that controls TALSPEAK (and related processes based on the application of actinide-selective holdback reagents). In the following report, advances in understanding of the fundamental chemistry of TALSPEAK that have occurred during the past decade will be reviewed and discussed.« less

The chemistry of TALSPEAK: A review of the science

DOE PAGES

Nash, Kenneth L.

2014-11-13

Here, the TALSPEAK Process (Trivalent Actinide Lanthanide Separation with Phosphorus-reagent Extraction from Aqueous Komplexes) was originally developed at Oak Ridge National Laboratory by B. Weaver and F.A. Kappelmann in the 1960s. It was envisioned initially as an alternative to the TRAMEX process (selective extraction of trivalent actinides by tertiary or quaternary amines over fission product lanthanides from concentrated LiCl solutions). TALSPEAK proposed the selective extraction of trivalent lanthanides away from the actinides, which are retained in the aqueous phase as aminopolycarboxylate complexes. After several decades of research and development, the conventional TALSPEAK process (based on di-(2-ethylhexyl) phosphoric acid (extractant) inmore » 1,4-di-isopropylbenzene (diluent) and a concentrated lactate buffer containing diethylenetriamine-N,N,N',N",N"-pentaacetic acid (actinide-selective holdback reagent)) has become a widely recognized benchmark for advanced aqueous partitioning of the trivalent 4f/5f elements. TALSPEAK chemistry has also been utilized as an actinide-selective stripping agent (Reverse TALSPEAK) with some notable success. Under ideal conditions, conventional TALSPEAK separates Am 3+ from Nd 3+ (the usual limiting pair) with a single-stage separation factor of about 100; both lighter and heavier lanthanides are more completely separated from Am 3+. Despite this apparent efficiency, TALSPEAK has not seen enthusiastic adoption for advanced partitioning of nuclear fuels at process scale for two principle reasons: 1) all adaptations of TALSPEAK chemistry to process scale applications require rigid pH control within a narrow range of pH, and 2) phase transfer kinetics are often slower than ideal. To compensate for these effects, high concentrations of the buffer (0.5-2 M H/Na lactate) are required. Acknowledgement of these complications in TALSPEAK process development has inspired significant research activities dedicated to improving understanding of the basic chemistry that controls TALSPEAK (and related processes based on the application of actinide-selective holdback reagents). In the following report, advances in understanding of the fundamental chemistry of TALSPEAK that have occurred during the past decade will be reviewed and discussed.« less

Chemical analysis kit for the presence of explosives

DOEpatents

Eckels, Joel Del [Livermore, CA; Nunes,; Peter, J [Danville, CA; Alcaraz, Armando [Livermore, CA; Whipple, Richard E [Livermore, CA

2011-05-10

A tester for testing for explosives associated with a test location comprising a first explosives detecting reagent; a first reagent holder, the first reagent holder containing the first explosives detecting reagent; a second explosives detecting reagent; a second reagent holder, the second reagent holder containing the second explosives detecting reagent; a sample collection unit for exposure to the test location, exposure to the first explosives detecting reagent, and exposure to the second explosives detecting reagent; and a body unit containing a heater for heating the sample collection unit for testing the test location for the explosives.

Cell type-specific termination of transcription by transposable element sequences.

PubMed

Conley, Andrew B; Jordan, I King

2012-09-30

Transposable elements (TEs) encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS) genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3' UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are evolutionarily young. The extent of transcription termination by TEs seen here, along with the preference for sense-oriented TE insertions to provide TTS, is consistent with the observed antisense orientation bias of human TEs.

External Guide Sequences Targeting the aac(6′)-Ib mRNA Induce Inhibition of Amikacin Resistance▿

PubMed Central

Bistué, Alfonso J. C. Soler; Ha, Hongphuc; Sarno, Renee; Don, Michelle; Zorreguieta, Angeles; Tolmasky, Marcelo E.

2007-01-01

The dissemination of AAC(6′)-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6′)-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6′)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6′)-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 μg of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 μg of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin. PMID:17387154

Antisense oligonucleotide-mediated correction of transcriptional dysregulation is correlated with behavioral benefits in the YAC128 mouse model of Huntington's disease.

PubMed

Stanek, Lisa M; Yang, Wendy; Angus, Stuart; Sardi, Pablo S; Hayden, Michael R; Hung, Gene H; Bennett, C Frank; Cheng, Seng H; Shihabuddin, Lamya S

2013-01-01

Huntington's disease (HD) is a neurological disorder caused by mutations in the huntingtin (HTT) gene, the product of which leads to selective and progressive neuronal cell death in the striatum and cortex. Transcriptional dysregulation has emerged as a core pathologic feature in the CNS of human and animal models of HD. It is still unclear whether perturbations in gene expression are a consequence of the disease or importantly, contribute to the pathogenesis of HD. To examine if transcriptional dysregulation can be ameliorated with antisense oligonucleotides that reduce levels of mutant Htt and provide therapeutic benefit in the YAC128 mouse model of HD. Quantitative real-time PCR analysis was used to evaluate dysregulation of a subset of striatal genes in the YAC128 mouse model. Transcripts were then evaluated following ICV delivery of antisense oligonucleotides (ASO). Rota rod and Porsolt swim tests were used to evaluate phenotypic deficits in these mice following ASO treatment. Transcriptional dysregulation was detected in the YAC128 mouse model and appears to progress with age. ICV delivery of ASOs directed against mutant Htt resulted in reduction in mutant Htt levels and amelioration in behavioral deficits in the YAC128 mouse model. These improvements were correlated with improvements in the levels of several dysregulated striatal transcripts. The role of transcriptional dysregulation in the pathogenesis of Huntington's disease is not well understood, however, a wealth of evidence now strongly suggests that changes in transcriptional signatures are a prominent feature in the brains of both HD patients and animal models of the disease. Our study is the first to show that a therapeutic agent capable of improving an HD disease phenotype is concomitantly correlated with normalization of a subset of dysregulated striatal transcripts. Our data suggests that correction of these disease-altered transcripts may underlie, at least in part, the therapeutic efficacy shown associated with ASO-mediated correction of HD phenotypes and may provide a novel set of early biomarkers for evaluating future therapeutic concepts for HD.

Inhibition of Ophiognomonia clavigignenti-juglandacearum by Juglans species bark extracts

Treesearch

M.E. Ostry; M. Moore

2013-01-01

A rapid and reliable screening technique is needed for selecting trees with resistance to butternut canker. In a laboratory assay, reagent grade naphthoquinones and crude bark extracts of Juglans species variously inhibited spore germination and growth of Ophiognomonia clavigignenti-juglandacearum, the causal fungus of butternut...

Technology Performance Review: Selecting And Using Solidification/Stabilization Treatment For Site Remediation

EPA Science Inventory

Solidification/Stabilization (S/S) is a widely used treatment technology to prevent migration and exposure of contaminants from a contaminated media (i.e., soil, sludge and sediment). Solidification refers to a process that binds a contaminated media with a reagent changing its ...

Investigations on blood coagulation in the green iguana (Iguana iguana).

PubMed

Kubalek, S; Mischke, R; Fehr, M

2002-05-01

The prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, kaolin clotting time (KCT), dilute Russell's viper venom time (DRVVT) and reptilase time, as well as five different plasma fibrinogen assays [gravimetry, Jacobsson method (extinction at 280 nm), Millar method (heat precipitation), kinetic turbidometry, Clauss method] and resonance thrombography were performed in 26 clinically healthy green iguanas. All assays were carried out in comparison with pooled normal canine plasma. In iguana plasma, the PT [median (x0.50) = 453-831 s, dependent on the reagent], APTT (x0.50 = 170-242 s, dependent on the reagent), thrombin time (x0.50 = 118 - > 1000 s, dependent on thrombin activity), KCT (x0.50 = 274 s), DRVVT (x0.50 = 349 s) and reptilase time (all samples > 1000 s) were widely scattered at the limit of measurability. Only fibrinogen concentrations measured using the Jacobsson method (x0.50 = 4.40 g/l) correlated well (r = 0.91) with gravimetry (x0.50 = 4.22 g/l). The results of this study indicate a limited suitability and a confined diagnostic significance of the selected methods in the green iguana. This may be caused by the species specificity of certain components of the reagents used, as well as a less optimal test system, i.e. relationship of test reagent to clotting factor concentrations in iguana plasma.

The influence of reagent type on the kinetics of ultrafine coal flotation

USGS Publications Warehouse

Read, R.B.; Camp, L.R.; Summers, M.S.; Rapp, D.M.

1989-01-01

A kinetic study has been conducted to determine the influence of reagent type on flotation rates of ultrafine coal. Two ultrafine coal samples, the Illinois No. 5 (Springfield) and Pittsburgh No. 8, have been evaluated with various reagent types in order to derive the rate constants for coal (kc), ash (ka), and pyrite (kc). The reagents used in the study include anionic surfactants, anionic surfactant-alcohol mixtures, and frothing alcohols. In general, the surfactant-alcohol mixtures tend to float ultrafine coal at a rate three to four times faster than either pure alcohols or pure anionic surfactants. Pine oil, a mixture of terpene alcohols and hydrocarbons, was an exception to this finding; it exhibited higher rate constants than the pure aliphatic alcohols or other pure anionic surfactants studied; this may be explained by the fact that the sample of pine oil used (70% alpha-terpineol) acted as a frother/collector system similar to alcohol/kerosene. The separation efficiencies of ash and pyrite from coal, as evidenced by the ratios of kc/ka or kc/kp, tend to indicate, however, that commercially available surfactant-alcohol mixtures are not as selective as pure alcohols such as 2-ethyl-1-hexanol or methylisobutylcarbinol. Some distinct differences in various rate constants, or their ratios, were noted between the two coals studied, and are possibly attributable to surface chemistry effects. ?? 1989.

Directed evolution of PDZ variants to generate high-affinity detection reagents.

PubMed

Ferrer, Marc; Maiolo, Jim; Kratz, Patricia; Jackowski, Jessica L; Murphy, Dennis J; Delagrave, Simon; Inglese, James

2005-04-01

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.

Anisotropic surface chemistry properties and adsorption behavior of silicate mineral crystals.

PubMed

Xu, Longhua; Tian, Jia; Wu, Houqin; Fang, Shuai; Lu, Zhongyuan; Ma, Caifeng; Sun, Wei; Hu, Yuehua

2018-03-07

Anisotropic surface properties of minerals play an important role in a variety of fields. With a focus on the two most intensively investigated silicate minerals (i.e., phyllosilicate minerals and pegmatite aluminosilicate minerals), this review highlights the research on their anisotropic surface properties based on their crystal structures. Four surface features comprise the anisotropic surface chemistry of minerals: broken bonds, energy, wettability, and charge. Analysis of surface broken bond and energy anisotropy helps to explain the cleavage and growth properties of mineral crystals, and understanding surface wettability and charge anisotropy is critical to the analysis of minerals' solution behavior, such as their flotation performance and rheological properties. In a specific reaction, the anisotropic surface properties of minerals are reflected in the adsorption strengths of reagents on different mineral surfaces. Combined with the knowledge of mineral crushing and grinding, a thorough understanding of the anisotropic surface chemistry properties and the anisotropic adsorption behavior of minerals will lead to the development of effective relational models comprising their crystal structure, surface chemistry properties, and targeted reagent adsorption. Overall, such a comprehensive approach is expected to firmly establish the connection between selective cleavage of mineral crystals for desired surfaces and designing novel reagents selectively adsorbed on the mineral surfaces. As tools to characterize the anisotropic surface chemistry properties of minerals, DLVO theory, atomic force microscopy (AFM), and molecular dynamics (MD) simulations are also reviewed. Copyright © 2017 Elsevier B.V. All rights reserved.

Kinetics and selectivity of permanganate chemiluminescence: a study of hydroxyl and amino disubstituted benzene positional isomers.

PubMed

Slezak, Teo; Smith, Zoe M; Adcock, Jacqui L; Hindson, Christopher M; Barnett, Neil W; Nesterenko, Pavel N; Francis, Paul S

2011-11-30

Examination of the chemiluminescence reactions of dihydroxybenzenes, aminophenols and phenylenediamines with acidic potassium permanganate has provided a new understanding of the relationships between analyte structure, reaction conditions, kinetics of the light-producing pathway and emission intensity, with broad implications for this widely utilised chemiluminescence detection system. Using a permanganate reagent prepared in a polyphosphate solution and adjusted to pH 2.5, large differences in the rate of reaction with different positional isomers were observed, with the meta-substituted forms reacting far slower and therefore exhibiting much lower chemiluminescence intensities in flow analysis systems. The preliminary partial reduction of permanganate to form significant concentrations of Mn(III) increased the rate of reaction with all analytes tested, resulting in comparable or (in the case of aminophenol and phenylenediamine) even greater emission intensities for the meta-isomers, demonstrating the opportunity to tune the selectivity of the reagent towards certain classes of compound or even specific positional isomers of the same compound. Using more acidic permanganate reagents, in which polyphosphates are not required, the discrepancy between the chemiluminescence intensities was still observed, but was less prominent due to the generally faster rates of reaction. The enhancement of these chemiluminescence reactions by on-line addition of formic acid or formaldehyde can in part also be attributed to the generation of significant pools of the key Mn(III) precursor to the emitting species. Copyright © 2011 Elsevier B.V. All rights reserved.

Synthesis of Quaternary Carbon Stereogenic Centers through Enantioselective Cu-Catalyzed Allylic Substitutions with Vinylaluminum Reagents

PubMed Central

Gao, Fang; McGrath, Kevin P.; Lee, Yunmi; Hoveyda, Amir H.

2010-01-01

Catalytic enantioselective allylic substitution (EAS) reactions, which involve the use of alkyl- or aryl-substituted vinylaluminum reagents and afford 1,4-dienes containing a quaternary carbon stereogenic center at their C-3 site, are disclosed. The C–C bond forming transformations are promoted by 0.5–2.5 mol % of sulfonate bearing chiral bidentate N-heterocyclic carbene (NHC) complexes, furnishing the desired products efficiently (66–97% yield of isolated products) and in high site- (>98% SN2′) and enantioselectivity [up to 99:1 enantiomer ratio (er)]. To the best of our knowledge, the present report puts forward the first cases of allylic substitution reactions that result in the generation of all-carbon quaternary stereogenic centers through the addition of a vinyl unit. The aryl- and vinyl-substituted vinylaluminum reagents, which cannot be prepared in high efficiency through direct reaction with diisobutylaluminum hydride, are accessed through a recently introduced Ni-catalyzed reaction of the corresponding terminal alkynes with the same inexpensive metal-hydride agent. Sequential Ni-catalyzed hydrometallations and Cu-catalyzed C–C bond forming reactions allow for efficient and selective synthesis of a range of enantiomerically enriched EAS products, which cannot cannot be accessed by previously disclosed strategies (due to inefficient vinylmetal synthesis or low reactivity and/or selectivity with Si-substituted derivatives). The utility of the protocols developed is demonstrated through a concise enantioselective synthesis of natural product bakuchiol. PMID:20860365

Oligonucleotides as antivirals: dream or realistic perspective?

PubMed

Van Aerschot, Arthur

2006-09-01

Many reports have been published on antiviral activity of synthetic oligonucleotides, targeted to act either by a true antisense effect or via non-sequence specific interactions. This short review will try to evaluate the current status of the field by focusing on the effects as reported for inhibition of either HSV-1, HCMV or HIV-1. Following an introduction with a historical background and a brief discussion on the different types of constructs and mechanisms of action, the therapeutic potential of antisense oligonucleotides as antivirals, as well as possible pitfalls upon their evaluation will be discussed.

FlyPrimerBank: An Online Database for Drosophila melanogaster Gene Expression Analysis and Knockdown Evaluation of RNAi Reagents

PubMed Central

Hu, Yanhui; Sopko, Richelle; Foos, Marianna; Kelley, Colleen; Flockhart, Ian; Ammeux, Noemie; Wang, Xiaowei; Perkins, Lizabeth; Perrimon, Norbert; Mohr, Stephanie E.

2013-01-01

The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo. PMID:23893746

A perspective on reagent diversity and non-covalent binding of reactive carbonyl species (RCS) and effector reagents in nonenzymatic glycation (NEG): Mechanistic considerations and implications for future research

NASA Astrophysics Data System (ADS)

Rodnick, Kenneth J.; Holman, R. W.; Ropski, Pamela S.; Huang, Mingdong; Swislocki, Arthur L. M.

2017-06-01

This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS), initial binding in the nonenzymatic glycation (NEG) process, and nonenzymatic covalent protein modification (here termed NECPM). While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA) and human serum albumin (HSA) are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.); here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.). Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.

Qualification of a select one-stage activated partial thromboplastin time-based clotting assay and two chromogenic assays for the post-administration monitoring of nonacog beta pegol.

PubMed

Tiefenbacher, S; Bohra, R; Amiral, J; Bowyer, A; Kitchen, S; Lochu, A; Rosén, S; Ezban, M

2017-10-01

Essentials Nonacog beta pegol (N9-GP) is an extended half-life, recombinant human factor IX (FIX). One-stage clotting (OSC) and chromogenic FIX activity assays were assessed for N9-GP recovery. OSC STA ® -Cephascreen ® , ROX FIX and BIOPHEN FIX chromogenic assays were qualified for N9-GP. Other extended half-life factor products should be assessed in a similar way prior to approval. Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factor IX that is under development for the prophylaxis and treatment of bleeding episodes in hemophilia B patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX Factor IX and BIOPHEN Factor IX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN Factor IX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective assay qualifications. Conclusion The one-stage clotting assay with the STA-Cephascreen APTT reagent, the ROX Factor IX chromogenic assay and the BIOPHEN Factor IX chromogenic assay are considered to be qualified for the measurement of N9-GP in 3.2% (0.109 m) citrated human plasma. © 2017 International Society on Thrombosis and Haemostasis.

Switching and optimizing control for coal flotation process based on a hybrid model

PubMed Central

Dong, Zhiyong; Wang, Ranfeng; Fan, Minqiang; Fu, Xiang

2017-01-01

Flotation is an important part of coal preparation, and the flotation column is widely applied as efficient flotation equipment. This process is complex and affected by many factors, with the froth depth and reagent dosage being two of the most important and frequently manipulated variables. This paper proposes a new method of switching and optimizing control for the coal flotation process. A hybrid model is built and evaluated using industrial data. First, wavelet analysis and principal component analysis (PCA) are applied for signal pre-processing. Second, a control model for optimizing the set point of the froth depth is constructed based on fuzzy control, and a control model is designed to optimize the reagent dosages based on expert system. Finally, the least squares-support vector machine (LS-SVM) is used to identify the operating conditions of the flotation process and to select one of the two models (froth depth or reagent dosage) for subsequent operation according to the condition parameters. The hybrid model is developed and evaluated on an industrial coal flotation column and exhibits satisfactory performance. PMID:29040305

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation.

PubMed

Keates, Tracy; Cooper, Christopher D O; Savitsky, Pavel; Allerston, Charles K; Phillips, Claire; Hammarström, Martin; Daga, Neha; Berridge, Georgina; Mahajan, Pravin; Burgess-Brown, Nicola A; Müller, Susanne; Gräslund, Susanne; Gileadi, Opher

2012-06-15

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome. Copyright © 2011 Elsevier B.V. All rights reserved.

Recyclable fluorescent gold nanocluster membrane for visual sensing of copper(II) ion in aqueous solution.

PubMed

Lin, Zhijin; Luo, Fenqiang; Dong, Tongqing; Zheng, Liyan; Wang, Yaxian; Chi, Yuwu; Chen, Guonan

2012-05-21

Recently, metal-selective fluorescent chemosensors have attracted intense attention for their simple and real-time tracking of metal ions in environmental samples. However, most of the existing fluorescent sensors are one-off sensors and thus suffer from large amount of reagent consumption, significant experimental cost and raising the risk of environmental pollution. In this paper, we developed a green (low reagent consumption, low-toxicity reagent use), recyclable, and visual sensor for Cu(2+) in aqueous solution by using a fluorescent gold nanoclusters membrane (FGM) as the sensing unit, basing on our findings on gold nanoclusters (Au NCs) that the bovine serum albumin (BSA)-coated Au NCs exhibit excellent membrane-forming ability under the isoelectric point of BSA, and thus enable us to obtain a new type of sensing membrane (i.e. FGM) by denaturing Au NCs; the fluorescence of FGM can be significantly quenched by Cu(2+) ion, and the quenched fluorescence can be totally recovered by histidine; the as-prepared FGM is very stable and recyclable, which makes it an ideal sensing material.

Organometallic Palladium Reagents for Cysteine Bioconjugation

PubMed Central

Vinogradova, Ekaterina V.; Zhang, Chi; Spokoyny, Alexander M.; Pentelute, Bradley L.; Buchwald, Stephen L.

2015-01-01

Transition-metal based reactions have found wide use in organic synthesis and are used frequently to functionalize small molecules.1,2 However, there are very few reports of using transition-metal based reactions to modify complex biomolecules3,4, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature, and mild pH) and the existence of multiple, reactive functional groups found in biopolymers. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation reactions. The bioconjugation reaction is rapid and robust under a range of biocompatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants, and external thiol nucleophiles. The broad utility of the new bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as a new set of benchtop reagents for diverse bioconjugation applications. PMID:26511579

Proton and non-proton activation of ASIC channels

PubMed Central

Gautschi, Ivan; van Bemmelen, Miguel Xavier; Schild, Laurent

2017-01-01

The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH values below 7.0. By contrast, non-proton ligands are able to trigger sustained ASIC currents at physiological pHs. To analyze the functional basis of the ASIC desensitizing and sustained currents, we have used ASIC1a and ASIC2a mutants with a cysteine in the pore vestibule for covalent binding of different sulfhydryl reagents. We found that ASIC1a and ASIC2a exhibit two distinct currents, a proton-induced desensitizing current and a sustained current triggered by sulfhydryl reagents. These currents differ in their pH dependency, their sensitivity to the sulfhydryl reagents, their ionic selectivity and their relative magnitude. We propose a model for ASIC1 and ASIC2 activity where the channels can function in two distinct modes, a desensitizing mode and a sustained mode depending on the activating ligands. The pore vestibule of the channel represents a functional site for binding non-proton ligands to activate ASIC1 and ASIC2 at neutral pH and to prevent channel desensitization. PMID:28384246

Expressing the human proteome for affinity proteomics: optimising expression of soluble protein domains and in vivo biotinylation

PubMed Central

Keates, Tracy; Cooper, Christopher D.O.; Savitsky, Pavel; Allerston, Charles K.; Phillips, Claire; Hammarström, Martin; Daga, Neha; Berridge, Georgina; Mahajan, Pravin; Burgess-Brown, Nicola A.; Müller, Susanne; Gräslund, Susanne; Gileadi, Opher

2012-01-01

The generation of affinity reagents to large numbers of human proteins depends on the ability to express the target proteins as high-quality antigens. The Structural Genomics Consortium (SGC) focuses on the production and structure determination of human proteins. In a 7-year period, the SGC has deposited crystal structures of >800 human protein domains, and has additionally expressed and purified a similar number of protein domains that have not yet been crystallised. The targets include a diversity of protein domains, with an attempt to provide high coverage of protein families. The family approach provides an excellent basis for characterising the selectivity of affinity reagents. We present a summary of the approaches used to generate purified human proteins or protein domains, a test case demonstrating the ability to rapidly generate new proteins, and an optimisation study on the modification of >70 proteins by biotinylation in vivo. These results provide a unique synergy between large-scale structural projects and the recent efforts to produce a wide coverage of affinity reagents to the human proteome. PMID:22027370

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

1991-10-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

1991-01-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

Remarkably selective iridium catalysts for the elaboration of aromatic C-H bonds.

PubMed

Cho, Jian-Yang; Tse, Man Kin; Holmes, Daniel; Maleczka, Robert E; Smith, Milton R

2002-01-11

Arylboron compounds have intriguing properties and are important building blocks for chemical synthesis. A family of Ir catalysts now enables the direct synthesis of arylboron compounds from aromatic hydrocarbons and boranes under "solventless" conditions. The Ir catalysts are highly selective for C-H activation and do not interfere with subsequent in situ transformations, including Pd-mediated cross-couplings with aryl halides. By virtue of their favorable activities and exceptional selectivities, these Ir catalysts impart the synthetic versatility of arylboron reagents to C-H bonds in aromatic and heteroaromatic hydrocarbons.

Treating respiratory viral diseases with chemically modified, second generation intranasal siRNAs.

PubMed

Barik, Sailen

2009-01-01

Chemically synthesized short interfering RNA (siRNA) of pre-determined sequence has ushered a new era in the application of RNA interference (RNAi) against viral genes. We have paid particular attention to respiratory viruses that wreak heavy morbidity and mortality worldwide. The clinically significant ones include respiratory syncytial virus (RSV), parainfluenza virus (PIV) and influenza virus. As the infection by these viruses is clinically restricted to the respiratory tissues, mainly the lungs, the logical route for the application of the siRNA was also the same, i.e., via the nasal route. Following the initial success of intranasal siRNA against RSV, second-generation siRNAs were made against the viral polymerase large subunit (L) that were chemically modified and screened for improved stability, activity and pharmacokinetics. 2'-O-methyl (2'-O-Me) and 2'-deoxy-2'-fluoro (2'-F) substitutions in the ribose ring were incorporated in different positions of the sense and antisense strands and the resultant siRNAs were tested with various transfection reagents intranasally against RSV. Based on these results, we propose the following consensus for designing intranasal antiviral siRNAs: (i) modified 19-27 nt long double-stranded siRNAs are functional in the lung, (ii) excessive 2'-OMe and 2'-F modifications in either or both strands of these siRNAs reduce efficacy, and (iii) limited modifications in the sense strand are beneficial, although their precise efficacy may be position-dependent.

High- and low-temperature-stable thermite composition for producing high-pressure, high-velocity gases

DOEpatents

Halcomb, Danny L.; Mohler, Jonathan H.

1990-10-16

A high- and low-temperature-stable thermite composition for producing high-pressure and high-velocity gases comprises an oxidizable metal, an oxidizing reagent, and a high-temperature-stable gas-producing additive selected from the group consisting of metal carbides and metal nitrides.

Stibathiolanes: Synthesis, solid state structure, and solution behavior

NASA Astrophysics Data System (ADS)

Fisher, Richard A.; Nielsen, Ralph B.; Davis, William M.; Buchwald, Stephen L.

1990-06-01

Interest in organometallic compounds of the main group metals has recently grown tremendously, due in part to the wide variety of applications of these compounds in the materials sciences. Despite this new activity, the synthetic strategies for main group organometallics have remained relatively undeveloped. The majority of syntheses of these compounds involve classical metathesis reactions between a main group halide and an organometallic compound such as an organolithium or Grignard reagent and are limited by a lack of selectivity and by the availability of suitable organometallic precursors. The latter limitation is severe for main group metallacycles because of the paucity of suitable 1, n(n=3,4,5)-dianionic reagents or their equivalents, which are most often used for the synthesis of this class of molecules.

Pentobarbital quantitation using EMIT serum barbiturate assay reagents: application to monitoring of high-dose pentobarbital therapy.

PubMed

Pape, B E; Cary, P L; Clay, L C; Godolphin, W

1983-01-01

Pentobarbital serum concentrations associated with a high-dose therapeutic regimen were determined using EMIT immunoassay reagents. Replicate analyses of serum controls resulted in a within-assay coefficient of variation of 5.0% and a between-assay coefficient of variation of 10%. Regression analysis of 44 serum samples analyzed by this technique (y) and a reference procedure (x) were y = 0.98x + 3.6 (r = 0.98; x = ultraviolet spectroscopy) and y = 1.04x + 2.4 (r = 0.96; x = high-performance liquid chromatography). Clinical evaluation of the results indicates the immunoassay is sufficiently sensitive and selective for pentobarbital to allow accurate quantitation within the therapeutic range associated with high-dose therapy.

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry

PubMed Central

Ornatsky, Olga I.; Kinach, Robert; Bandura, Dmitry R.; Lou, Xudong; Tanner, Scott D.; Baranov, Vladimir I.; Nitz, Mark; Winnik, Mitchell A.

2008-01-01

Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping. PMID:19122859

Heat shock protein-90 beta is expressed at the surface of multipotential mesenchymal precursor cells: generation of a novel monoclonal antibody, STRO-4, with specificity for mesenchymal precursor cells from human and ovine tissues.

PubMed

Gronthos, Stan; McCarty, Rosa; Mrozik, Krzysztof; Fitter, Stephen; Paton, Sharon; Menicanin, Danijela; Itescu, Silviu; Bartold, P Mark; Xian, Cory; Zannettino, Andrew C W

2009-11-01

Mesenchymal stromal cells (MSCs) and their precursor cells (MPCs) can proliferate and differentiate into multiple mesodermal and some ectodermal and endodermal tissues. Culture-expanded MSCs are currently being evaluated as a possible cell therapy to replace/repair injured or diseased tissues. While a number of mAb reagents with specificity to human MSCs, including STRO-1, STRO-3 (BLK ALP), CD71 (SH2, SH3), CD106 (VCAM-1), CD166, and CD271, have facilitated the isolation of purified populations of human MSCs from primary tissues, few if any mAb reagents have been described that can be used to isolate equivalent cells from other species. This is of particular relevance when assessing the tissue regenerative efficacy of MSCs in large immunocompetent, preclinical animal models of disease. In light of this, we sought to generate novel monoclonal antibodies (mAb) with specific reactivity against a cell surface molecule that is expressed at high levels by MSCs from different species. Using CD106 (VCAM-1)-selected ovine MSCs as an immunogen, mAb-producing hybridomas were selected for their reactivity to both human and ovine MSCs. One such hybridoma, termed STRO-4, produced an IgG mAb that reacted with <5% of human and ovine bone marrow (BM) mononuclear cells. As a single selection reagent, STRO-4 mAb was able to enrich colony-forming fibroblasts (CFU-F) in both human and ovine BM by 16- and 8-folds, respectively. Cells isolated with STRO-4 exhibited reactivity with markers commonly associated with MSCs isolated by plastic adherence including CD29, CD44, and CD166. Moreover, when placed in inductive culture conditions in vitro, STRO-4(+) MSCs exhibited multilineage differentiation potential and were capable of forming a mineralized matrix, lipid-filled adipocytes, and chondrocytes capable of forming a glycosaminoglycan-rich matrix. Biochemical analysis revealed that STRO-4 identified the beta isoform of heat shock protein-90 (Hsp90beta). In addition to identifying an antibody reagent that identifies a highly conserved epitope expressed by MSCs from different species, our study also points to a potential role for Hsp90beta in MSC biology.

Knockdown of synaptic scaffolding protein Homer 1b/c attenuates secondary hyperalgesia induced by complete Freund's adjuvant in rats.

PubMed

Yao, Yong-Xing; Jiang, Zhen; Zhao, Zhi-Qi

2011-12-01

Previous studies have demonstrated that Homer 1b/c, a postsynaptic molecular scaffolding protein that binds and clusters metabotropic glutamate receptors at neuronal synapses, has an important role in the metabotropic glutamate receptor signaling process. In the current study, we investigated the possible involvement of Homer 1b/c in secondary hyperalgesia induced by complete Freund's adjuvant (CFA). Chronic inflammation was induced by injecting CFA into the left hind ankle of Wistar rats. Homer 1b/c antisense or missense oligonucleotides were intrathecally administrated (antisense, 10 μg/10 μL, 5 μg/10 μL, or 2.5 μg/10 μL, once a day; missense, 10 μg/10 μL) from 5 to 8 days after the onset of inflammation. The withdrawal threshold and withdrawal latency to mechanical or thermal stimuli were determined before and after the intrathecal administration. The expression and distribution of Homer 1b/c were examined in the spinal cord using immunological techniques. Mechanical allodynia and thermal hyperalgesia were induced within 24 hours and maintained for >2 weeks after the CFA injection. The expression of Homer 1b/c reached the highest level 7 days after inflammation and returned to baseline at day 28. Intrathecal administration of Homer 1b/c antisense oligonucleotides markedly reduced the expression of Homer 1b/c protein in the spinal cord. Additionally, administration of Homer 1b/c antisense oligonucleotides attenuated secondary mechanical hypersensitization on days 2 to 5 and reduced thermal hypersensitization on days 3 to 4. There were no effects of missense oligonucleotides on hypersensitization and the expression of Homer 1b/c. In the naïve rats, Homer 1b/c antisense oligonucleotides did not affect the mechanical and thermal responses or locomotor activity. These novel results demonstrate that Homer 1b/c in the spinal cord contributes to the maintenance of secondary hyperalgesia induced by CFA and suggest that Homer 1b/c may be a novel target for pain therapy.

Secretion of prebeta HDL increases with the suppression of cholesteryl ester transfer protein in Hep G2 cells.

PubMed

Sawada, S; Sugano, M; Makino, N; Okamoto, H; Tsuchida, K

1999-10-01

Prebeta HDL are small, protein rich lipoproteins that are predominantly composed of apo A-I, without apo A-II. Prebeta HDL are secreted from the liver as nascent HDL and/or are produced in the incubated plasma by cholesteryl ester transfer protein (CETP). However, the role of CETP in the secretion of HDL from the liver has yet to be determined. In the present study, we examined the effect of the suppression of hepatic CETP by antisense oligodeoxynucleotides (ODNs) against CETP targeted to the liver on the secretion of apo A-I using a Hep G2 cell culture. The ODNs against CETP were coupled to asialoglycoprotein (ASOR) carrier molecules, which serve as an important method for the regulation of liver gene expression. Hep G2 cells were cultured in DMEM supplemented with 10 FBS. After 2 days, the medium was changed to DMEM with EGF and the cells were divided into three groups. The control group received saline, while the sense group was mixed with the sense ODNs complex and the antisense group was mixed with the antisense ODNs complex, respectively, for 2 days. Both the hepatic CETP mRNA and the CETP mass in the medium in the antisense group decreased significantly more than in the sense and the control groups (CETP mass: 1.697 + /- 0.410 ng/mg cell protein vs. 2.367 + /- 0.22 and 2.360 + /- 0.139, n = 3 in each determination). In contrast, both the hepatic apo A-I mRNA and the apo A-I mass in the medium in the antisense group were significantly higher than those in the sense and the control groups (apo A-I mass; 1.877 + /- 0.215 micro/mg cell protein vs. 1.213 + /- 0.282 and 1.097 + /- 0.144, n = 3 in each determination). The increase in apo A-I was mainly due to the increase in prebeta apo A-I. These findings may partly explain why HDL and apo A-I increase in patients with CETP deficiency, while also indicating the possibility that the original level of prebeta HDL is sufficient in such patients.

Audiogenic seizure activity following HSV-1 GAD65 sense or antisense injection into inferior colliculus of Long-Evans rat.

PubMed

Coleman, James R; Thompson, Karen C; Wilson, Marlene A; Wilson, Steven P

2017-06-01

Herpes virus technology involving manipulation of GAD65 was used to study effects on audiogenic seizures (AGS). Audiogenic seizure behaviors were examined following injections of replication-defective herpes simplex virus (HSV-1) vectors incorporating sense or antisense toward GAD65 along with 10% lac-Z into the central nucleus of inferior colliculus (CNIC) of Long-Evans rats. In seizure-sensitive animals developmentally primed by intense sound exposure, injection of GAD65 in the sense orientation increased wild running latencies and reduced incidence of clonus compared with lac-Z only, unoperated, and vehicle seizure groups. In contrast, infection of CNIC with GAD65 antisense virus resulted in 100% incidence of wild running and clonus behaviors in AGS animals. Unprimed animals not operated continued to show uniform absence of seizure activity. Administration of GAD65 antisense virus into CNIC produced novel wild running and clonus behaviors in some unprimed animals. Staining for β-galactosidase in all vector animals revealed no differences in pattern or numbers of immunoreactive cells at injection sites. Qualitatively, typical small and medium multipolar/stellate and medium fusiform neurons appeared in the CNIC of vector animals. These results demonstrate that HSV-1 vector constructs implanted into the CNIC can predictably influence incidence and severity of AGS and suggest that viral vectors can be useful in studying GABA mechanisms with potential for therapeutic application in epilepsy. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic". Copyright © 2016 Elsevier Inc. All rights reserved.

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

PubMed

Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

2016-04-15

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit.

PubMed Central

D'Aoust, M A; Yelle, S; Nguyen-Quoc, B

1999-01-01

The role of sucrose synthase (SuSy) in tomato fruit was studied in transgenic tomato (Lycopersicon esculentum) plants expressing an antisense fragment of fruit-specific SuSy RNA (TOMSSF) under the control of the cauliflower mosaic virus 35S promoter. Constitutive expression of the antisense RNA markedly inhibited SuSy activity in flowers and fruit pericarp tissues. However, inhibition was only slight in the endosperm and was undetectable in the embryo, shoot, petiole, and leaf tissues. The activity of sucrose phosphate synthase decreased in parallel with that of SuSy, but acid invertase activity did not increase in response to the reduced SuSy activity. The only effect on the carbohydrate content of young fruit was a slight reduction in starch accumulation. The in vitro sucrose import capacity of fruits was not reduced by SuSy inhibition at 23 days after anthesis, and the rate of starch synthesized from the imported sucrose was not lessened even when SuSy activity was decreased by 98%. However, the sucrose unloading capacity of 7-day-old fruit was substantially decreased in lines with low SuSy activity. In addition, the SuSy antisense fruit from the first week of flowering had a slower growth rate. A reduced fruit set, leading to markedly less fruit per plant at maturity, was observed for the plants with the least SuSy activity. These results suggest that SuSy participates in the control of sucrose import capacity of young tomato fruit, which is a determinant for fruit set and development. PMID:10590167

Bcl-2 inhibitors potentiate the cytotoxic effects of radiation in Bcl-2 overexpressing radioresistant tumor cells

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

Hara, Takamitsu; Omura-Minamisawa, Motoko; Chao Cheng

Purpose: Bcl-2, an inhibitor of apoptosis frequently shows elevated expression in human tumors, thus resulting in resistance to radiation therapy. Therefore, inhibiting Bcl-2 function may enhance the radiosensitivity of tumor cells. Tetrocarcin A (TC-A) and bcl-2 antisense oligonucleotides exhibit antitumor activity by inhibiting Bcl-2 function and transcription, respectively. We investigated whether these antitumor agents would enhance the cytotoxic effects of radiation in tumor cells overexpressing Bcl-2. Methods and materials: We used HeLa/bcl-2 cells, a stable Bcl-2-expressing cell line derived from wild-type HeLa (HeLa/wt) cells. Cells were incubated with TC-A and bcl-2 antisense oligonucleotides for 24 h after irradiation, and cellmore » viability was then determined. Apoptotic cells were quantified by flow cytometric assay. Results: The HeLa/bcl-2 cells were more resistant to radiation than HeLa/wt cells. At concentrations that are not inherently cytotoxic, both TC-A and bcl-2 antisense oligonucleotides increased the cytotoxic effects of radiation in HeLa/bcl-2 cells, but not in HeLa/wt cells. However, in HeLa/bcl-2 cells, additional treatment with TC-A in combination with radiation did not significantly increase apoptosis. Conclusions: The present results suggest that TC-A and bcl-2 antisense oligonucleotides reduce radioresistance of tumor cells overexpressing Bcl-2. Therefore, a combination of radiotherapy and Bcl-2 inhibitors may prove to be a useful therapeutic approach for treating tumors that overexpress Bcl-2.« less

Hfq restructures RNA-IN and RNA-OUT and facilitates antisense pairing in the Tn10/IS10 system

PubMed Central

Ross, Joseph A.; Ellis, Michael J.; Hossain, Shahan; Haniford, David B.

2013-01-01

Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base-pairing of mRNAs and trans-encoded sRNAs that share partial sequence complementarity. It is less clear if Hfq is required for pairing of cis-encoded RNAs (i.e., antisense RNAs) with their target mRNAs. In the current work, we have characterized the interactions between Escherichia coli Hfq and the components of the Tn10/IS10 antisense system, RNA-IN and RNA-OUT. We show that Hfq interacts with RNA-OUT through its proximal RNA-binding surface, as is typical for Hfq and trans-encoded sRNAs. In contrast, RNA-IN binds both proximal and distal RNA-binding surfaces in Hfq with a higher affinity for the latter, as is typical for mRNA interactions in canonical sRNA-mRNA pairs. Importantly, an amino acid substitution in Hfq that interferes with RNA binding to the proximal site negatively impacts RNA-IN:OUT pairing in vitro and suppresses the ability of Hfq to negatively regulate IS10 transposition in vivo. We also show that Hfq binding to RNA-IN and RNA-OUT alters secondary structure elements in both of these RNAs and speculate that this could be important in how Hfq facilitates RNA-IN:OUT pairing. Based on the results presented here, we suggest that Hfq could be involved in regulating RNA pairing in other antisense systems, including systems encoded by other transposable elements. PMID:23510801

Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes.

PubMed

Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

2015-09-30

Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3' maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

RNA-targeted therapeutics in cancer clinical trials: Current status and future directions.

PubMed

Barata, Pedro; Sood, Anil K; Hong, David S

2016-11-01

Recent advances in RNA delivery and target selection provide unprecedented opportunities for cancer treatment, especially for cancers that are particularly hard to treat with existing drugs. Small interfering RNAs, microRNAs, and antisense oligonucleotides are the most widely used strategies for silencing gene expression. In this review, we summarize how these approaches were used to develop drugs targeting RNA in human cells. Then, we review the current state of clinical trials of these agents for different types of cancer and outcomes from published data. Finally, we discuss lessons learned from completed studies and future directions for this class of drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Improved silencing properties using small internally segmented interfering RNAs

PubMed Central

Bramsen, Jesper B.; Laursen, Maria B.; Damgaard, Christian K.; Lena, Suzy W.; Ravindra Babu, B.; Wengel, Jesper; Kjems, Jørgen

2007-01-01

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo. PMID:17726057

Identification of functional features of synthetic SINEUPs, antisense lncRNAs that specifically enhance protein translation

PubMed Central

Kozhuharova, Ana; Sharma, Harshita; Ohyama, Takako; Fasolo, Francesca; Yamazaki, Toshio; Cotella, Diego; Santoro, Claudio; Zucchelli, Silvia; Gustincich, Stefano; Carninci, Piero

2018-01-01

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest. PMID:29414979

Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

PubMed Central

Wong, Michael; Daly, Seth M.; Greenberg, David E.; Toprak, Erdal

2016-01-01

The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities. PMID:27631336

Discrimination of heterogenous mRNAs encoding strychnine-sensitive glycine receptors in Xenopus oocytes by antisense oligonucleotides.

PubMed Central

Akagi, H; Patton, D E; Miledi, R

1989-01-01

Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of approximately 10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (approximately 2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors. Images PMID:2479016

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice.

PubMed

Zinker, Bradley A; Rondinone, Cristina M; Trevillyan, James M; Gum, Rebecca J; Clampit, Jill E; Waring, Jeffrey F; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E; Reilly, Regina M; Koterski, Sandra; Opgenorth, Terry J; Ulrich, Roger G; Crosby, Seth; Butler, Madeline; Murray, Susan F; McKay, Robert A; Bhanot, Sanjay; Monia, Brett P; Jirousek, Michael R

2002-08-20

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

PubMed Central

Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

2002-01-01

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

Asymmetric localization of natural antisense RNA of neuropeptide sensorin in Aplysia sensory neurons during aging and activity.

PubMed

Kadakkuzha, Beena M; Liu, Xin-An; Narvaez, Maria; Kaye, Alexandra; Akhmedov, Komolitdin; Puthanveettil, Sathyanarayanan V

2014-01-01

Despite the advances in our understanding of transcriptome, regulation and function of its non-coding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN), a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail Aplysia californica. Sensorin (SRN) has a key role in learning and long-term memory storage in Aplysia. We have now identified NAT-SRN in the central nervous system (CNS) and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro-dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduction in levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.

Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.

PubMed

Crosby, Jeff R; Zhao, Chenguang; Jiang, Chong; Bai, Dong; Katz, Melanie; Greenlee, Sarah; Kawabe, Hiroshi; McCaleb, Michael; Rotin, Daniela; Guo, Shuling; Monia, Brett P

2017-11-01

Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Intravenous administration of stabilized antisense lipid particles (SALP) leads to activation and expansion of liver natural killer cells.

PubMed

Bramson, J L; Bodner, C A; Johnson, J; Semple, S; Hope, M J

2000-06-01

Stabilized antisense lipid particles (SALP) have been developed for the systemic delivery of oligonucleotides. The impact of intravenous SALP administration was measured with respect to activation of natural killer (NK) and NK1.1+ T (NKT) cells in the livers of immunocompetent mice. Treatment with a SALP containing a highly mitogenic oligonucleotide (INX-6295) generated an increase in NK cytolytic activity and cell number within the liver but did not appear to affect the number of hepatic NKT cells or their cytolytic activity. The same results were observed after intravenous administration of the mitogenic oligonucleotide alone. Interestingly, treatment with a SALP containing a weakly mitogenic oligonucleotide (INX-6300) also activated the liver NK cells, whereas the oligonucleotide alone was unable to elicit these effects. The NK stimulatory activity of a SALP containing INX-6300 required both lipid and oligonucleotide components. These results demonstrate that in addition to modifying the pharmacokinetics and biodistribution of intravenously administered oligonucleotides, SALP possess immunostimulatory activity independent of oligonucleotide mitogenicity, which can serve as an adjuvant to antisense therapies for cancer.

Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase.

PubMed

Coetzer, C; Corsini, D; Love, S; Pavek, J; Tumer, N

2001-02-01

Polyphenol oxidase (PPO) activity of Russet Burbank potato was inhibited by sense and antisense PPO RNAs expressed from a tomato PPO cDNA under the control of the 35S promoter from the cauliflower mosaic virus. Transgenic Russet Burbank potato plants from 37 different lines were grown in the field. PPO activity and the level of enzymatic browning were measured in the tubers harvested from the field. Of the tubers from 28 transgenic lines that were sampled, tubers from 5 lines exhibited reduced browning. The level of PPO activity correlated with the reduction in enzymatic browning in these lines. These results indicate that expression of tomato PPO RNA in sense or antisense orientation inhibits PPO activity and enzymatic browning in the major commercial potato cultivar. Expression of tomato PPO RNA in sense orientation led to the greatest decrease in PPO activity and enzymatic browning, possibly due to cosuppression. These results suggest that expression of closely related heterologous genes can be used to prevent enzymatic browning in a wide variety of food crops without the application of various food additives.

Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B.

PubMed

Crooke, Stanley T; Geary, Richard S

2013-08-01

Mipomersen is a second generation antisense oligonucleotide that targets apolipoprotein B. It has been studied thoroughly in clinical trials (more than 800 subjects), including four randomized double-blind placebo controlled phase 3 studies involving 391 patients, and is in registration for the treatment of severe hypercholesterolaemia. The pharmacokinetic and pharmacodynamic properties of mipomersen are well characterized. Mipomersen is rapidly and extensively absorbed after subcutaneous administration and has an elimination half-life of approximately 30 days across species. It is cleared by nuclease metabolism and renal excretion of the metabolites. Mipomersen reduces all apolipoprotein B containing atherogenic particles and displays dose dependent reductions between 50-400 mg week⁻¹ , both as a single agent and in the presence of maximal lipid lowering therapy. No drug-drug interactions have been identified. Mipomersen is a representative of second generation antisense drugs, all of which have similar properties, and is thus representative of the behaviour of the class of drugs. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

Cross-Electrophile Coupling: Principles of Reactivity and Selectivity

PubMed Central

2015-01-01

A critical overview of the catalytic joining of two different electrophiles, cross-electrophile coupling (XEC), is presented with an emphasis on the central challenge of cross-selectivity. Recent synthetic advances and mechanistic studies have shed light on four possible methods for overcoming this challenge: (1) employing an excess of one reagent; (2) electronic differentiation of starting materials; (3) catalyst–substrate steric matching; and (4) radical chain processes. Each method is described using examples from the recent literature. PMID:24820397

Selective and metal-free epoxidation of terminal alkenes by heterogeneous polydioxirane in mild conditions

NASA Astrophysics Data System (ADS)

Kazemnejadi, M.; Shakeri, A.; Nikookar, M.; Shademani, R.; Mohammadi, M.

2018-05-01

Polydioxirane (PDOX) was prepared by the treatment of polysalicylaldehyde with Oxone and was found as a selective, highly efficient and heterogeneous reagent for epoxidation of alkenes which can be successfully isolated. This work also introduced a simpler, safer and milder way for epoxidation of alkenes with dioxirane groups than before. PDOX can be simply recovered from the reaction mixture by plain filtration and reused for eight runs without significant reactivity loss.

Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

PubMed Central

Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

2015-01-01

We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

Reaction-Mediated Desorption of Macromolecules: Novel Phenomenon Enabling Simultaneous Reaction and Separation.

PubMed

Isakari, Yu; Kishi, Yuhi; Yoshimoto, Noriko; Yamamoto, Shuichi; Podgornik, Aleš

2018-02-02

Combining chemical reaction with separation offers several advantages. In this work possibility to induce spontaneous desorption of adsorbed macromolecules, once being PEGylated, through adjustment of the reagent composition is investigated. Bovine serum albumin (BSA) and activated oligonucleotide, 9T, are used as the test molecules and 20 kDa linear activated PEG is used for their PEGylation. BSA solid-phase PEGylation is performed on Q Sepharose HP. Distribution coefficient of BSA and PEG-BSA as a function of NaCl is determined using linear gradient elution (LGE) experiments and Yamamoto model. According to the distribution coefficient the selectivity between BSA and PEG - BSA of around 15 is adjusted by using NaCl. Spontaneous desorption of PEG - BSA is detected with no presence of BSA. However, due to a rather low selectivity, also desorption of BSA occurred at high elution volume. A similar procedure is applied for activated 9T oligonucleotide, this time using monolithic CIM QA disk monolithic column for adsorption. Selectivity of over 2000 is obtained by proper adjustment of PEG reagent composition. High selectivity enables spontaneous desorption of PEG-9T without any desorption of activated 9T. Both experiments demonstrates that reaction-mediated desorption of macromolecules is possible when the reaction conditions are properly tuned. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antisense Masking of an hnRNP A1/A2 Intronic Splicing Silencer Corrects SMN2 Splicing in Transgenic Mice

PubMed Central

Hua, Yimin; Vickers, Timothy A.; Okunola, Hazeem L.; Bennett, C. Frank; Krainer, Adrian R.

2008-01-01

survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA. PMID:18371932

2′-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo

PubMed Central

Prakash, Thazha P.; Johnston, Joseph F.; Graham, Mark J.; Condon, Thomas P.; Manoharan, Muthiah

2004-01-01

Synthesis and antisense activity of oligonucleotides modified with 2′-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2′-O-DMAOE) are described. The 2′-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide ‘gapmers’, with 2′-O-DMAOE- modified nucleoside residues at the ends and 2′-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. ‘Gapmer’ oligonucleotides have one or two 2′-O-modified regions and a 2′-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2′-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression was observed. Oligonucleotides with 2′-O-DMAOE modifications throughout the sequences reduced the intercellular adhesion molecule-1 (ICAM-1) protein expression very efficiently in HUVEC cells with an IC50 of 1.8 nM. The inhibition of ICAM-1 protein expression by these uniformly modified 2′-O-DMAOE oligonucleotides may be due to selective interference with the formation of the translational initiation complex. These results demonstrate that 2′-O-DMAOE- modified oligonucleotides are useful for antisense-based therapeutics when either RNase H-dependent or RNase H-independent target reduction mechanisms are employed. PMID:14762210

THE ROLE OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN (MRP) IN THE BLOOD-BRAIN BARRIER AND OPIOID ANALGESIA

PubMed Central

Su, Wendy; Pasternak, Gavril W.

2013-01-01

The blood brain barrier protects the brain from circulating compounds and drugs. The ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) is involved with the barrier, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood, raising the possibility of a similar role for other transporters. Multidrug resistance associated protein (MRP), a 190 kDa protein similar to Pgp is also ABC transport that has been implicated in the blood brain barrier. The current study explores its role in opioid action. Immunohistochemically, it is localized in the choroid plexus in ratsand can be selectively downregulated by antisense treatment at both the level of mRNA, as shown by RT-PCR, and protein, as demonstrated immunohistochemically. Behaviorally, downregulation of MRP significantly enhances the analgesic potency of systemic morphine in MRP knockout mice and in antisense-treated rats by lowering the blood brain barrier. Following intracerebroventricular administration, a number of compounds, including some opioids, are rapidly secreted from the brain into the blood where they contribute to the overall analgesic effects by activating peripheral systems. MRP plays a role in this efflux. Downregulating MRP expression leads to a corresponding decrease in the transport and a diminished analgesic response from opioids administered intracerebroventricularly. Thus, the transporter protein MRP plays a role in maintaining the blood-brain barrier and modulates the activity of opioids. PMID:23508590

Stimulated-single fiber electromyography monitoring of anti-sense induced changes in experimental autoimmune myasthenia gravis.

PubMed

Boneva, Neli; Hamra-Amitay, Yasmine; Wirguin, Itzhak; Brenner, Talma

2006-05-01

The neuromuscular weakness associated with myasthenia gravis (MG) can be transiently relieved by pharmacological inhibitors of acetylcholinesterase (AChE). Here, we expand the anticholinesterase repertoire to include 2'-O-methyl-protected antisense oligonucleotides targeted to AChE mRNA (EN101). Using stimulated-single fiber electromyography, we show that EN101 treatment of rats with experimental autoimmune myasthenia gravis (EAMG), improved the mean consecutive difference (MCD) and blocking for 24h. This treatment was more efficient than pyridostigmine and was accompanied by marked improvement in stamina and clinical profile.

Spot test kit for explosives detection

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

Pagoria, Philip F; Whipple, Richard E; Nunes, Peter J

An explosion tester system comprising a body, a lateral flow membrane swab unit adapted to be removeably connected to the body, a first explosives detecting reagent, a first reagent holder and dispenser operatively connected to the body, the first reagent holder and dispenser containing the first explosives detecting reagent and positioned to deliver the first explosives detecting reagent to the lateral flow membrane swab unit when the lateral flow membrane swab unit is connected to the body, a second explosives detecting reagent, and a second reagent holder and dispenser operatively connected to the body, the second reagent holder and dispensermore » containing the second explosives detecting reagent and positioned to deliver the second explosives detecting reagent to the lateral flow membrane swab unit when the lateral flow membrane swab unit is connected to the body.« less

Development Of Antibody-Based Fiber-Optic Sensors

NASA Astrophysics Data System (ADS)

Tromberg, Bruce J.; Sepaniak, Michael J.; Vo-Dinh, Tuan

1988-06-01

The speed and specificity characteristic of immunochemical complex formation has encouraged the development of numerous antibody-based analytical techniques. The scope and versatility of these established methods can be enhanced by combining the principles of conventional immunoassay with laser-based fiber-optic fluorimetry. This merger of spectroscopy and immunochemistry provides the framework for the construction of highly sensitive and selective fiber-optic devices (fluoroimmuno-sensors) capable of in-situ detection of drugs, toxins, and naturally occurring biochemicals. Fluoroimmuno-sensors (FIS) employ an immobilized reagent phase at the sampling terminus of a single quartz optical fiber. Laser excitation of antibody-bound analyte produces a fluorescence signal which is either directly proportional (as in the case of natural fluorophor and "antibody sandwich" assays) or inversely proportional (as in the case of competitive-binding assays) to analyte concentration. Factors which influence analysis time, precision, linearity, and detection limits include the nature (solid or liquid) and amount of the reagent phase, the method of analyte delivery (passive diffusion, convection, etc.), and whether equilibrium or non-equilibrium assays are performed. Data will be presented for optical fibers whose sensing termini utilize: (1) covalently-bound solid antibody reagent phases, and (2) membrane-entrapped liquid antibody reagents. Assays for large-molecular weight proteins (antigens) and small-molecular weight, carcinogenic, polynuclear aromatics (haptens) will be considered. In this manner, the influence of a system's chemical characteristics and measurement requirements on sensor design, and the consequence of various sensor designs on analytical performance will be illustrated.

KENNEDY SPACE CENTER, FLA. - The Minus Eighty Lab Freezer for ISS (MELFI), provided as Laboratory Support Equipment by the European Space Agency for the International Space Station, is seen in the Space Station Processing Facility. The lab will provide cooling and storage for reagents, samples and perishable materials in four insulated containers called dewars with independently selectable temperatures of -80°C, -26°C, and +4°C. It also will be used to transport samples to and from the station. The MELFI is planned for launch on the ULF-1 mission.

NASA Image and Video Library

2003-09-08

KENNEDY SPACE CENTER, FLA. - The Minus Eighty Lab Freezer for ISS (MELFI), provided as Laboratory Support Equipment by the European Space Agency for the International Space Station, is seen in the Space Station Processing Facility. The lab will provide cooling and storage for reagents, samples and perishable materials in four insulated containers called dewars with independently selectable temperatures of -80°C, -26°C, and +4°C. It also will be used to transport samples to and from the station. The MELFI is planned for launch on the ULF-1 mission.

KENNEDY SPACE CENTER, FLA. - After removing its cover, technicians look over the Minus Eighty Lab Freezer for ISS (MELFI), provided as Laboratory Support Equipment by the European Space Agency for the International Space Station. The lab will provide cooling and storage for reagents, samples and perishable materials in four insulated containers called dewars with independently selectable temperatures of -80°C, -26°C, and +4°C. It also will be used to transport samples to and from the station. The MELFI is planned for launch on the ULF-1 mission.

NASA Image and Video Library

2003-09-08

KENNEDY SPACE CENTER, FLA. - After removing its cover, technicians look over the Minus Eighty Lab Freezer for ISS (MELFI), provided as Laboratory Support Equipment by the European Space Agency for the International Space Station. The lab will provide cooling and storage for reagents, samples and perishable materials in four insulated containers called dewars with independently selectable temperatures of -80°C, -26°C, and +4°C. It also will be used to transport samples to and from the station. The MELFI is planned for launch on the ULF-1 mission.

KENNEDY SPACE CENTER, FLA. - In the Space Station Processing Facility, technicians remove the cover from the Minus Eighty Lab Freezer for ISS(MELFI) provided as Laboratory Support Equipment by the European Space Agency for the International Space Station. The lab will provide cooling and storage for reagents, samples and perishable materials in four insulated containers called dewars with independently selectable temperatures of -80°C, -26°C, and +4°C. It also will be used to transport samples to and from the station. The MELFI is planned for launch on the ULF-1 mission.

NASA Image and Video Library

2003-09-08

KENNEDY SPACE CENTER, FLA. - In the Space Station Processing Facility, technicians remove the cover from the Minus Eighty Lab Freezer for ISS(MELFI) provided as Laboratory Support Equipment by the European Space Agency for the International Space Station. The lab will provide cooling and storage for reagents, samples and perishable materials in four insulated containers called dewars with independently selectable temperatures of -80°C, -26°C, and +4°C. It also will be used to transport samples to and from the station. The MELFI is planned for launch on the ULF-1 mission.

Successful immunotherapy of canine flea allergy with injected Actinomycetales preparations.

PubMed

Marro, Alicia; Pirles, Mónica; Schiaffino, Laura; Bin, Liliana; Dávila, Héctor; Bottasso, Oscar A; McIntyre, Graham; Ripley, Paul R; Stanford, Cynthia A; Stanford, John L

2011-08-01

Can heat-killed, borate-buffered suspensions of Gordonia bronchialis, Rhodococcus coprophilus or Tsukamurella inchonensis be used to treat canine flea allergy? Organisms cultured on Sauton's medium into stationary phase were autoclaved in borate-buffered saline and stored at 10 mg wet weight/ml. Intradermal injections of 0.1 ml containing 1 mg of bacilli were administered on the first and 20th days of the study. G. bronchialis and R. coprophilus were most effective in a pilot study of a small number of dogs with flea allergy. A larger number of affected dogs were then randomized to receive placebo or either of the two selected reagents. The extent and severity of allergic signs and symptoms were scored and blood samples were collected just before the first injection and 28 days after the second. Both selected reagents reduced the extent and severity of lesions (p < 0.001) and reduced scratching. Eosinophil numbers were reduced (p < 0.0001) between the first and second assessment. Injections of G. bronchialis or R. coprophilus effectively reduce the signs and symptoms of flea allergy in dogs.

ortho-Methoxyphenols as Convenient Oxidative Bioconjugation Reagents with Application to Site-Selective Heterobifunctional Cross-Linkers.

PubMed

ElSohly, Adel M; MacDonald, James I; Hentzen, Nina B; Aanei, Ioana L; El Muslemany, Kareem M; Francis, Matthew B

2017-03-15

The synthesis of complex protein-based bioconjugates has been facilitated greatly by recent developments in chemoselective methods for biomolecular modification. The oxidative coupling of o-aminophenols or catechols with aniline functional groups is chemoselective, mild, and rapid; however, the oxidatively sensitive nature of the electron-rich aromatics and the paucity of commercial sources pose some obstacles to the general use of these reactive strategies. Herein, we identify o-methoxyphenols as air-stable, commercially available derivatives that undergo efficient oxidative couplings with anilines in the presence of periodate as oxidant. Mechanistic considerations informed the development of a preoxidation protocol that can greatly reduce the amount of periodate necessary for effective coupling. The stability and versatility of these reagents was demonstrated through the synthesis of complex protein-protein bioconjugates using a site-selective heterobifunctional cross-linker comprising both o-methoxyphenol and 2-pyridinecarboxaldehyde moieties. This compound was used to link epidermal growth factor to genome-free MS2 viral capsids, affording nanoscale delivery vectors that can target a variety of cancer cell types.

α-Fluorovinyl Weinreb Amides and α- Fluoroenones from a Common Fluorinated Building Block

PubMed Central

Ghosh, Arun K.; Banerjee, Shaibal; Sinha, Saikat; Kang, Soon Bang; Zajc, Barbara

2009-01-01

Synthesis and reactivity of N-methoxy-N-methyl-(1,3-benzothiazol-2-ylsulfonyl)fluoroacetamide, a building block for Julia olefination, is reported. This reagent undergoes condensation reactions with aldehydes and cyclic ketones, to give α-fluorovinyl Weinreb amides. Olefination reactions proceed under mild, DBU-mediated conditions, or in the presence of NaH. DBU-mediated condensations proceed with either E or Z-selectivity, depending upon reaction conditions, whereas NaH-mediated reactions are ≥98% Z-stereoselective. Conversion of the Weinreb amide moiety in N-methoxy-N-methyl-(1,3-benzothiazol-2-ylsulfanyl)fluoroacetamide to ketones, followed by oxidation, resulted in another set of olefination reagents, namely (1,3-benzothiazol-2-ylsulfonyl)fluoromethyl phenyl and propyl ketones. In the presence of DBU, these compounds react with aldehydes tested to give α-fluoroenones with high Z-selectivity. The use of N-methoxy-N-methyl-(1,3-benzothiazol-2-ylsulfanyl)fluoroacetamide as a common fluorinated intermediate in the synthesis of α-fluorovinyl Weinreb amides and α-fluoroenones has been demonstrated. Application of the Weinreb amide to α-fluoro allyl amine synthesis is also shown. PMID:19361189

Solvent extraction: the coordination chemistry behind extractive metallurgy.

PubMed

Wilson, A Matthew; Bailey, Phillip J; Tasker, Peter A; Turkington, Jennifer R; Grant, Richard A; Love, Jason B

2014-01-07

The modes of action of the commercial solvent extractants used in extractive hydrometallurgy are classified according to whether the recovery process involves the transport of metal cations, M(n+), metalate anions, MXx(n-), or metal salts, MXx into a water-immiscible solvent. Well-established principles of coordination chemistry provide an explanation for the remarkable strengths and selectivities shown by most of these extractants. Reagents which achieve high selectivity when transporting metal cations or metal salts into a water-immiscible solvent usually operate in the inner coordination sphere of the metal and provide donor atom types or dispositions which favour the formation of particularly stable neutral complexes that have high solubility in the hydrocarbons commonly used in recovery processes. In the extraction of metalates, the structures of the neutral assemblies formed in the water-immiscible phase are usually not well defined and the cationic reagents can be assumed to operate in the outer coordination spheres. The formation of secondary bonds in the outer sphere using, for example, electrostatic or H-bonding interactions are favoured by the low polarity of the water-immiscible solvents.

Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.

PubMed

Kitchen, S; Kershaw, G; Tiefenbacher, S

2016-07-01

The recent development of modified recombinant factor VIII (FVIII) and factor IX (FIX) therapeutic products with extended half-lives will create challenges for the haemostasis laboratory in obtaining recovery estimates of these products in clinical samples using existing assays. The new long-acting therapeutic concentrates contain molecular modifications of Fc fusion, site-specific of polyethylene glycol or albumin fusion. The optimum methods for monitoring each new product will need to be assessed individually and laboratories should select an assay which gives similar results to the assay used to assign potency to the product in question. For some extended half-life FVIII and FIX products some one stage assays are entirely unsuitable for monitoring purposes. For most products and assay reagents studied so far, and reviewed in this manuscript, chromogenic FVIII or FIX assays can be safely used with conventional plasma standards. If one stage assays are used then they should be performed using carefully selected reagents/methods which have been shown to recover activity close to the labelled potency for the specific product being monitored. © 2016 John Wiley & Sons Ltd.