Autumn orientation behaviour of paddyfield warblers, Acrocephalus agricola, from a recently expanded breeding range on the western Black Sea coast.

PubMed

Zehtindjiev, Pavel; Ilieva, Mihaela; Akesson, Susanne

2010-10-01

The paddyfield warbler, Acrocephalus agricola, has extended its breeding range from Central Asia to the western Black Sea coast. The Balkan population offers a unique chance to test the effect of breeding range expansion on the genetically programmed migratory direction. We studied 21 paddyfield warblers at Durankulak Lake, NE Bulgaria, by recording their autumn migratory orientation in circular orientation cages. Our data show that the preferred migratory orientation is directed along a NE-SW axis. Paddyfield warblers seem to avoid direct crossing of the Black Sea by following the western coast. The mean bearing was parallel to the nearest coastline and corresponds to the direction of the historical breeding range expansion of the species. In our experiment many individuals showed south-western orientation in autumn, a course which would potentially lead the birds to exploratory movements outside the current breeding range. An axial orientation response has been often shown in circular cage tests, and can be due to factors such as coastal orientation or reverse orientation triggered by the physiological condition of some individuals. However, it might also be one of the driving mechanisms for range expansion. Hence, we can expect the future expansion of Balkan paddyfield warblers to continue towards south-west. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Hydrothermal syntheses, crystal structures and characterization of new vanadoborates: The novel decorated cage cluster [V₆B₂₂O₄₄(OH)₁₀

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

Liu, Xing; Zhou, Jian, E-mail: Jianzhou888888@yahoo.com.cn; College of Chemistry and Material, Yulin Normal University, Yulin 537000

2013-05-01

Three new vanadoborates [H₃teta]₃[V₁₂B₁₈O₅₄(OH)₆(H₂O)]·(H₃O)·5H₂O (1, teta=triethylenetetramine), [H₄teta]₂[V₆B₂₂O₄₄(OH)₁₀] ·4.5H₂O (2) and [H₄tepa]₂[V₆B₂₂O₄₄(OH)₁₀] ·4H₂O (3, tepa=tetraethylenepentamine) have been hydrothermally synthesized and structurally characterized. 1 contains cage cluster [V₁₂B₁₈O₅₄(OH)₆(H₂O)]¹⁰⁻ based on one puckered B₁₈O₃₆(OH)₆ ring sandwiched between two V₆O₁₅ clusters. Both 2 and 3 contain new decorated cage cluster [V₆B₂₂O₄₄(OH)₁₀]⁸⁻ built up from a central band of [V₆O₁₈] capped top and bottom by two raft-like polyborate [B₁₁O₂₀(OH)₄] units. The magnetic susceptibility of 3 demonstrates the presence of antiferromagnetic interaction between V{sup IV} cations. The theoretical band structure and spectroscopic properties have been also studied. - Graphical abstract: The circular [V₆O₁₈] fragment sandwiched bymore » the two raft-like polyborate [B₁₁O₂₀(OH)₄] units via sharing O atoms form a new cage cluster [V₆B₂₂O₄₄(OH)₁₀]⁸⁻, which represents a new type. Highlights: • Novel decorated cage cluster [V₆B₂₂O₄₄(OH)₁₀] is observed. • The mixed valence vanadoborate cluster is found. • The theoretical band structure and spectroscopic properties have been also studied.« less

Barnes maze testing strategies with small and large rodent models.

PubMed

Rosenfeld, Cheryl S; Ferguson, Sherry A

2014-02-26

Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g. bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g. distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e. random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g. shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.

Molecular biology of viroid-host interactions and disease control strategies.

PubMed

Kovalskaya, Natalia; Hammond, Rosemarie W

2014-11-01

Viroids are single-stranded, covalently closed, circular, highly structured noncoding RNAs that cause disease in several economically important crop plants. They replicate autonomously and move systemically in host plants with the aid of the host machinery. In addition to symptomatic infections, viroids also cause latent infections where there is no visual evidence of infection in the host; however, transfer to a susceptible host can result in devastating disease. While there are non-hosts for viroids, no naturally occurring durable resistance has been observed in most host species. Current effective control methods for viroid diseases include detection and eradication, and cultural controls. In addition, heat or cold therapy combined with meristem tip culture has been shown to be effective for elimination of viroids for some viroid-host combinations. An understanding of viroid-host interactions, host susceptibility, and non-host resistance could provide guidance for the design of viroid-resistant plants. Efforts to engineer viroid resistance into host species have been underway for several years, and include the use of antisense RNA, antisense RNA plus ribozymes, a dsRNase, and siRNAs, among others. The results of those efforts and the challenges associated with creating viroid resistant plants are summarized in this review. Published by Elsevier Ireland Ltd.

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.

A pair of novel Cd(II) enantiomers based on lactate derivatives: Synthesis, crystal structures and properties

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

Xu, Zhong-Xuan, E-mail: xuzhongxuan1974@163.com; State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, the Chinese Academy of Sciences, Fuzhou, Fujian 350002; Ao, Ke-Hou

A pair of novel 3D homochiral metal−organic frameworks (HMOFs), namely [Cd{sub 2.5}((R)-CIA){sub 6}(1,4-DIB)(H{sub 2}O){sub 2}]·((CH{sub 3}){sub 2}NH{sub 2})·H{sub 2}O (1-D), [Cd{sub 2.5}((S)-CIA){sub 6}(1,4-DIB)(H{sub 2}O){sub 2}]·((CH{sub 3}){sub 2}NH{sub 2})·H{sub 2}O (1-L), have been synthesized using lactic acid derivative ligands ((R)-H{sub 3}CIA and (S)-H{sub 3}CIA) and 1,4-DIB. Crystallographic analyses indicate that the complexes 1-D and 1-L are packed by cage substructures. Some physical characteristics, such as solid-state circular dichroism (CD), thermal stabilities and photoluminescent properties are also investigated. Our results highlight the effective method to apply lactic acid derivative ligands to form interesting HMOFs. - Graphical abstract: Using lactic acid derivative ligandsmore » ((R)-H{sub 3}CIA and (S)-H{sub 3}CIA) and 1,4-DIB to assemble with Cd{sup 2+} ions, a pair of novel 3D homochiral metal-organic frameworks (HMOFs) with cage substructures have been synthesized. Display Omitted - Highlights: • Lactic acid derivative ligands • Cage substructure • Enantiomers.« less

Border Enforcement and Return Migration by Documented and Undocumented Mexicans

PubMed Central

Massey, Douglas S.; Durand, Jorge; Pren, Karen A.

2014-01-01

Using data from the Mexican Migration Project we compute probabilities of departure and return for first and later trips to the United States in both documented and undocumented status. We then estimate statistical models to analyze the determinants of departure and return according to legal status. Prior to 1986, Mexico-U.S. migration was characterized by great circularity, but since then circularity has declined markedly for undocumented migrants but increased dramatically for documented migrants. Whereas return migration by undocumented migrants dropped in response to the massive increase in border enforcement, that of documented migrants did not. At present, the Mexico-U.S. migration system has reached a new equilibrium in which undocumented migrants are caged in as long term settlers in the United States while documented migrants increasingly range freely and circulate back and forth across the border within rising frequency. PMID:26273210

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

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.

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.

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.

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

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

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

Pharmacology of Antisense Drugs.

PubMed

Bennett, C Frank; Baker, Brenda F; Pham, Nguyen; Swayze, Eric; Geary, Richard S

2017-01-06

Recent studies have led to a greater appreciation of the diverse roles RNAs play in maintaining normal cellular function and how they contribute to disease pathology, broadening the number of potential therapeutic targets. Antisense oligonucleotides are the most direct means to target RNA in a selective manner and have become an established platform technology for drug discovery. There are multiple molecular mechanisms by which antisense oligonucleotides can be used to modulate RNAs in cells, including promoting the degradation of the targeted RNA or modulating RNA function without degradation. Antisense drugs utilizing various antisense mechanisms are demonstrating therapeutic potential for the treatment of a broad variety of diseases. This review focuses on some of the advances that have taken place in translating antisense technology from the bench to the clinic.

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.

Computational study for the circular redox reaction of N2O with CO catalyzed by fullerometallic cations C60Fe+ and C70Fe.

PubMed

Anafcheh, Maryam; Naderi, Fereshteh; Khodadadi, Zahra; Ektefa, Fatemeh; Ghafouri, Reza; Zahedi, Mansour

2017-03-01

We applied density functional calculations to study the circular redox reaction mechanism of N 2 O with CO catalyzed by fullerometallic cations C 60 Fe + and C 70 Fe + . The on-top sites of six-membered rings (η 6 ) of fullerene cages are the most preferred binding sites for Fe + cation, and the hexagon to pentagon migration of Fe + is unlikely under ambient thermodynamic conditions. The initial ion/molecule reaction, N 2 O rearrangement and N 2 abstraction on the considered fullerometallic cations are easier than those on the bare Fe + cation in the gas phase. Generally, our results indicate that fullerometallic ions, C 60 Fe + and C 70 Fe + , are more favorable substrates for redox reaction of N 2 O with CO in comparison to the other previously studied carbon nanostructures such as graphene and nanotubes. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiple Isoforms of ANRIL in Melanoma Cells: Structural Complexity Suggests Variations in Processing.

PubMed

Sarkar, Debina; Oghabian, Ali; Bodiyabadu, Pasani K; Joseph, Wayne R; Leung, Euphemia Y; Finlay, Graeme J; Baguley, Bruce C; Askarian-Amiri, Marjan E

2017-06-27

The long non-coding RNA ANRIL , antisense to the CDKN2B locus, is transcribed from a gene that encompasses multiple disease-associated polymorphisms. Despite the identification of multiple isoforms of ANRIL , expression of certain transcripts has been found to be tissue-specific and the characterisation of ANRIL transcripts remains incomplete. Several functions have been associated with ANRIL . In our judgement, studies on ANRIL functionality are premature pending a more complete appreciation of the profusion of isoforms. We found differential expression of ANRIL exons, which indicates that multiple isoforms exist in melanoma cells. In addition to linear isoforms, we identified circular forms of ANRIL ( circANRIL ). Further characterisation of circANR IL in two patient-derived metastatic melanoma cell lines (NZM7 and NZM37) revealed the existence of a rich assortment of circular isoforms. Moreover, in the two melanoma cell lines investigated, the complements of circANRIL isoforms were almost completely different. Novel exons were also discovered. We also found the family of linear ANRIL was enriched in the nucleus, whilst the circular isoforms were enriched in the cytoplasm and they differed markedly in stability. With respect to the variable processing of circANRIL species, bioinformatic analysis indicated that intronic Arthrobacter luteus (Alu) restriction endonuclease inverted repeats and exon skipping were not involved in selection of back-spliced exon junctions. Based on our findings, we hypothesise that " ANRIL " has wholly distinct dual sets of functions in melanoma. This reveals the dynamic nature of the locus and constitutes a basis for investigating the functions of ANRIL in melanoma.

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

An integrative pharmacological approach to radio telemetry and blood sampling in pharmaceutical drug discovery and safety assessment.

PubMed

Litwin, Dennis C; Lengel, David J; Kamendi, Harriet W; Bialecki, Russell A

2011-01-18

A successful integration of the automated blood sampling (ABS) and telemetry (ABST) system is described. The new ABST system facilitates concomitant collection of physiological variables with blood and urine samples for determination of drug concentrations and other biochemical measures in the same rat without handling artifact. Integration was achieved by designing a 13 inch circular receiving antenna that operates as a plug-in replacement for the existing pair of DSI's orthogonal antennas which is compatible with the rotating cage and open floor design of the BASi Culex® ABS system. The circular receiving antenna's electrical configuration consists of a pair of electrically orthogonal half-toroids that reinforce reception of a dipole transmitter operating within the coil's interior while reducing both external noise pickup and interference from other adjacent dipole transmitters. For validation, measured baclofen concentration (ABST vs. satellite (μM): 69.6 ± 23.8 vs. 76.6 ± 19.5, p = NS) and mean arterial pressure (ABST vs. traditional DSI telemetry (mm Hg): 150 ± 5 vs.147 ± 4, p = NS) variables were quantitatively and qualitatively similar between rats housed in the ABST system and traditional home cage approaches. The ABST system offers unique advantages over traditional between-group study paradigms that include improved data quality and significantly reduced animal use. The superior within-group model facilitates assessment of multiple physiological and biochemical responses to test compounds in the same animal. The ABST also provides opportunities to evaluate temporal relations between parameters and to investigate anomalous outlier events because drug concentrations, physiological and biochemical measures for each animal are available for comparisons.

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.

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

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.

Structure and properties of some chiralanes and chirolanes

NASA Astrophysics Data System (ADS)

Novak, Igor

2018-06-01

The molecular structures, spectra and properties of six chiralanes and chirolanes (approximately spheroidal, saturated, cage hydrocarbons) have been determined by density functional theory (DFT) quantum chemistry calculations. The main features determined are: molecular geometry, partial atomic charges, standard enthalpy of formation, IR, nuclear magnetic resonance (NMR) and circular dichroism (CD) spectra. On the basis of the calculated standard enthalpies of formation and highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) gaps, we suggest that chiralanes/chirolanes are potential synthetic targets. We have calculated the anomalously large downfield 13C-NMR shifts for endohedral carbons in the spectra of [5.5] and [5.7]chiralanes.

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.

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.

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

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.

Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. x Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation.

PubMed

Kayim, M; Ceccardi, T L; Berretta, M J G; Barthe, G A; Derrick, K S

2004-11-01

The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause. The function of p12 is not known, but sequence analysis indicates it may be related to expansins. In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system. Homogeneous beta-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent. Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses. In addition, 20 sense and 18 antisense shoots were rooted. The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting. Northern blots showed the expected RNA in the sense and antisense plants. A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants. At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants. Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.

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.

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…

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.

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.

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.

PubMed

Nuzzo, Francesca; Radu, Claudia; Baralle, Marco; Spiezia, Luca; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2013-11-28

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

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.

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

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.

Drosophila increase exploration after visually detecting predators.

PubMed

de la Flor, Miguel; Chen, Lijian; Manson-Bishop, Claire; Chu, Tzu-Chun; Zamora, Kathya; Robbins, Danielle; Gunaratne, Gemunu; Roman, Gregg

2017-01-01

Novel stimuli elicit behaviors that are collectively known as specific exploration. These behaviors allow the animal to become more familiar with the novel objects within its environment. Specific exploration is frequently suppressed by defensive reactions to predator cues. Herein, we examine if this suppression occurs in Drosophila melanogaster by measuring the response of these flies to wild harvested predators. The flies used in our experiments have been cultured and had not lived under predator threat for multiple decades. In a circular arena with centrally-caged predators, wild type Drosophila actively avoided the pantropical jumping spider, Plexippus paykulli, and the Texas unicorn mantis, Phyllovates chlorophaena, indicating an innate defensive reaction to these predators. Interestingly, wild type Drosophila males also avoided a centrally-caged mock spider, and the avoidance of the mock spider became exaggerated when it was made to move within the cage. Visually impaired Drosophila failed to detect and avoid the Plexippus paykulli and the moving mock spider, while the broadly anosmic orco2 mutants were fully capable of detecting and avoiding Plexippus paykulli, indicating that these flies principally relied upon vison to perceive the predator stimuli. During early exploration of the arena, exploratory activity increased in the presence of Plexippus paykulli and the moving mock spider. The elevated activity induced by Plexippus paykulli disappeared after the fly had finished exploring, suggesting the flies were capable of habituating the predator cues. Taken together, these results indicate that despite being isolated from predators for decades Drosophila will visually detect these predators, retain innate defensive behaviors, respond by increasing exploratory activity in the arena rather than suppressing activity, and may habituate to normal predator cues.

Drosophila increase exploration after visually detecting predators

PubMed Central

Manson-Bishop, Claire; Chu, Tzu-Chun; Zamora, Kathya; Robbins, Danielle; Gunaratne, Gemunu

2017-01-01

Novel stimuli elicit behaviors that are collectively known as specific exploration. These behaviors allow the animal to become more familiar with the novel objects within its environment. Specific exploration is frequently suppressed by defensive reactions to predator cues. Herein, we examine if this suppression occurs in Drosophila melanogaster by measuring the response of these flies to wild harvested predators. The flies used in our experiments have been cultured and had not lived under predator threat for multiple decades. In a circular arena with centrally-caged predators, wild type Drosophila actively avoided the pantropical jumping spider, Plexippus paykulli, and the Texas unicorn mantis, Phyllovates chlorophaena, indicating an innate defensive reaction to these predators. Interestingly, wild type Drosophila males also avoided a centrally-caged mock spider, and the avoidance of the mock spider became exaggerated when it was made to move within the cage. Visually impaired Drosophila failed to detect and avoid the Plexippus paykulli and the moving mock spider, while the broadly anosmic orco2 mutants were fully capable of detecting and avoiding Plexippus paykulli, indicating that these flies principally relied upon vison to perceive the predator stimuli. During early exploration of the arena, exploratory activity increased in the presence of Plexippus paykulli and the moving mock spider. The elevated activity induced by Plexippus paykulli disappeared after the fly had finished exploring, suggesting the flies were capable of habituating the predator cues. Taken together, these results indicate that despite being isolated from predators for decades Drosophila will visually detect these predators, retain innate defensive behaviors, respond by increasing exploratory activity in the arena rather than suppressing activity, and may habituate to normal predator cues. PMID:28746346

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)

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

Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

NASA Astrophysics Data System (ADS)

Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

1988-08-01

Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

PRAPI: post-transcriptional regulation analysis pipeline for Iso-Seq.

PubMed

Gao, Yubang; Wang, Huiyuan; Zhang, Hangxiao; Wang, Yongsheng; Chen, Jinfeng; Gu, Lianfeng

2018-05-01

The single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) based on Pacific Bioscience (PacBio) platform has received increasing attention for its ability to explore full-length isoforms. Thus, comprehensive tools for Iso-Seq bioinformatics analysis are extremely useful. Here, we present a one-stop solution for Iso-Seq analysis, called PRAPI to analyze alternative transcription initiation (ATI), alternative splicing (AS), alternative cleavage and polyadenylation (APA), natural antisense transcripts (NAT), and circular RNAs (circRNAs) comprehensively. PRAPI is capable of combining Iso-Seq full-length isoforms with short read data, such as RNA-Seq or polyadenylation site sequencing (PAS-seq) for differential expression analysis of NAT, AS, APA and circRNAs. Furthermore, PRAPI can annotate new genes and correct mis-annotated genes when gene annotation is available. Finally, PRAPI generates high-quality vector graphics to visualize and highlight the Iso-Seq results. The Dockerfile of PRAPI is available at http://www.bioinfor.org/tool/PRAPI. lfgu@fafu.edu.cn.

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.

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.

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.

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)

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.

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.

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

Biomechanical Analysis of Porous Additive Manufactured Cages for Lateral Lumbar Interbody Fusion: A Finite Element Analysis.

PubMed

Zhang, Zhenjun; Li, Hui; Fogel, Guy R; Liao, Zhenhua; Li, Yang; Liu, Weiqiang

2018-03-01

A porous additive manufactured (AM) cage may provide stability similar to that of traditional solid cages and may be beneficial to bone ingrowth. The biomechanical influence of various porous cages on stability, subsidence, stresses in cage, and facet contact force has not been fully described. The purpose of this study was to verify biomechanical effects of porous AM cages. The surgical finite element models with various cages were constructed. The partially porous titanium (PPT) cages and fully porous titanium (FPT) cages were applied. The mechanical parameters of porous materials were obtained by mechanical test. Then the porous AM cages were compared with solid titanium (TI) cage and solid polyetheretherketone (PEEK) cage. The 4 motion modes were simulated. Range of motion (ROM), cage stress, end plate stress, and facet joint force (FJF) were compared. For all the surgical models, ROM decreased by >90%. Compared with TI and PPT cages, PEEK and FPT cages substantially reduced the maximum stresses in cage and end plate in all motion modes. Compared with PEEK cages, the stresses in cage and end plate for FPT cages decreased, whereas the ROM increased. Comparing FPT cages, the stresses in cage and end plate decreased with increasing porosity, whereas ROM increased with increasing porosity. After interbody fusion, FJF was substantially reduced in all motion modes except for flexion. Fully porous cages may offer an alternative to solid PEEK cages in lateral lumbar interbody fusion. However, it may be prudent to further increase the porosity of the cage. Copyright © 2017 Elsevier Inc. All rights reserved.

Trapping two types of particles using a double-ring-shaped radially polarized beam

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

Zhang Yaoju; Ding Biaofeng; Suyama, Taikei

An optical-trap method based on the illumination of a double-ring-shaped radially polarized beam (R-TEM{sub 11}*) is proposed. The numerical results based on the vector diffraction theory show that a highly focused R-TEM{sub 11}* beam not only can produce a bright spot but also can form an optical cage in the focal region by changing the truncation parameter {beta}, defined as the ratio of the radius of the aperture to the waist of the beam. The radiation forces acting on Rayleigh particles are calculated by using the Rayleigh scattering theory. The bright spot generated by the R-TEM{sub 11}* beam with amore » {beta} value close to 2 can three-dimensionally trap a particle with a refractive index larger than that of the ambient. An optical cage or three-dimensional dark spot generated by the R-TEM{sub 11}* beam with a {beta} value close to 1.3 can three-dimensionally trap a particle with refractive index smaller than that of the ambient. Because the adjustment of the truncation parameter can be actualized by simply changing the radius of a circular aperture inserted in the front of the lens, only one optical-trap system in the present method can be used to three-dimensionally trap two types of particles with different refractive indices.« less

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.

On the role of methacrylic acid copolymers in the intracellular delivery of antisense oligonucleotides.

PubMed

Yessine, Marie-Andrée; Meier, Christian; Petereit, Hans-Ulrich; Leroux, Jean-Christophe

2006-05-01

The delivery of active biomacromolecules to the cytoplasm is a major challenge as it is generally hindered by the endosomal/lysosomal barrier. Synthetic titratable polyanions can overcome this barrier by destabilizing membrane bilayers at pH values typically found in endosomes. This study investigates how anionic polyelectrolytes can enhance the cytoplasmic delivery of an antisense oligonucleotide (ODN). Novel methacrylic acid (MAA) copolymers were examined for their pH-sensitive properties and ability to destabilize cell membranes in a pH-dependent manner. Ternary complex formulations prepared with the ODN, a cationic lipid and a MAA copolymer were systematically characterized with respect to their size, zeta potential, antisense activity, cytotoxicity and cellular uptake using the A549 human lung carcinoma cell line. The MAA copolymer substantially increased the activity of the antisense ODN in inhibiting the expression of protein kinase C-alpha. Uptake, cytotoxicity and antisense activity were strongly dependent on copolymer concentration. Metabolic inhibitors demonstrated that endocytosis was the major internalization pathway of the complexes, and that endosomal acidification was essential for ODN activity. Confocal microscopy analysis of cells incubated with fluorescently-labeled complexes revealed selective delivery of the ODN, but not of the copolymer, to the cytoplasm/nucleus. This study provides new insight into the mechanisms of intracellular delivery of macromolecular drugs, using synthetic anionic polyelectrolytes.

Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas.

PubMed

Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco

2010-01-01

Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.

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

Emlen funnel experiments revisited: methods update for studying compass orientation in songbirds.

PubMed

Bianco, Giuseppe; Ilieva, Mihaela; Veibäck, Clas; Öfjäll, Kristoffer; Gadomska, Alicja; Hendeby, Gustaf; Felsberg, Michael; Gustafsson, Fredrik; Åkesson, Susanne

2016-10-01

Migratory songbirds carry an inherited capacity to migrate several thousand kilometers each year crossing continental landmasses and barriers between distant breeding sites and wintering areas. How individual songbirds manage with extreme precision to find their way is still largely unknown. The functional characteristics of biological compasses used by songbird migrants has mainly been investigated by recording the birds directed migratory activity in circular cages, so-called Emlen funnels. This method is 50 years old and has not received major updates over the past decades. The aim of this work was to compare the results from newly developed digital methods with the established manual methods to evaluate songbird migratory activity and orientation in circular cages.We performed orientation experiments using the European robin ( Erithacus rubecula ) using modified Emlen funnels equipped with thermal paper and simultaneously recorded the songbird movements from above. We evaluated and compared the results obtained with five different methods. Two methods have been commonly used in songbirds' orientation experiments; the other three methods were developed for this study and were based either on evaluation of the thermal paper using automated image analysis, or on the analysis of videos recorded during the experiment.The methods used to evaluate scratches produced by the claws of birds on the thermal papers presented some differences compared with the video analyses. These differences were caused mainly by differences in scatter, as any movement of the bird along the sloping walls of the funnel was recorded on the thermal paper, whereas video evaluations allowed us to detect single takeoff attempts by the birds and to consider only this behavior in the orientation analyses. Using computer vision, we were also able to identify and separately evaluate different behaviors that were impossible to record by the thermal paper.The traditional Emlen funnel is still the most used method to investigate compass orientation in songbirds under controlled conditions. However, new numerical image analysis techniques provide a much higher level of detail of songbirds' migratory behavior and will provide an increasing number of possibilities to evaluate and quantify specific behaviors as new algorithms will be developed.

Furnished Cage System and Hen Well-Being: Comparative Effects of Furnished Cages and Battery Cages on Behavioral Exhibitions in White Leghorn Chickens

USDA-ARS?s Scientific Manuscript database

The battery cage system is being banned in the European Union before or by 2012; and the furnished cage system will be the only cage system allowed after 2012. This study was conducted to examine the different effects of caging systems, furnished cages vs. battery cages, on bird behaviors. One hundr...

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

The polymethyl methacrylate cervical cage for treatment of cervical disk disease Part III. Biomechanical properties.

PubMed

Chen, Jyi-Feng; Lee, Shih-Tseng

2006-10-01

In a previous article, we used the PMMA cervical cage in the treatment of single-level cervical disk disease and the preliminary clinical results were satisfactory. However, the mechanical properties of the PMMA cage were not clear. Therefore, we designed a comparative in vitro biomechanical study to determine the mechanical properties of the PMMA cage. The PMMA cervical cage and the Solis PEEK cervical cage were compressed in a materials testing machine to determine the mechanical properties. The compressive yield strength of the PMMA cage (7030 +/- 637 N) was less than that of the Solis polymer cervical cage (8100 +/- 572 N). The ultimate compressive strength of the PMMA cage (8160 +/- 724 N) was less than that of the Solis cage (9100 +/- 634 N). The stiffness of the PMMA cervical cage (8106 +/- 817 N/mm) was greater than that of the Solis cage (6486 +/- 530 N/mm). The elastic modulus of the PMMA cage (623 +/- 57 MPa) was greater than that of the Solis cage (510 +/- 42 MPa). The elongation of PMMA cage (43.5 +/- 5.7%) was larger than that of the Solis cage (36.1 +/- 4.3%). Although the compressive yield strength and ultimate compressive strength of the PMMA cervical cage were less than those of the Solis polymer cage, the mechanical properties are better than those of the cervical vertebral body. The PMMA cage is strong and safe for use as a spacer for cervical interbody fusion. Compared with other cage materials, the PMMA cage has many advantages and no obvious failings at present. However, the PMMA cervical cage warrants further long-term clinical study.

Noncoding RNA:RNA Regulatory Networks in Cancer

PubMed Central

Chan, Jia Jia; Tay, Yvonne

2018-01-01

Noncoding RNAs (ncRNAs) constitute the majority of the human transcribed genome. This largest class of RNA transcripts plays diverse roles in a multitude of cellular processes, and has been implicated in many pathological conditions, especially cancer. The different subclasses of ncRNAs include microRNAs, a class of short ncRNAs; and a variety of long ncRNAs (lncRNAs), such as lincRNAs, antisense RNAs, pseudogenes, and circular RNAs. Many studies have demonstrated the involvement of these ncRNAs in competitive regulatory interactions, known as competing endogenous RNA (ceRNA) networks, whereby lncRNAs can act as microRNA decoys to modulate gene expression. These interactions are often interconnected, thus aberrant expression of any network component could derail the complex regulatory circuitry, culminating in cancer development and progression. Recent integrative analyses have provided evidence that new computational platforms and experimental approaches can be harnessed together to distinguish key ceRNA interactions in specific cancers, which could facilitate the identification of robust biomarkers and therapeutic targets, and hence, more effective cancer therapies and better patient outcome and survival. PMID:29702599

Progress and Prospects of Anti-HBV Gene Therapy Development

PubMed Central

Maepa, Mohube B.; Roelofse, Ilke; Ely, Abdullah; Arbuthnot, Patrick

2015-01-01

Despite the availability of an effective vaccine against hepatitis B virus (HBV), chronic infection with the virus remains a major global health concern. Current drugs against HBV infection are limited by emergence of resistance and rarely achieve complete viral clearance. This has prompted vigorous research on developing better drugs against chronic HBV infection. Advances in understanding the life cycle of HBV and improvements in gene-disabling technologies have been impressive. This has led to development of better HBV infection models and discovery of new drug candidates. Ideally, a regimen against chronic HBV infection should completely eliminate all viral replicative intermediates, especially covalently closed circular DNA (cccDNA). For the past few decades, nucleic acid-based therapy has emerged as an attractive alternative that may result in complete clearance of HBV in infected patients. Several genetic anti-HBV strategies have been developed. The most studied approaches include the use of antisense oligonucleotides, ribozymes, RNA interference effectors and gene editing tools. This review will summarize recent developments and progress made in the use of gene therapy against HBV. PMID:26263978

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

Testing the neurovascular hypothesis of Alzheimer's disease: LRP-1 antisense reduces blood-brain barrier clearance, increases brain levels of amyloid-beta protein, and impairs cognition.

PubMed

Jaeger, Laura B; Dohgu, Shinya; Hwang, Mark C; Farr, Susan A; Murphy, M Paul; Fleegal-DeMotta, Melissa A; Lynch, Jessica L; Robinson, Sandra M; Niehoff, Michael L; Johnson, Steven N; Kumar, Vijaya B; Banks, William A

2009-01-01

Decreased clearance is the main reason amyloid-beta protein (Abeta) is increased in the brains of patients with Alzheimer's disease (AD). The neurovascular hypothesis states that this decreased clearance is caused by impairment of low density lipoprotein receptor related protein-1 (LRP-1), the major brain-to-blood transporter of Abeta at the blood-brain barrier (BBB). As deletion of the LRP-1 gene is a lethal mutation, we tested the neurovascular hypothesis by developing a cocktail of phosphorothioate antisenses directed against LRP-1 mRNA. We found these antisenses in comparison to random antisense selectively decreased LRP-1 expression, reduced BBB clearance of Abeta42, increased brain levels of Abeta42, and impaired learning ability and recognition memory in mice. These results support dysfunction of LRP-1 at the BBB as a mechanism by which brain levels of Abeta could increase and AD would be promoted.

Modulation of lipoprotein metabolism by antisense technology: preclinical drug discovery methodology.

PubMed

Crooke, Rosanne M; Graham, Mark J

2013-01-01

Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs.

Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

PubMed

Ramazeilles, C; Mishra, R K; Moreau, S; Pascolo, E; Toulmé, J J

1994-08-16

We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.

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.

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.

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...

Furnished cage system and hen well-being: Comparative effects of furnished cages and battery cages on behavioral exhibitions in White Leghorn chickens.

PubMed

Pohle, K; Cheng, H-W

2009-08-01

The battery cage system is being banned in the European Union before or by 2012, and the furnished cage system will be the only cage system allowed after 2012. This study was conducted to examine the different effects of caging systems, furnished cages vs. battery cages, on bird behaviors. One hundred ninety-two 1-d-old non-beak-trimmed Hy-Line W-36 White Leghorn chicks were reared using standard management practices in raised wire cages. At 19 wk of age, the birds were randomly assigned into battery cages or furnished cages. The battery cages were commercial wire cages containing 6 birds per cage, providing 645 cm(2) of floor space per birds. The furnished cages had wire floors and solid metal walls, with perches, a dustbathing area, scratch pads, and a nestbox area with a concealment curtain. Based on the company recommendations, 10 birds were housed per cage, providing a stocking density of 610 cm(2) of floor space per bird. Behavioral observations were conducted using the Noldus Observer software package. The birds were observed at 5-min intervals for the entire light period. The birds housed in battery cages had higher posture and behavioral transitions and increased time spent walking and performing exploratory behavior (P < 0.05, 0.01, respectively), which may indicate they were stressed, resulting in restlessness, whereas the birds housed in furnished cages had higher levels of preening (P < 0.05). Preening has been considered as a comfort behavior in birds. These results may suggest that furnished cages may be a favorable alternative system for housing birds by allowing them to perform certain natural behaviors.

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.

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.

Temperature dependence of polyhedral cage volumes in clathrate hydrates

USGS Publications Warehouse

Chakoumakos, B.C.; Rawn, C.J.; Rondinone, A.J.; Stern, L.A.; Circone, S.; Kirby, S.H.; Ishii, Y.; Jones, C.Y.; Toby, B.H.

2003-01-01

The polyhedral cage volumes of structure I (sI) (carbon dioxide, methane, trimethylene oxide) and structure II (sII) (methane-ethane, propane, tetrahydrofuran, trimethylene oxide) hydrates are computed from atomic positions determined from neutron powder-diffraction data. The ideal structural formulas for sI and sII are, respectively, S2L6 ?? 46H2O and S16L???8 ?? 136H2O, where S denotes a polyhedral cage with 20 vertices, L a 24-cage, and L??? a 28-cage. The space-filling polyhedral cages are defined by the oxygen atoms of the hydrogen-bonded network of water molecules. Collectively, the mean cage volume ratio is 1.91 : 1.43 : 1 for the 28-cage : 24-cage : 20-cage, which correspond to equivalent sphere radii of 4.18, 3.79, and 3.37 A??, respectively. At 100 K, mean polyhedral volumes are 303.8, 227.8, and 158.8 A??3 for the 28-cage, 24-cage, and 20-cage, respectively. In general, the 20-cage volume for a sII is larger than that of a sI, although trimethylene oxide is an exception. The temperature dependence of the cage volumes reveals differences between apparently similar cages with similar occupants. In the case of trimethylene oxide hydrate, which forms both sI and sII, the 20-cages common to both structures contract quite differently. From 220 K, the sII 20-cage exhibits a smooth monotonic reduction in size, whereas the sI 20-cage initially expands upon cooling to 160 K, then contracts more rapidly to 10 K, and overall the sI 20-cage is larger than the sII 20-cage. The volumes of the large cages in both structures contract monotonically with decreasing temperature. These differences reflect reoriented motion of the trimethyelene oxide molecule in the 24-cage of sI, consistent with previous spectroscopic and calorimetric studies. For the 20-cages in methane hydrate (sI) and a mixed methane-ethane hydrate (sII), both containing methane as the guest molecule, the temperature dependence of the 20-cage volume in sII is much less than that in sI, but sII is overall larger in volume.

Effects of separation of resources on behaviour, physical condition and production of laying hens in furnished cages.

PubMed

Shimmura, T; Azuma, T; Eguchi, Y; Uetake, K; Tanaka, T

2009-01-01

1. Based on our previous studies, we designed a medium-sized furnished cage with a dust bath and nest box on both sides of the cage (MFS) and evaluated its usefulness. 2. We used 180 White Leghorn layers. At the age of 17 weeks, the birds were distributed at random into one of the 4 cage designs: conventional cages (CC; 6 cages and 5 hens per cage), small (SF; 6 cages and 5 hens per cage) and medium furnished cages (MFL; 6 cages and 10 hens per cage) with a 'localised' dust bath and nest box on one side of the cage, and MFS (6 cages and 10 hens per cage). The total allocation of resources per bird was similar for all furnished cage designs. Behaviour, physical condition and production were measured in each cage. 3. Moving was more frequent in MFS and MFL than in CC and SF. The proportion of hens performing aggressive pecking and severe feather pecking was higher in MFL than CC and SF. These aggressive interactions occurred frequently in the dust bath area in MFL; however, these tendencies were not found in MFS. Egg production and egg mass were lower in MFL than in SF, while the production in MFS was similar to those in CC and SF. MFS hens laid eggs on the cage floor more often than in MFL. 4. In conclusion, these results demonstrate the possible usefulness of MFS. However, some inconsistent results and ways of improving MFS design were also identified.

Polymers containing borane or carborane cage compounds and related applications

DOEpatents

Bowen, III, Daniel E; Eastwood, Eric A

2013-04-23

Polymers comprising residues of cage compound monomers having at least one polyalkoxy silyl substituent are provided. The cage compound monomers are selected from borane cage compound monomers comprising at least 7 cage atoms and/or carborane cage compound monomers comprising 7 to 11 cage compound monomers. Such polymers can further comprise one or more reactive matrices and/or co-monomers covalently bound with the cage compound monomer residues. Articles of manufacture comprising such polymers are also disclosed.

Effect of housing arrangement on fecal-oral transmission of avian hepatitis E virus in chicken flocks.

PubMed

Liu, Baoyuan; Sun, Yani; Chen, Yiyang; Du, Taofeng; Nan, Yuchen; Wang, Xinjie; Li, Huixia; Huang, Baicheng; Zhang, Gaiping; Zhou, En-Min; Zhao, Qin

2017-09-07

Avian hepatitis E virus (HEV) infection is common in chicken flocks in China, as currently no measures exist to prevent the spread of the disease. In this study, we analyzed the effect of caged versus cage-free housing arrangements on avian HEV transmission. First, 127 serum and 110 clinical fecal samples were collected from 4 chicken flocks including the two arrangements in Shaanxi Province, China and tested for HEV antibodies and/or virus. Concurrently, 36 specific-pathogen-free chickens were divided equally into four experimental living arrangement groups, designated cage-free (Inoculated), caged (Inoculated), cage-free (Negative) and caged (Negative) groups. In caged groups, three cages contained 3 chickens each. Three chickens each from cage-free (Inoculated) and caged (Inoculated) groups (one chicken of each cage) were inoculated by cutaneous ulnar vein with the same dose of avian HEV, respectively. The cage-free (Negative) and caged (Negative) groups served as negative control. Serum and fecal samples were collected at 1 to 7 weeks post-inoculation (wpi) and liver lesions were scored at 7 wpi. The results of serology showed that the avian HEV infection rate (54.10%) of the cage-free chickens was significantly higher than the one (12.12%) for caged chickens (P < 0.05). Also, the rate of detection of avian HEV RNA in the clinical fecal samples was significantly higher in the cage-free (22.80%, 13/57) than caged birds (5.66%, 3/53). Moreover, under experimental conditions, the infected number of uninoculated cage-free chickens (6) was significantly higher than the one for the uninoculated caged birds (2), as evidenced by seroconversion, fecal virus shedding, viremia and gross and microscopic liver lesions. These results suggest that reduction of contact with feces as seen in the caged arrangement of housing chickens can reduce avian HEV transmission. This study provides insights for prevention and control of avian HEV infection in chicken flocks.

Protein cage assembly across multiple length scales.

PubMed

Aumiller, William M; Uchida, Masaki; Douglas, Trevor

2018-05-21

Within the materials science community, proteins with cage-like architectures are being developed as versatile nanoscale platforms for use in protein nanotechnology. Much effort has been focused on the functionalization of protein cages with biological and non-biological moieties to bring about new properties of not only individual protein cages, but collective bulk-scale assemblies of protein cages. In this review, we report on the current understanding of protein cage assembly, both of the cages themselves from individual subunits, and the assembly of the individual protein cages into higher order structures. We start by discussing the key properties of natural protein cages (for example: size, shape and structure) followed by a review of some of the mechanisms of protein cage assembly and the factors that influence it. We then explore the current approaches for functionalizing protein cages, on the interior or exterior surfaces of the capsids. Lastly, we explore the emerging area of higher order assemblies created from individual protein cages and their potential for new and exciting collective properties.

Microclimatic Variation Within Sleeve Cages Used in Ecological Studies

PubMed Central

Nelson, Lori A.; Rieske, Lynne K.

2014-01-01

Abstract Sleeve cages for enclosing or excluding arthropods are essential components of field studies evaluating trophic interactions. Microclimatic variation in sleeve cages was evaluated to characterize its potential effects on subsequent long-term experiments. Two sleeve cage materials, polyester and nylon, and two cage sizes, 400 and 6000 cm 2 , were tested on eastern hemlock, Tsuga canadensis (L.) Carrière. Temperature and relative humidity inside and outside cages, and the cost and durability of the cage materials, were compared. Long-term effects of the sleeve cages were observed by measuring new growth on T. canadensis branches. The ultimate goal was to identify a material that minimizes bag-induced microclimatic variation. Bagged branches whose microclimates mimic those of surrounding unbagged branches should have minimal effects on plant growth and may prove ideal venues for assessing herbivore and predator behavior under natural conditions. No differences were found in temperature or humidity between caging materials. Small cages had higher average temperatures than large cages, especially in the winter, but this difference was confounded by the fact that small cages were positioned higher in trees than large cages. Differences in plant growth were detected. Eastern hemlock branches enclosed within polyester cages produced fewer new growth tips than uncaged controls. Both polyester and nylon cages reduced the length of new shoot growth relative to uncaged branches. In spite of higher costs, nylon cages were superior to polyester with respect to durability and ease of handling. PMID:25368083

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.

Rational disruption of the oligomerization of the mini-ferritin E. coli DPS through protein-protein interface mutation

PubMed Central

Zhang, Yu; Fu, Jing; Chee, Sze Y; Ang, Emmiline X W; Orner, Brendan P

2011-01-01

DNA-binding protein from starved cells (DPS), a mini-ferritin capable of self-assembling into a 12-meric nano-cage, was chosen as the basis for an alanine-shaving mutagenesis study to investigate the importance of key amino acid residues, located at symmetry-related protein-protein interfaces, in controlling protein stability and self-assembly. Nine mutants were designed through simple inspection, synthesized, and subjected to transmission electron microscopy, circular dichroism, size exclusion chromatography, and “virtual alanine scanning” computational analysis. The data indicate that many of these residues may be hot spot residues. Most remarkably, two residues, R83 and R133, were observed to shift the oligomerization state to ˜50% dimer. Based on the hypothesis that these two residues constitute a “hot strip,” located at the ferritin-like threefold axis, the double mutant was generated which completely shuts down detectable formation of 12-mer in solution, favoring a cooperatively folded dimer. The fact that this effect logically builds upon the single mutants emphasizes that complex self-assembly has the potential to be manipulated rationally. This study should have an impact on the fundamental understanding of the assembly of DPS protein cages specifically and protein quaternary structure in general. In addition, as there is much interest in applying these and similar systems to the templation of nano-materials and drug delivery, the ability to control this ferritin's oligomerization state and stability could prove especially valuable. PMID:21898653

Response of Olive Fruit Fly (Diptera: Tephritidae) to an Attract-and-Kill Trap in Greenhouse Cage Tests

PubMed Central

Yokoyama, Victoria Y.

2014-01-01

Abstract A novel attract-and-kill trap for olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), was constructed with yellow corrugated plastic in an inverted cylindrical pan shape formed from a disk and collar. The trap components were tested under three greenhouse temperatures and humidities of warm, hot, and very hot for attractiveness to caged young or older adults. A greater proportion of adults regardless of age were found underneath the devices including disks, cylindrical pans, and pans with pheromone lures and test units of cylindrical pans sprayed with water, insecticidal bait spray, and with lures. The effect was related to lower temperatures on the underside compared with the top and the intolerance of the pest to heat. A circular collar added to the perimeter of the disk that formed the top of the inverted cylinder made the attract-and-kill trap more attractive to adults than the disk alone. Pheromone lures or bait sprays did not increase adult attraction, so were not needed for efficacy. The cylindrical pan was especially attractive to adults when temperatures were high by providing shelter from the heat. At very high temperatures, the pan became unattractive, possibly due to heating of the construction materials. Cylindrical pans sprayed with water on the underside attracted the highest number of adults especially at high temperatures. Greenhouse tests showed that the inverted cylindrical pan design has potential as an attract-and-kill device for olive fruit fly control. PMID:25368094

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.

Computational comparison of three posterior lumbar interbody fusion techniques by using porous titanium interbody cages with 50% porosity.

PubMed

Lee, Yung-Heng; Chung, Chi-Jen; Wang, Chih-Wei; Peng, Yao-Te; Chang, Chih-Han; Chen, Chih-Hsien; Chen, Yen-Nien; Li, Chun-Ting

2016-04-01

This study investigated the biomechanical response of porous cages and lumbar spine segments immediately after surgery and after bone fusion, in addition to the long-term effects of various posterior lumbar interbody fusion (PLIF) techniques, by using the finite element method. Lumbar L3-L4 models based on three PLIF techniques (a single cage at the center of the intervertebral space, a single cage half-anterior to the intervertebral space, and two cages bilateral to the intervertebral space) with and without bone ingrowth were used to determine the biomechanical response of porous cages and lumbar segments instrumented with porous titanium cages (cage porosity=50%, pore diameter=1mm). The results indicated that bone fusion enhanced the stability of the lumbar segments with porous cages without any posterior instrumentation and reduced the peak von Mises stress in the cortical bones and porous cages. Two cages placed bilateral to the intervertebral space achieved the highest structural stability in the lumbar segment and lowest von Mises stress in the cages under both bone fusion conditions. Under identical loading (2-Nm), the range of motion in the single cage at the center of the intervertebral space with bone fusion decreased by 11% (from 1.18° to 1.05°) during flexion and by 66.5% (from 4.46° to 1.5°) during extension in the single cage half-anterior to the intervertebral space with bone fusion compared with no-fusion models. Thus, two porous titanium cages with 50% porosity can achieve high stability of a lumbar segment with PLIF. If only one cage is available, placing the cage half-anterior to the intervertebral space is recommended for managing degenerated lumbar segments. Copyright © 2016 Elsevier Ltd. All rights reserved.

Do position and size matter? An analysis of cage and placement variables for optimum lordosis in PLIF reconstruction.

PubMed

Landham, Priyan R; Don, Angus S; Robertson, Peter A

2017-11-01

To examine monosegmental lordosis after posterior lumbar interbody fusion (PLIF) surgery and relate lordosis to cage size, shape, and placement. Eighty-three consecutive patients underwent single-level PLIF with paired identical lordotic cages involving a wide decompression and bilateral facetectomies. Cage parameters relating to size (height, lordosis, and length) and placement (expressed as a ratio relative to the length of the inferior vertebral endplate) were recorded. Centre point ratio (CPR) was the distance to the centre of both cages and indicated mean position of both cages. Posterior gap ratio (PGR) was the distance to the most posterior cage and indicated position and cage length indirectly. Relationships between lordosis and cage parameters were explored. Mean lordosis increased by 5.98° (SD 6.86°). The cages used varied in length from 20 to 27 mm, in lordosis from 10° to 18°, and in anterior cage height from 10 to 17 mm. The mean cage placement as determined by CPR was 0.54 and by PGR was 0.16. The significant correlations were: both CPR and PGR with lordosis gain at surgery (r = 0.597 and 0.537, respectively, p < 0.001 both), cage lordosis with the final lordosis (r = 0.234, p < 0.05), and anterior cage height was negatively correlated with a change in lordosis (r = -0.297, p < 0.01). Cage size, shape, and position, in addition to surgical technique, determine lordosis during PLIF surgery. Anterior placement with sufficient "clear space" behind the cages is recommended. In addition, cages should be of moderate height and length, so that they act as an effective pivot for lordosis.

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.

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.

Quantitative Proteomics Reveals the Regulatory Networks of Circular RNA CDR1as in Hepatocellular Carcinoma Cells.

PubMed

Yang, Xue; Xiong, Qian; Wu, Ying; Li, Siting; Ge, Feng

2017-10-06

Circular RNAs (circRNAs), a class of widespread endogenous RNAs, play crucial roles in diverse biological processes and are potential biomarkers in diverse human diseases and cancers. Cerebellar-degeneration-related protein 1 antisense RNA (CDR1as), an oncogenic circRNA, is involved in human tumorigenesis and is dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CDR1as functions in HCC remain unclear. Here we explored the functions of CDR1as and searched for CDR1as-regulated proteins in HCC cells. A quantitative proteomics strategy was employed to globally identify CDR1as-regulated proteins in HCC cells. In total, we identified 330 differentially expressed proteins (DEPs) upon enhanced CDR1as expression in HepG2 cells, indicating that they could be proteins regulated by CDR1as. Bioinformatic analysis revealed that many DEPs were involved in cell proliferation and the cell cycle. Further functional studies of epidermal growth factor receptor (EGFR) found that CDR1as exerts its effects on cell proliferation at least in part through the regulation of EGFR expression. We further confirmed that CDR1as could inhibit the expression of microRNA-7 (miR-7). EGFR is a validated target of miR-7; therefore, CDR1as may exert its function by regulating EGFR expression via targeting miR-7 in HCC cells. Taken together, we revealed novel functions and underlying mechanisms of CDR1as in HCC cells. This study serves as the first proteome-wide analysis of a circRNA-regulated protein in cells and provides a reliable and highly efficient method for globally identifying circRNA-regulated proteins.

Characterization of a new caged proton capable of inducing large pH jumps.

PubMed Central

Barth, Andreas; Corrie, John E T

2002-01-01

A new caged proton, 1-(2-nitrophenyl)ethyl sulfate (caged sulfate), is characterized by infrared spectroscopy and compared with a known caged, proton 2-hydroxyphenyl 1-(2-nitrophenyl)ethyl phosphate (caged HPP). In contrast to caged HPP, caged sulfate can induce large pH jumps and protonate groups that have pK values as low as 2.2. The photolysis mechanism of caged sulfate is analogous to that of P(3)-[1-(2-nitrophenyl)ethyl] ATP (caged ATP), and the photolysis efficiency is similar. The utility of this new caged compound for biological studies was demonstrated by its ability to drive the acid-induced conformational change of metmyoglobin. This transition from the native conformation to a partially unfolded form takes place near pH 4 and was monitored by near-UV absorption spectroscopy. PMID:12414718

The challenge of pelvic discontinuity: cup-cage reconstruction does better than conventional cages in mid-term.

PubMed

Abolghasemian, M; Tangsaraporn, S; Drexler, M; Barbuto, R; Backstein, D; Safir, O; Kuzyk, P; Gross, A

2014-02-01

The use of ilioischial cage reconstruction for pelvic discontinuity has been replaced by the Trabecular Metal (Zimmer, Warsaw, Indiana) cup-cage technique in our institution, due to the unsatisfactory outcome of using a cage alone in this situation. We report the outcome of 26 pelvic discontinuities in 24 patients (20 women and four men, mean age 65 years (44 to 84)) treated by the cup-cage technique at a mean follow-up of 82 months (12 to 113) and compared them with a series of 19 pelvic discontinuities in 19 patients (18 women and one man, mean age 70 years (42 to 86)) treated with a cage at a mean follow-up of 69 months (1 to 170). The clinical and radiological outcomes as well as the survivorship of the groups were compared. In all, four of the cup-cage group (15%) and 13 (68%) of the cage group failed due to septic or aseptic loosening. The seven-year survivorship was 87.2% (95% confidence interval (CI) 71 to 103) for the cup-cage group and 49.9% (95% CI 15 to 84) for the cage-alone group (p = 0.009). There were four major complications in the cup-cage group and nine in the cage group. Radiological union of the discontinuity was found in all successful cases in the cup-cage group and three of the successful cage cases. Three hips in the cup-cage group developed early radiological migration of the components, which stabilised with a successful outcome. Cup-cage reconstruction is a reliable technique for treating pelvic discontinuity in mid-term follow-up and is preferred to ilioischial cage reconstruction. If the continuity of the bone graft at the discontinuity site is not disrupted, early migration of the components does not necessarily result in failure.

Reproductive Performance of Mice in Disposable and Standard Individually Ventilated Cages

PubMed Central

Ferguson, Danielle R; Bailey, Michele M

2013-01-01

This study assessed the reproductive performance of mice housed in 2 types of individually ventilated caging systems. Breeding pairs from 48 female and 24 male mice of 3 established transgenic mouse breeding colonies were placed in either a standard or disposable ventilated caging system. For 3 breeding cycles, the number of pups born, pup survival rate to weaning, time interval between litters, and pup weights were monitored for each breeding pair. Disposable and standard cages were maintained in the same location during breeding. Environmental parameters included intracage temperature, humidity, and ammonia and carbon dioxide levels and room light intensity and sound. Overall, 776 offspring were produced. Breeding performance did not differ significantly between the 2 cage types. By 11 wk of age, the weights of pups from both cage types were equivalent. The intracage temperature was 1.1 °F warmer and light intensity at the site of the nest was 34 lx dimmer in disposable cages than in standard caging. The difference in lighting likely was due to nest location; the nests in the disposable cages were at the back of the cages and away from the anterior air supply, whereas in standard caging, nests were at the front of the cages, with the air supply at the rear. Under these husbandry conditions, mice housed in disposable caging systems have comparable breeding performance to those housed in standard individually ventilated cages. PMID:23849403

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

Effects of wind speed on aerosol spray penetration in adult mosquito bioassay cages.

PubMed

Hoffmann, W Clint; Fritz, Bradley K; Farooq, Muhammad; Cooperband, Miriam F

2008-09-01

Bioassay cages are commonly used to assess efficacy of insecticides against adult mosquitoes in the field. To correlate adult mortality readings to insecticidal efficacy and/or spray application parameters properly, it is important to know how the cage used in the bioassay interacts with the spray cloud containing the applied insecticide. This study compared the size of droplets, wind speed, and amount of spray material penetrating cages and outside of cages in a wind tunnel at different wind speeds. Two bioassay cages, Center for Medical, Agricultural and Veterinary Entomology (CMAVE) and Circle, were evaluated. The screen materials used on these cages reduced the size of droplets, wind speed, and amount of spray material inside the cages as compared to the spray cloud and wind velocity outside of the cages. When the wind speed in the dispersion tunnel was set at 0.6 m/sec (1.3 mph), the mean wind speed inside of the CMAVE Bioassay Cage and Circle Cage was 0.045 m/sec (0.10 mph) and 0.075 m/sec (0.17 mph), respectively. At air velocities of 2.2 m/sec (4.9 mph) in the dispersion tunnel, the mean wind speed inside of the CMAVE Bioassay Cage and Circle Cage was 0.83 m/sec (1.86 mph) and 0.71 m/sec (1.59 mph), respectively. Consequently, there was a consistent 50-70% reduction of spray material penetrating the cages compared to the spray cloud that approached the cages. These results provide a better understanding of the impact of wind speed, cage design, and construction on ultra-low-volume spray droplets.

Long-term effects of placing one or two cages in instrumented posterior lumbar interbody fusion.

PubMed

Zhang, Mingzheng; Pu, Fang; Xu, Liqiang; Zhang, Linlin; Yao, Jie; Li, Deyu; Wang, Yu; Fan, Yubo

2016-06-01

Posterior lumbar interbody fusion (PLIF) is an established surgical procedure for spine stabilization after the removal of an intervertebral disc. Researches have shown that inserting a single oblique cage has a similar immediate effect to coupled cages, and it has been proposed that single-cage PLIF is a useful alternative to traditional two-cage PLIF. However, it is not clear whether placing one or two cages represents the best choice for long-term fusion. The aim of this study is to examine how cage placement affects bone remodeling after PLIF surgery, and how this consequently impacts the long-term fusion process. A finite element model of a L3-L4 lumbar spine with PLIF was developed. The spinal segment was modeled with a partial laminectomy and a discectomy with partial facetectomy, and implanted with posterior pedicle screws. Two models were analyzed, one with coupled parallel cages and one with a single oblique cage. Adaptive bone remodeling was simulated according to Huiskes' criterion. The results showed that in the initial state prior to any bone remodeling, cage stress, cage subsidence and cage dislodgement in the single cage model were all greater than in the coupled cage model. In the final state after significant bone remodeling had taken place, these parameters had decreased in both models and the differences between the two models were reduced. Also, the single cage model demonstrated superior bone development in the bone graft when placed under a constant 400 N axial compressive load. Based on the long-term results, instrumented PLIF with a single cage could also be encouraged in clinical practice.

A novel test cage with an air ventilation system as an alternative to conventional cages for the efficacy testing of mosquito repellents.

PubMed

Obermayr, U; Rose, A; Geier, M

2010-11-01

We have developed a novel test cage and improved method for the evaluation of mosquito repellents. The method is compatible with the United States Environmental Protection Agency, 2000 draft OPPTS 810.3700 Product Performance Test Guidelines for Testing of Insect Repellents. The Biogents cages (BG-cages) require fewer test mosquitoes than conventional cages and are more comfortable for the human volunteers. The novel cage allows a section of treated forearm from a volunteer to be exposed to mosquito probing through a window. This design minimizes residual contamination of cage surfaces with repellent. In addition, an air ventilation system supplies conditioned air to the cages after each single test, to flush out and prevent any accumulation of test substances. During biting activity tests, the untreated skin surface does not receive bites because of a screen placed 150 mm above the skin. Compared with the OPPTS 810.3700 method, the BG-cage is smaller (27 liters, compared with 56 liters) and contains 30 rather than hundreds of blood-hungry female mosquitoes. We compared the performance of a proprietary repellent formulation containing 20% KBR3023 with four volunteers on Aedes aegypti (L.) (Diptera: Culicidae) in BG- and conventional cages. Repellent protection time was shorter in tests conducted with conventional cages. The average 95% protection time was 4.5 +/- 0.4 h in conventional cages and 7.5 +/- 0.6 h in the novel BG-cages. The protection times measured in BG-cages were more similar to the protection times determined with these repellents in field tests.

Influence of 5 Different Caging Types and the Use of Cage-Changing Stations on Mouse Allergen Exposure

PubMed Central

Feistenauer, Susan; Sander, Ingrid; Schmidt, Jörg; Zahradnik, Eva; Raulf, Monika; Brielmeier, Markus

2014-01-01

Animal allergens constitute a serious health risk in laboratory animal facilities. To assess possibilities for allergen reduction by technical and organizational measures, we studied personnel exposure to mouse urinary aeroallergens in an animal facility with a holding capacity of 30,000 cages. Short-term (2 h) and intermediate-term (12 h) stationary samples (n = 107) and short-term (2 h) personnel samples (n = 119) were collected on polytetrafluorethylene filters by using air pumps. Long-term (14 d) stationary dust samples containing airborne allergens (n = 165) were collected with electrostatic dust fall collectors (EDC). Mouse allergens were quantified by ELISA. Personnel samples were collected during bedding disposal and refilling of clean cages as well as during cage changing with and without use of cage-changing station. Animal rooms were equipped with either open cages, cages with a soft filter top, cages with a rigid filter top (static microisolation caging), or with individually ventilated cages (IVC) with either a sealed or nonsealed lid, each in positive- or negative-pressure mode. Highest personnel allergen exposure was detected during cage change and emptying of soiled cages. Allergen concentrations were lowest in rooms with sealed IVC under positive or negative pressure, with unsealed IVC under negative pressure, and with static microisolation caging. The use of cage-changing stations and a vacuum bedding-disposal system reduced median personnel exposures 14- to 25-fold, respectively. Using sealed IVC and changing stations minimized allergen exposure, indicating that state-of-the-art equipment reduces exposure to mouse allergens and decreases health risks among animal facility personnel. PMID:25199090

Flexibility and fatigue evaluation of oblique as compared with anterior lumbar interbody cages with integrated endplate fixation.

PubMed

Freeman, Andrew L; Camisa, William J; Buttermann, Glenn R; Malcolm, James R

2016-01-01

This study was undertaken to quantify the in vitro range of motion (ROM) of oblique as compared with anterior lumbar interbody devices, pullout resistance, and subsidence in fatigue. Anterior and oblique cages with integrated plate fixation (IPF) were tested using lumbar motion segments. Flexibility tests were conducted on the intact segments, cage, cage + IPF, and cage + IPF + pedicle screws (6 anterior, 7 oblique). Pullout tests were then performed on the cage + IPF. Fatigue testing was conducted on the cage + IPF specimens for 30,000 cycles. No ROM differences were observed in any test group between anterior and oblique cage constructs. The greatest reduction in ROM was with supplemental pedicle screw fixation. Peak pullout forces were 637 ± 192 N and 651 ± 127 N for the anterior and oblique implants, respectively. The median cage subsidence was 0.8 mm and 1.4 mm for the anterior and oblique cages, respectively. Anterior and oblique cages similarly reduced ROM in flexibility testing, and the integrated fixation prevented device displacement. Subsidence was minimal during fatigue testing, most of which occurred in the first 2500 cycles.

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

Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans.

PubMed

Graham, Mark J; Lee, Richard G; Bell, Thomas A; Fu, Wuxia; Mullick, Adam E; Alexander, Veronica J; Singleton, Walter; Viney, Nick; Geary, Richard; Su, John; Baker, Brenda F; Burkey, Jennifer; Crooke, Stanley T; Crooke, Rosanne M

2013-05-24

Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

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

Comparison of Expandable and Fixed Interbody Cages in a Human Cadaver Corpectomy Model: Fatigue Characteristics.

PubMed

Pekmezci, Murat; Tang, Jessica A; Cheng, Liu; Modak, Ashin; McClellan, Robert T; Buckley, Jenni M; Ames, Christopher P

2016-11-01

In vitro cadaver biomechanics study. The goal of this study is to compare the in situ fatigue life of expandable versus fixed interbody cage designs. Expandable cages are becoming more popular, in large part, due to their versatility; however, subsidence and catastrophic failure remain a concern. This in vitro analysis investigates the fatigue life of expandable and fixed interbody cages in a single level human cadaver corpectomy model by evaluating modes of subsidence of expandable and fixed cages as well as change in stiffness of the constructs with cyclic loading. Nineteen specimens from 10 human thoracolumbar spines (T10-L2, L3-L5) were biomechanically evaluated after a single level corpectomy that was reconstructed with an expandable or fixed cage and anterior dual rod instrumentation. All specimens underwent 98 K cycles to simulate 3 months of postoperative weight bearing. In addition, a third group with hyperlordotic cages was used to simulate catastrophic failure that is observed in clinical practice. Three fixed and 2 expandable cages withstood the cyclic loading despite perfect sagittal and coronal plane fitting of the endcaps. The majority of the constructs settled in after initial subsidence. The catastrophic failures that were observed in clinical practice could not be reproduced with hyperlordotic cages. However, all cages in this group subsided, and 60% resulted in endplate fractures during deployment of the cage. Despite greater surface contact area, expandable cages have a trend for higher subsidence rates when compared with fixed cages. When there is edge loading as in the hyperlordotic cage scenario, there is a higher risk of subsidence and intraoperative fracture during deployment of expandable cages.

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

Design and modeling of Faraday cages for substrate noise isolation

NASA Astrophysics Data System (ADS)

Wu, Joyce H.; del Alamo, Jesús A.

2013-07-01

A Faraday cage structure using through-substrate vias is an effective strategy to suppress substrate crosstalk, particularly at high frequencies. Faraday cages can reduce substrate noise by 32 dB at 10 GHz, and 26 dB at 50 GHz. We have developed lumped-element, equivalent circuit models of the Faraday cages and test structures to better understand the performance of the Faraday cages. These models compare well to measured results and show that the vias of the Faraday cage act as an RLC shunt to ground that draws substrate current. Designing a Faraday cage to achieve optimum isolation requires low via impedance and mitigation of via sidewall capacitance. The Faraday cage inductance is correlated to the number of vias and via spacing of the cage and can be optimized for the frequency of operation.

[Experiences with cage combinations for guinea pigs].

PubMed

von Zychlinski, J

1989-01-01

Special cage units described in 1982 for guinea pigs have been used either as cages for small groups of breeding animals or for caging of growing animals. By using these cages the following advantages have been noted; the cage size can be adapted to number, age and body weight of the animals; aggression and panic are avoided by corners, walls and tunnels; economic use of breeding males by mating with more females.

Ammonia and Carbon Dioxide Concentrations in Disposable and Reusable Ventilated Mouse Cages

PubMed Central

Silverman, Jerald; Bays, David W; Cooper, Sheldon F; Baker, Stephen P

2008-01-01

This study compares resuable and disposable individually ventilated mouse cages in terms of the formation of intracage CO2 and NH3. Crl:CD-1(ICR) female mice were placed in either disposable or reusable ventilated cages in a positive pressure animal rack. Intracage CO2 and NH3 were measured once daily for 9 d; temperature and relative humidity were monitored for the first 7 d. Results indicated higher CO2 levels in the rear of the disposable cages and in the front of the reusable cages. This pattern corresponded to where the mice tended to congregate. However, CO2 concentrations did not differ significantly between the 2 cage types. Average CO2 levels in both cage types never exceeded approximately 3000 ppm. Intracage NH3 began to rise in the reusable cages on day 4, reached approximately 50 ppm by day 5 and by day 9 was greater than 150 ppm at the cages' rear sampling port while remaining at approximately 70 ppm at the front sampling port. Intracage NH3 levels in the disposable cages remained less than or equal to 3.2 ppm. Intracage temperature and relative humidity were approximately the same in both cage types. We concluded that the disposable ventilated cage performed satisfactorily under the conditions of the study. PMID:18351723

Effects of Furnished Cage Type on Behavior and Welfare of Laying Hens.

PubMed

Li, Xiang; Chen, Donghua; Li, Jianhong; Bao, Jun

2016-06-01

This study was conducted to compare the effects of layout of furniture (a perch, nest, and sandbox) in cages on behavior and welfare of hens. Two hundred and sixteen Hyline Brown laying hens were divided into five groups (treatments) with four replicates per group: small furnished cages (SFC), medium furnished cages type I (MFC-I), medium furnished cages type II (MFC-II), and medium furnished cages type III (MFC-III) and conventional cages (CC). The experiment started at 18 week of age and finished at 52 week of age. Hens' behaviors were filmed during the following periods: 8:00 to 10:00; 13:00 to 14:00; 16:00 to 17:00 on three separate days and two hens from each cage were measured for welfare parameters at 50 wk of age. The results showed that feeding and laying of all hens showed no effect by cage type (p>0.05), and the hens in the furnished cages had significantly lower standing and higher walking than CC hens (p<0.05). The birds in MFC-III had significant higher preening, scratching and feather-pecking behavior than in the other cages (p<0.05). No difference in nesting behavior was found in the hens between the furnished cages (p>0.05). The hens in MFC-I, -II, and -III showed a significant higher socializing behavior than SFC and CC (p<0.05). The lowest perching was for the hens in SFC and the highest perching found for the hens in MFC-III. Overall, the hens in CC showed poorer welfare conditions than the furnished cages, in which the feather condition score, gait score and tonic immobility duration of the hens in CC was significantly higher than SFC, MFC-I, MFC-II, and MFC-III (p<0.05). In conclusion, the furnished cage design affected both behavior and welfare states of hens. Overall, MFC-III cage design was better than SFC, MFC-I, and MFC-II cage designs.

Effects of Furnished Cage Type on Behavior and Welfare of Laying Hens

PubMed Central

Li, Xiang; Chen, Donghua; Li, Jianhong; Bao, Jun

2016-01-01

This study was conducted to compare the effects of layout of furniture (a perch, nest, and sandbox) in cages on behavior and welfare of hens. Two hundred and sixteen Hyline Brown laying hens were divided into five groups (treatments) with four replicates per group: small furnished cages (SFC), medium furnished cages type I (MFC-I), medium furnished cages type II (MFC-II), and medium furnished cages type III (MFC-III) and conventional cages (CC). The experiment started at 18 week of age and finished at 52 week of age. Hens’ behaviors were filmed during the following periods: 8:00 to 10:00; 13:00 to 14:00; 16:00 to 17:00 on three separate days and two hens from each cage were measured for welfare parameters at 50 wk of age. The results showed that feeding and laying of all hens showed no effect by cage type (p>0.05), and the hens in the furnished cages had significantly lower standing and higher walking than CC hens (p<0.05). The birds in MFC-III had significant higher preening, scratching and feather-pecking behavior than in the other cages (p<0.05). No difference in nesting behavior was found in the hens between the furnished cages (p>0.05). The hens in MFC-I, −II, and −III showed a significant higher socializing behavior than SFC and CC (p<0.05). The lowest perching was for the hens in SFC and the highest perching found for the hens in MFC-III. Overall, the hens in CC showed poorer welfare conditions than the furnished cages, in which the feather condition score, gait score and tonic immobility duration of the hens in CC was significantly higher than SFC, MFC-I, MFC-II, and MFC-III (p<0.05). In conclusion, the furnished cage design affected both behavior and welfare states of hens. Overall, MFC-III cage design was better than SFC, MFC-I, and MFC-II cage designs. PMID:26954171

One and two hydrogen molecules in the large cage of the structure II clathrate hydrate: quantum translation-rotation dynamics close to the cage wall.

PubMed

Sebastianelli, Francesco; Xu, Minzhong; Kanan, Dalal K; Bacić, Zlatko

2007-07-19

We have performed a rigorous theoretical study of the quantum translation-rotation (T-R) dynamics of one and two H2 and D2 molecules confined inside the large hexakaidecahedral (5(12)6(4)) cage of the sII clathrate hydrate. For a single encapsulated H2 and D2 molecule, accurate quantum five-dimensional calculations of the T-R energy levels and wave functions are performed that include explicitly, as fully coupled, all three translational and the two rotational degrees of freedom of the hydrogen molecule, while the cage is taken to be rigid. In addition, the ground-state properties, energetics, and spatial distribution of one and two p-H2 and o-D2 molecules in the large cage are calculated rigorously using the diffusion Monte Carlo method. These calculations reveal that the low-energy T-R dynamics of hydrogen molecules in the large cage are qualitatively different from that inside the small cage, studied by us recently. This is caused by the following: (i) The large cage has a cavity whose diameter is about twice that of the small cage for the hydrogen molecule. (ii) In the small cage, the potential energy surface (PES) for H2 is essentially flat in the central region, while in the large cage the PES has a prominent maximum at the cage center, whose height exceeds the T-R zero-point energy of H2/D2. As a result, the guest molecule is excluded from the central part of the large cage, its wave function localized around the off-center global minimum. Peculiar quantum dynamics of the hydrogen molecule squeezed between the central maximum and the cage wall manifests in the excited T-R states whose energies and wave functions differ greatly from those for the small cage. Moreover, they are sensitive to the variations in the hydrogen-bonding topology, which modulate the corrugation of the cage wall.

Sagittal Plane Correction Using the Lateral Transpsoas Approach: A Biomechanical Study on the Effect of Cage Angle and Surgical Technique on Segmental Lordosis.

PubMed

Melikian, Rojeh; Yoon, Sangwook Tim; Kim, Jin Young; Park, Kun Young; Yoon, Caroline; Hutton, William

2016-09-01

Cadaveric biomechanical study. To determine the degree of segmental correction that can be achieved through lateral transpsoas approach by varying cage angle and adding anterior longitudinal ligament (ALL) release and posterior element resection. Lordotic cage insertion through the lateral transpsoas approach is being used increasingly for restoration of sagittal alignment. However, the degree of correction achieved by varying cage angle and ALL release and posterior element resection is not well defined. Thirteen lumbar motion segments between L1 and L5 were dissected into single motion segments. Segmental angles and disk heights were measured under both 50 N and 500 N compressive loads under the following conditions: intact specimen, discectomy (collapsed disk simulation), insertion of parallel cage, 10° cage, 30° cage with ALL release, 30° cage with ALL release and spinous process (SP) resection, 30° cage with ALL release, SP resection, facetectomy, and compression with pedicle screws. Segmental lordosis was not increased by either parallel or 10° cages as compared with intact disks, and contributed small amounts of lordosis when compared with the collapsed disk condition. Placement of 30° cages with ALL release increased segmental lordosis by 10.5°. Adding SP resection increased lordosis to 12.4°. Facetectomy and compression with pedicle screws further increased lordosis to approximately 26°. No interventions resulted in a decrease in either anterior or posterior disk height. Insertion of a parallel or 10° cage has little effect on lordosis. A 30° cage insertion with ALL release resulted in a modest increase in lordosis (10.5°). The addition of SP resection and facetectomy was needed to obtain a larger amount of correction (26°). None of the cages, including the 30° lordotic cage, caused a decrease in posterior disk height suggesting hyperlordotic cages do not cause foraminal stenosis. N/A.

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.

The Transcriptome of Exophiala dermatitidis during Ex-vivo Skin Model Infection

PubMed Central

Poyntner, Caroline; Blasi, Barbara; Arcalis, Elsa; Mirastschijski, Ursula; Sterflinger, Katja; Tafer, Hakim

2016-01-01

The black yeast Exophiala dermatitidis is a widespread polyextremophile and human pathogen, that is found in extreme natural habitats and man-made environments such as dishwashers. It can cause various diseases ranging from phaeohyphomycosis and systemic infections, with fatality rates reaching 40%. While the number of cases in immunocompromised patients are increasing, knowledge of the infections, virulence factors and host response is still scarce. In this study, for the first time, an artificial infection of an ex-vivo skin model with Exophiala dermatitidis was monitored microscopically and transcriptomically. Results show that Exophiala dermatitidis is able to actively grow and penetrate the skin. The analysis of the genomic and RNA-sequencing data delivers a rich and complex transcriptome where circular RNAs, fusion transcripts, long non-coding RNAs and antisense transcripts are found. Changes in transcription strongly affect pathways related to nutrients acquisition, energy metabolism, cell wall, morphological switch, and known virulence factors. The L-Tyrosine melanin pathway is specifically upregulated during infection. Moreover the production of secondary metabolites, especially alkaloids, is increased. Our study is the first that gives an insight into the complexity of the transcriptome of Exophiala dermatitidis during artificial skin infections and reveals new virulence factors. PMID:27822460

Integrative analysis of circRNAs acting as ceRNAs involved in ethylene pathway in tomato.

PubMed

Wang, Yunxiang; Wang, Qing; Gao, Lipu; Zhu, Benzhong; Luo, Yunbo; Deng, Zhiping; Zuo, Jinhua

2017-11-01

Circular RNAs (circRNAs) are a large class of non-coding endogenous RNAs that could act as competing endogenous RNAs (ceRNAs) to terminate the mRNA targets' suppression of miRNAs. To elucidate the intricate regulatory roles of circRNAs in the ethylene pathway in tomato fruit, deep sequencing and bioinformatics methods were performed. After strict screening, a total of 318 circRNAs were identified. Among these circRNAs, 282 were significantly differentially expressed among wild-type and sense-/antisense-LeERF1 transgenic tomato fruits. Besides, 1254 target genes were identified and a large amount of them were found to be involved in ethylene pathway. In addition, a sophisticated regulatory model consisting of circRNAs, target genes and ethylene was set up. Importantly, 61 circRNAs were found to be potential ceRNAs to combine with miRNAs and some of the miRNAs had been revealed to participate in the ethylene signaling pathway. This research further raised the possibility that the ethylene pathway in tomato fruit may be under the regulation of various circRNAs and provided a new perspective of the roles of circRNAs. © 2017 Scandinavian Plant Physiology Society.

Therapeutic nucleic acids: current clinical status

PubMed Central

Sridharan, Kannan

2016-01-01

Abstract Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are simple linear polymers that have been the subject of considerable research in the last two decades and have now moved into the realm of being stand‐alone therapeutic agents. Much of this has stemmed from the appreciation that they carry out myriad functions that go beyond mere storage of genetic information and protein synthesis. Therapy with nucleic acids either uses unmodified DNA or RNA or closely related compounds. From both a development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. This review addresses therapeutic uses of DNA based on antisense oligonucleotides, DNA aptamers and gene therapy; and therapeutic uses of RNA including micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs. With their specificity, functional diversity and limited toxicity, therapeutic nucleic acids hold enormous promise. However, challenges that need to be addressed include targeted delivery, mass production at low cost, sustaining efficacy and minimizing off‐target toxicity. Technological developments will hold the key to this and help accelerate drug approvals in the years to come. PMID:27111518

Nasal Histopathology and Intracage Ammonia Levels in Female Groups and Breeding Mice Housed in Static Isolation Cages

PubMed Central

Mexas, Angela M; Brice, Angela K; Caro, Adam C; Hillanbrand, Troy S

2015-01-01

Many factors influence ammonia levels in rodent cages, and high intracage ammonia has been associated with specific types of abnormal nasal pathology in mice. The use of autoclaved corncob bedding and the maintenance of low room humidity reduce the accumulation of ammonia in mouse cages. However, there are no engineering standards that define the limits of ammonia exposure for mice housed in static isolation cages. Regulatory guidance indicates that solid bottom cages must be sanitized at least weekly and that cage components in direct contact with animals must be sanitized at least every 2 wk. Common practice is to replace the bottoms and bedding of static isolation cages once weekly. To determine whether changing static isolation cages once weekly is an appropriate performance standard for mice, we prospectively evaluated the relationship between ammonia levels, nasal histopathology, and housing densities in various grouping strategies of mice housed in static isolation cages. Here, we report that the average nasal pathology score per cage and intracage ammonia levels were correlated, but nasal pathology scores did not differ among mice housed in breeding pairs, breeding trios, or female groups. In light of ammonia levels and histopathology scores as performance standards, these results suggest that a weekly cage-change frequency for static isolation cages does not result in adverse effects. Our results provide evidence to support current practices in the use of static isolation cages for housing laboratory mice in modern vivaria. PMID:26424245

Disparities in Ammonia, Temperature, Humidity, and Airborne Particulate Matter between the Micro-and Macroenvironments of Mice in Individually Ventilated Caging

PubMed Central

Rosenbaum, Matthew D; VandeWoude, Susan; Volckens, John; Johnson, Thomas E

2010-01-01

Animal room environmental parameters typically are monitored with the assumption that the environment within the cage closely mirrors the room environment. This study evaluated that premise by examining macro- (room) and microenvironmental (cage) parameters in individually ventilated cages housing mice with variable amounts of bedding over a period of 17 d without cage changes. Intracage ammonia levels remained within recommended human guidelines but were higher than room levels, confirming that microisolation caging is efficient at preventing ammonia generated from animal waste from escaping into the room. Humidity and temperature within cages were consistently higher than room levels. Particles in the room predominantly consisted of fine particles (diameter less than 2.5 µm), presumably from the ambient atmosphere; some of these particles were found in the cage microenvironment. In addition, mouse activity within cages produced larger particles, and these particles contributed to substantially higher aerosol mass concentrations within the cage. These findings demonstrate that, although cage and room environmental parameters differ, knowledge of room environmental conditions can be used to predict certain conditions within the cage. This association is relevant in that typical animal care standard operating procedures rely on room measurements, not intracage measurements, which arguably are more important for assessing animal welfare. Further, location and ambient climate can influence particle concentrations in the room, and consequently within the animal cage, suggesting local weather patterns and air quality may account for variability among studies conducted at sites that are geographically divergent. PMID:20353692

Elimination of Subsidence with 26-mm-Wide Cages in Extreme Lateral Interbody Fusion.

PubMed

Lang, Gernot; Navarro-Ramirez, Rodrigo; Gandevia, Lena; Hussain, Ibrahim; Nakhla, Jonathan; Zubkov, Micaella; Härtl, Roger

2017-08-01

Extreme lateral interbody fusion (ELIF) has gained popularity as a minimally invasive technique for indirect decompression. However, graft subsidence potentially threatens long-term success of ELIF. This study evaluated whether 26-mm-wide cages can eliminate subsidence and subsequent loss of decompression in ELIF. Patients undergoing ELIF surgery using a 26-mm-wide cage were analyzed retrospectively. Patient demographics and perioperative data for radiographic and clinical outcomes were recorded. Radiographic parameters included regional sagittal lumbar lordosis and foraminal and disc height. Clinical parameters were evaluated using the Oswestry Disability Index and visual analog scale. Subsidence of 26-mm-wide cages was compared with previous outcomes of patients undergoing ELIF using 18-mm-wide and 22-mm-wide cages. There were 21 patients and 28 spinal segments analyzed. Radiographic outcome measures such as disc and foraminal height revealed significant improvement at follow-up compared with before surgery (P = 0.001). Postoperative to last follow-up cage subsidence translated into 0.34 mm ± 0.26 and -0.55 mm ± 0.64 in disc and foraminal height loss, respectively. Patients with 26-mm-wide cages experienced less subsidence by means of disc (26 mm vs. 18 mm and 22 mm, P ≤ 0.05) and foraminal height (26 mm vs. 18 mm, P = 0.005; 26 mm vs. 22 mm, P = 0.208) loss compared with patients receiving 18-mm-wide and 22-mm-wide cages. The 26-mm-wide cages almost eliminated cage subsidence in ELIF. Compared with 18-mm-wide and 22-mm-wide cages, 26-mm-wide cages significantly reduced cage subsidence in ELIF at midterm follow-up. A 26-mm-wide cage should be used in ELIF to achieve sustained indirect decompression. Copyright © 2017. Published by Elsevier Inc.

Do Lordotic Cages Provide Better Segmental Lordosis Versus Nonlordotic Cages in Lateral Lumbar Interbody Fusion (LLIF)?

PubMed

Sembrano, Jonathan N; Horazdovsky, Ryan D; Sharma, Amit K; Yson, Sharon C; Santos, Edward R G; Polly, David W

2017-05-01

A retrospective comparative radiographic review. To evaluate the radiographic changes brought about by lordotic and nonlordotic cages on segmental and regional lumbar sagittal alignment and disk height in lateral lumbar interbody fusion (LLIF). The effects of cage design on operative level segmental lordosis in posterior interbody fusion procedures have been reported. However, there are no studies comparing the effect of sagittal implant geometry in LLIF. This is a comparative radiographic analysis of consecutive LLIF procedures performed with use of lordotic and nonlordotic interbody cages. Forty patients (61 levels) underwent LLIF. Average age was 57 years (range, 30-83 y). Ten-degree lordotic PEEK cages were used at 31 lumbar interbody levels, and nonlordotic cages were used at 30 levels. The following parameters were measured on preoperative and postoperative radiographs: segmental lordosis; anterior and posterior disk heights at operative level; segmental lordosis at supra-level and subjacent level; and overall lumbar (L1-S1) lordosis. Measurement changes for each cage group were compared using paired t test analysis. The use of lordotic cages in LLIF resulted in a significant increase in lordosis at operative levels (2.8 degrees; P=0.01), whereas nonlordotic cages did not (0.6 degrees; P=0.71) when compared with preoperative segmental lordosis. Anterior and posterior disk heights were significantly increased in both groups (P<0.01). Neither cage group showed significant change in overall lumbar lordosis (lordotic P=0.86 vs. nonlordotic P=0.25). Lordotic cages provided significant increase in operative level segmental lordosis compared with nonlordotic cages although overall lumbar lordosis remained unchanged. Anterior and posterior disk heights were significantly increased by both cages, providing basis for indirect spinal decompression.

Development of furnished cages for laying hens.

PubMed

Appleby, M C; Walker, A W; Nicol, C J; Lindberg, A C; Freire, R; Hughes, B O; Elson, H A

2002-09-01

1. A 3-year trial was carried out of cages for laying hens, occupying a full laying house. The main cage designs used were 5000 cm2 in area, 50 cm high at the rear and furnished with nests and perches. F cages had a front rollaway nest at the side, lined with artificial turf. FD cages also had a dust bath containing sand over the nest. H cages had two nest hollows at the side, one in front of the other. They were compared with conventional cages 2500 cm2 in area and 38 cm high at the rear. 2. Cages were stocked with from 4 to 8 ISA Brown hens per cage, resulting in varied allowances of area, feeder and perch per bird. No birds were beak trimmed. In F and FD cages two further treatments were applied: nests and dust baths were sometimes fitted with gates to exclude birds from dust baths in the morning and from both at night; elevated food troughs, with a lip 33 cm above the cage floor, were compared with standard troughs. 3. Management of the house was generally highly successful, with temperature control achieved by ventilation. Egg production was above breeders' standards and not significantly affected by cage design. More eggs per bird were collected when there were fewer birds per cage but food consumption also then tended to be higher. 4. The number of downgraded eggs was variable, with some tendency for more in furnished cages. Eggs laid in dust baths were often downgraded. Those laid at the back of the cage were frequently dirty because of accumulation of droppings. H nests were unsuccessful, with less than 50% of eggs laid in the nest hollows. However, up to 93% of eggs were laid in front rollaways, and few of these were downgraded. 5. Feather and foot damage were generally less in furnished than in conventional cages, greater where there were more birds per cage. With an elevated food trough there was less feather damage but more overgrowth of claws. In year 2, mortality was greater in cages with more birds. 6. Pre-laying behaviour was mostly settled in front rollaway nests. Dust baths were used more for pecking and scratching than for dust bathing. Comfort behaviour was more frequent in furnished cages than conventional, although still not frequent. Locomotion was strongly affected by number of birds per cage or by space per bird, being reduced by crowding. Most birds perched at night except in one treatment providing only 10.7 cm perch per bird. 7. Behaviour was more unrestricted and varied, and physical condition was better, in furnished than in conventional cages. However, egg production will cost more in furnished cages, partly because more eggs are downgraded. Dust baths must be fitted with gates that the birds cannot open from outside, but gates for nest boxes were found unnecessary. If a low perch is fitted it must be far enough from the back of the cage for birds to walk there. 8. Where there was less space per bird (more birds per cage) than the requirements in the 1999 European Commission Directive on laying hens, there were: fewer eggs per hen, but still above the breeders' target; lower food consumption; more feather and foot damage, but less than in conventional cages; higher mortality in one trial out of three; less freedom of movement. However, the results were still very good even with 8 birds per cage, and support the principle that furnished cages provide an acceptable way of protecting the welfare of laying hens.

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

Improving Breast Cancer Diagnosis by Antisense Targeting

DTIC Science & Technology

2007-08-01

aminohexanoic acid linker (21st Century Biochemicals, Mar- lboro, MA). The biotinylated cholesterol was synthesized by reacting biotinyl-3,6...radiolabel was placed on the MORF. The model carriers were a tat and a polyarginine peptide and cholesterol . The 25 mer MORF was selected as a suitable test...the MORF/streptavidin/ cholesterol accumulations were lower but stil1 significant). Furthermore, accumulations of the antisense MORF/streptavidin

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

Rearing Laying Hens in Aviaries Reduces Fearfulness following Transfer to Furnished Cages

PubMed Central

Brantsæter, Margrethe; Tahamtani, Fernanda M.; Moe, Randi O.; Hansen, Tone B.; Orritt, Rachel; Nicol, Christine; Janczak, Andrew M.

2016-01-01

Appropriate rearing is essential for ensuring the welfare and productivity of laying hens. Early experience has the potential to affect the development of fearfulness. This study tested whether rearing in aviaries, as opposed to cages, reduces the fearfulness of laying hens after transfer to furnished cages. Fear responses were recorded as avoidance of a novel object in the home cage. Lohmann Selected Leghorns were reared in an aviary system or conventional rearing cages and then transported to furnished cages at 16 weeks, before the onset of lay. Observations of a selection of birds were conducted at 19 (N = 50 independent cages) and 21 (N = 48 independent cages) weeks of age. At 19 and 21 weeks, cage-reared birds showed higher levels of fearfulness indicated by spending more time away from the novel object compared to aviary-reared birds. These results suggest that rearing in an enriched aviary environment reduces fearfulness up to the fifth week after transfer to a new housing system, compared to rearing in cages. PMID:26955634

Acclimatization of mice to different cage types and social groupings with respect to fecal secretion of IgA and corticosterone metabolites.

PubMed

Bundgaard, Cathrine Juel; Kalliokoski, Otto; Abelson, Klas S P; Hau, Jann

2012-01-01

Stress associated with transport and change of environment may have widespread effects on physiological parameters in laboratory animals. To investigate the time needed for mice to acclimatize to a new environment, based on fecal IgA and corticosterone excretion, eightweek-old BALB/c mice of both genders were housed either in groups of eight in different cage types in open conventional cages, in Individual Ventilated Cages (IVC), in open conventional cages inside a plastic isolator, or in different group sizes (8, 4, 8, 10 or 12 mice in each group) in open conventional cages. Feces were collected from each cage on routine cage changing. There was no significant difference in corticosterone excretion in feces between animals housed in the different cage types or between animals housed in different group sizes. IgA excretion for both males and females was found to be affected by transfer of mice into a novel cage, and it was found that it takes at least four weeks for the mice to acclimatize to a new environment with respect to this parameter.

Investigation of compressible vortex flow characteristics

NASA Technical Reports Server (NTRS)

Muirhead, V. U.

1977-01-01

The nature of intense air vortices was studied and the factors which determine the intensity and rate of decay of both single and pairs of vortices were investigated. Vortex parameters of axial pressure differential, circulation, outflow rates, separation distance and directions of rotation were varied. Unconfined vortices, generated by a single rotating cage, were intensified by an increasing axial pressure gradient. Breakdown occurred when the axial gradient became negligible. The core radius was a function of the axial gradient. Dual vortices, generated by two counterrotating cages, rotated opposite to the attached cages. With minimum spacing only one vortex was formed which rotated in a direction opposite to the attached cage. When one cage rotated at half the speed of the other cage, one vortex formed at the higher speed cage rotating in the cage direction.

Animal Welfare and Food Safety Aspects of Confining Broiler Chickens to Cages

PubMed Central

Shields, Sara; Greger, Michael

2013-01-01

Simple Summary In commercial chicken meat production, broiler chickens are usually kept on the floor in ware-house like buildings, but the use of cages is becoming more common. Confining chickens to cages is a welfare problem, as has been thoroughly demonstrated for laying hens used for egg production. Caged broiler chickens may suffer from poor bone strength due to lack of exercise, feather loss, and restriction of natural behavior. There are also potential food safety concerns associated with the use of cages. While cages may provide an economic advantage in some geographical regions of the world, the severe, inherent disadvantages should also be considered before cages are more widely adopted in the global broiler chicken industry. Abstract In most areas of the world, broiler chickens are raised in floor systems, but cage confinement is becoming more common. The welfare of broiler chickens in cages is affected by movement restriction, poor bone strength due to lack of exercise, and prevention of key behavioral patterns such as dustbathing and ground scratching. Cages for broiler chickens also have a long history of causing skin and leg conditions that could further compromise welfare, but a lack of controlled studies makes it difficult to draw conclusions about newer cage designs. Cage environments are usually stocked at a higher density than open floor systems, and the limited studies available suggest that caging may lead to increased levels of fear and stress in the birds. Further, birds reared on the floor appear less likely to harbor and shed Salmonella, as litter may serve as a seeding agent for competitive exclusion by other microorganisms. Cages for laying hens used in egg production have met with substantial opposition due to welfare concerns and caging broiler chickens will likely be subject to the same kinds of social disapproval. PMID:26487409

Ammonia Levels and Urine-Spot Characteristics as Cage-Change Indicators for High-Density Individually Ventilated Mouse Cages

PubMed Central

Washington, Ida M; Payton, Mark E

2016-01-01

Mouse cage and bedding changes are potentially stressful to mice and are also labor- and resource-intensive. These changes are often performed on a calendar-based schedule to maintain a clean microenvironment and limit the concentrations of ammonia to which mice and workers are exposed. The current study sought to establish a performance-based approach to mouse cage-changing that uses urine spot characteristics as visual indicators of intracage ammonia levels. Colorimetric ammonia indicators were used to measure ammonia levels in individually-ventilated cages (IVC) housing male or female mice (n =5 per cage) of various strains at 1 to 16 d after cage change. Urine spot characteristics were correlated with ammonia levels to create a visual indicator of the cage-change criterion of 25 ppm ammonia. Results demonstrated a consistent increase in ammonia levels with days since cage change, with cages reaching the cage-change criterion at approximately 10 d for IVC containing male mice and 16 d for those with female mice. Ammonia levels were higher for male than female mice but were not correlated with mouse age. However, urine spot diameter, color, and edge characteristics were strongly correlated with ammonia levels. Husbandry practices based on using urine spot characteristics as indicators of ammonia levels led to fewer weekly cage changes and concomitant savings in labor and resources. Therefore, urine spot characteristics can be used as visual indicators of intracage ammonia levels for use of a performance (urine spot)-based approach to cage-changing frequency that maintains animal health and wellbeing. PMID:27177558

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.

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays

PubMed Central

2011-01-01

Background With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles. Results RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts. Conclusions An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. PMID:21235785

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

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.

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

Compositions containing borane or carborane cage compounds and related applications

DOEpatents

Bowen, III, Daniel E; Eastwood, Eric A

2013-05-28

Compositions comprising a polymer-containing matrix and a filler comprising a cage compound selected from borane cage compounds, carborane cage compounds, metal complexes thereof, residues thereof, mixtures thereof, and/or agglomerations thereof, where the cage compound is not covalently bound to the matrix polymer. Methods of making and applications for using such compositions are also disclosed.

Compositions containing borane or carborane cage compounds and related applications

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

Bowen, III, Daniel E; Eastwood, Eric A

2014-11-11

Compositions comprising a polymer-containing matrix and a filler comprising a cage compound selected from borane cage compounds, carborane cage compounds, metal complexes thereof, residues thereof, mixtures thereof, and/or agglomerations thereof, where the cage compound is not covalently bound to the matrix polymer. Methods of making and applications for using such compositions are also disclosed.

Compositions containing borane or carborane cage compounds and related applications

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

Bowen, III, Daniel E.; Eastwood, Eric A.

2015-09-15

Compositions comprising a polymer-containing matrix and a filler comprising a cage compound selected from borane cage compounds, carborane cage compounds, metal complexes thereof, residues thereof, mixtures thereof, and/or agglomerations thereof, where the cage compound is not covalently bound to the matrix polymer. Methods of making and applications for using such compositions are also disclosed.

Cage Change Influences Serum Corticosterone and Anxiety-Like Behaviors in the Mouse

PubMed Central

Rasmussen, Skye; Miller, Melinda M.; Filipski, Sarah B.; Tolwani, Ravi J.

2011-01-01

Environmental variables and husbandry practices can influence physiology and alter behavior in mice. Our study evaluated the effects of cage change on serum corticosterone levels and anxiety-like behaviors in C57BL/6 male mice. We examined the effects of 3 different methods of performing cage transfer and of transferring mice to a clean or a dirty familiar cage microenvironment. The 3 different handling methods were forceps transfer, gentle transfer with gloved hands, and a passive transfer technique that did not involve active handling. Active handling methods and transfer to both clean and dirty cage microenvironments significantly increased serum corticosterone 15 min after cage change; however, at 60 min after cage change, levels were comparable to those of unmanipulated mice. Although the effects were transient, cage change altered anxiety-like behaviors in the open field when behavioral testing was performed on the same day. These results demonstrate that the timing of cage change can influence behavioral results, an effect that is an important consideration for rodent behavioral studies. PMID:21838975

Development of an integrated CAD-FEA system for patient-specific design of spinal cages.

PubMed

Zhang, Mingzheng; Pu, Fang; Xu, Liqiang; Zhang, Linlin; Liang, Hang; Li, Deyu; Wang, Yu; Fan, Yubo

2017-03-01

Spinal cages are used to create a suitable mechanical environment for interbody fusion in cases of degenerative spinal instability. Due to individual variations in bone structures and pathological conditions, patient-specific cages can provide optimal biomechanical conditions for fusion, strengthening patient recovery. Finite element analysis (FEA) is a valuable tool in the biomechanical evaluation of patient-specific cage designs, but the time- and labor-intensive process of modeling limits its clinical application. In an effort to facilitate the design and analysis of patient-specific spinal cages, an integrated CAD-FEA system (CASCaDeS, comprehensive analytical spinal cage design system) was developed. This system produces a biomechanical-based patient-specific design of spinal cages and is capable of rapid implementation of finite element modeling. By comparison with commercial software, this system was validated and proven to be both accurate and efficient. CASCaDeS can be used to design patient-specific cages with a superior biomechanical performance to commercial spinal cages.

Computed Tomography Measurement of Rib Cage Morphometry in Emphysema

PubMed Central

Sverzellati, Nicola; Colombi, Davide; Randi, Giorgia; Pavarani, Antonio; Silva, Mario; Walsh, Simon L.; Pistolesi, Massimo; Alfieri, Veronica; Chetta, Alfredo; Vaccarezza, Mauro; Vitale, Marco; Pastorino, Ugo

2013-01-01

Background Factors determining the shape of the human rib cage are not completely understood. We aimed to quantify the contribution of anthropometric and COPD-related changes to rib cage variability in adult cigarette smokers. Methods Rib cage diameters and areas (calculated from the inner surface of the rib cage) in 816 smokers with or without COPD, were evaluated at three anatomical levels using computed tomography (CT). CTs were analyzed with software, which allows quantification of total emphysema (emphysema%). The relationship between rib cage measurements and anthropometric factors, lung function indices, and %emphysema were tested using linear regression models. Results A model that included gender, age, BMI, emphysema%, forced expiratory volume in one second (FEV1)%, and forced vital capacity (FVC)% fit best with the rib cage measurements (R2 = 64% for the rib cage area variation at the lower anatomical level). Gender had the biggest impact on rib cage diameter and area (105.3 cm2; 95% CI: 111.7 to 98.8 for male lower area). Emphysema% was responsible for an increase in size of upper and middle CT areas (up to 5.4 cm2; 95% CI: 3.0 to 7.8 for an emphysema increase of 5%). Lower rib cage areas decreased as FVC% decreased (5.1 cm2; 95% CI: 2.5 to 7.6 for 10 percentage points of FVC variation). Conclusions This study demonstrates that simple CT measurements can predict rib cage morphometric variability and also highlight relationships between rib cage morphometry and emphysema. PMID:23935872

Influence of Tribology of Cage Material on Ball Bearing Cage Instability

NASA Astrophysics Data System (ADS)

Servais, S.; Duquenne, M.; Bozet, J.-L.

2013-09-01

By creating a solid lubricant thickness on both bearing races, a cage material of cryogenic ball bearing plays a significant role in the good dynamical behavior of the cage. This role is essential because of the lack of conventional lubricant into this kind of bearing.In this paper, a method able to identify if a particular potential cage material can correctly fulfill its function is described. In other words, if it can lead to a stable movement of the cage. From the identification of fundamental tribological parameters governing the cage behavior, this method presents an example of ranking of such materials. This is based on pin-on-disk tests and on a numerical approach.

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

Selective Androgen Receptor Down-Regulators (SARDs): A New Prostate Cancer Therapy

DTIC Science & Technology

2007-10-01

PCa (9). Thus far, the techniques that have been used to down-regulate the AR include antisense oligonucleotides (10, 11), ribozyme treatments (12...Our findings suggest that ICI may present a useful treatment option for patients with AR-dependent PCa. Unlike the ribozyme , antisense, siRNA, or...Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol

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

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.

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

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

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

25 CFR 542.14 - What are the minimum internal control standards for the cage?

Code of Federal Regulations, 2010 CFR

2010-04-01

... the cage shall be recorded on a cage accountability form on a per-shift basis. (6) Only cash, cash... identity, including photo identification. (8) A file for customers shall be prepared prior to acceptance of... cage shall be summarized on a cage accountability form on a per shift basis and shall be supported by...

Atypical gnawing and cage destructive behavior in one strain of laboratory rat.

PubMed

Jacobs, H K; Van Pelt, L F; Dunlap, B K

1984-06-01

Approximately 80% of a colony of 900 Long Evans rats, Blu:(LE), gnawed on polypropylene shoe box type cages around the automatic watering grommet. The gnawing led to cage destruction and escape of the animals. The addition of a large stainless steel shield between the original grommet backing and the cage prevented further gnawing on the cage.

Effects of a 28-Day Cage-Change Interval on Intracage Ammonia Levels, Nasal Histology, and Perceived Welfare of CD1 Mice

PubMed Central

Vogelweid, Catherine M; Zapien, Kathleen A; Honigford, Matthew J; Li, Linghui; Li, Hua; Marshall, Heather

2011-01-01

We measured daily intracage ammonia levels and performed weekly assessments of CD1 male, female, and breeder mice housed within disposable, ventilated cages that remained unchanged for 28 d. We tested housing groups comprising 1, 3, or 5 sex-matched mice per cage and breeder pairs with litters. Mice housed in cages with higher concentrations of ammonia developed degeneration and inflammatory lesions in the nasal passages. Mean ammonia exposure levels that caused rhinitis were 181 ppm for 18 d. Ammonia exposures of 93 ppm for 16 d caused necrosis of the olfactory epithelium, whereas 52 ppm for 13 d caused epithelial degeneration. Observers could not detect visible signs of rhinitis or identify cages with elevated ammonia levels, nor did they identify any sick or distressed mice. Observers consistently assigned poorer welfare scores as cages became dirtier. We conclude that we can extend the cage-change interval to at least 28 d for disposable, ventilated caging housing a single CD1 mouse. Cages containing 3 CD1 mice of either sex should be changed biweekly, and cages containing 5 CD1 mice or breeder pairs should be changed at least once weekly. PMID:22330779

Dynamical cage behaviour and hydrogen migration in hydrogen and hydrogen-tetrahydrofuran clathrate hydrates

NASA Astrophysics Data System (ADS)

Gorman, Paul D.; English, Niall J.; MacElroy, J. M. D.

2012-01-01

Classical equilibrium molecular dynamics simulations have been performed to investigate dynamical properties of cage radial breathing modes and intra- and inter-cage hydrogen migration in both pure hydrogen and mixed hydrogen-tetrahydrofuran sII hydrates at 0.05 kbar and up to 250 K. For the mixed H2-THF system in which there is single H2 occupation of the small cage (labelled "1SC 1LC"), we find that no H2 migration occurs, and this is also the case for pure H2 hydrate with single small-cavity occupation and quadruple occupancy for large cages (dubbed "1SC 4LC"). However, for the more densely filled H2-THF and pure-H2 systems, in which there is double H2 occupation in the small cage (dubbed "2SC 1LC" and "2SC 4LC," respectively), there is an onset of inter-cage H2 migration events from the small cages to neighbouring cavities at around 200 K, with an approximate Arrhenius temperature-dependence for the migration rate from 200 to 250 K. It was found that these "cage hopping" events are facilitated by temporary openings of pentagonal small-cage faces with the relaxation and reformation of key stabilising hydrogen bonds during and following passage. The cages remain essentially intact up to 250 K, save for transient hydrogen bond weakening and reformation during and after inter-cage hydrogen diffusion events in the 200-250 K range. The "breathing modes," or underlying frequencies governing the variation in the cavities' radii, exhibit a certain overlap with THF rattling motion in the case of large cavities, while there is some overlap of small cages' radial breathing modes with lattice acoustic modes.

Architecture of clathrin fullerene cages reflects a geometric constraint--the head-to-tail exclusion rule--and a preference for asymmetry.

PubMed

Schein, Stan

2009-03-27

Fullerene cages have n trivalent vertices, 12 pentagonal faces, and (n-20)/2 hexagonal faces. The smallest cage in which all of the pentagons are surrounded by hexagons and thus isolated from each other has 60 vertices and is shaped like a soccer ball. The protein clathrin self-assembles into fullerene cages of a variety of sizes and shapes, including smaller ones with adjacent pentagons as well as larger ones, but the variety is limited. To explain the range of clathrin architecture and how these fullerene cages self-assemble, we proposed a hypothesis, the "head-to-tail exclusion rule" (the "Rule"). Of the 5769 small clathrin cage isomers with n< or =60 vertices and adjacent pentagons, the Rule permits just 15, three identified in 1976 and 12 others. A "weak version" of the Rule permits another 99. Based on cryo-electron tomography, Cheng et al. reported six raw clathrin fullerene cages. One was among the three identified in 1976. Here, (1) we identify the remaining five. (2) Four are new and are among the 12 others permitted by the Rule. (3) One, also new, is among the 99 weak version cages. (4) Of particular note, none of the remaining 5565 excluded cages has been identified. These findings provide powerful experimental confirmation of the Rule and the principle on which it is based. (5) Surprisingly, the newly identified clathrin cages are among the least symmetric of those permitted. (6) By devising a method for counting assembly paths, (7) we show that asymmetric cages can be assembled by larger numbers of paths, thus providing a kinetic explanation for the prevalence of asymmetric cages. (8) Finally, we show that operation during cage growth of the Rule greatly increases the likelihood of producing a closed fullerene cage, specifically one of those permitted, but efficient assembly still appears to require internal remodeling.

The influence of fish cage aquaculture on pelagic carbon flow and water chemistry in tidally dominated mangrove estuaries of peninsular Malaysia.

PubMed

Alongi, D M; Chong, V C; Dixon, P; Sasekumar, A; Tirendi, F

2003-05-01

The impact of floating net cages culturing the seabass, Lates calcarifer, on planktonic processes and water chemistry in two heavily used mangrove estuaries in Malaysia was examined. Concentrations of dissolved inorganic and particulate nutrients were usually greater in cage vs. adjacent (approximately 100 m) non-cage waters, although most variability in water-column chemistry related to water depth and tides. There were few consistent differences in plankton abundance, production or respiration between cage and non-cage sites. Rates of primary production were low compared with rates of pelagic mineralization reflecting high suspended loads coupled with large inputs of organic matter from mangrove forests, fishing villages, fish cages, pig farms and other industries within the catchment. Our preliminary sampling did not reveal any large-scale eutrophication due to the cages. A crude estimate of the contribution of fish cage inputs to the estuaries shows that fish cages contribute only approximately 2% of C but greater percentages of N (32-36%) and P (83-99%) to these waters relative to phytoplankton and mangrove inputs. Isolating and detecting impacts of cage culture in such heavily used waterways--a situation typical of most mangrove estuaries in Southeast Asia--are constrained by a background of large, highly variable fluxes of organic material derived from extensive mangrove forests and other human activities. Copyright 2002 Elsevier Science B.V.

Solute diffusion through fibrotic tissue formed around protective cage system for implantable devices.

PubMed

Prihandana, Gunawan Setia; Ito, Hikaru; Tanimura, Kohei; Yagi, Hiroshi; Hori, Yuki; Soykan, Orhan; Sudo, Ryo; Miki, Norihisa

2015-08-01

This article presents the concept of an implantable cage system that can house and protect implanted biomedical sensing and therapeutic devices in the body. Cylinder-shaped cages made of porous polyvinyl alcohol (PVA) sheets with an 80-µm pore size and/or stainless steel meshes with 0.54-mm openings were implanted subcutaneously in the dorsal region of rats for 5 weeks. Analysis of the explanted cages showed the formation of fibrosis tissue around the cages. PVA cages had fibrotic tissue growing mostly along the outer surface of cages, while stainless steel cages had fibrotic tissue growing into the inside surface of the cage structure, due to the larger porosity of the stainless steel meshes. As the detection of target molecules with short time lags for biosensors and mass transport with low diffusion resistance into and out of certain therapeutic devices are critical for the success of such devices, we examined whether the fibrous tissue formed around the cages were permeable to molecules of our interest. For that purpose, bath diffusion and microfluidic chamber diffusion experiments using solutions containing the target molecules were performed. Diffusion of sodium, potassium and urea through the fibrosis tissue was confirmed, thus suggesting the potential of these cylindrical cages surrounded by fibrosis tissue to successfully encase implantable sensors and therapeutic apparatus. © 2014 Wiley Periodicals, Inc.

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.

Performance and welfare of rabbit does in various caging systems.

PubMed

Mikó, A; Matics, Zs; Gerencsér, Zs; Odermatt, M; Radnai, I; Nagy, I; Szendrő, K; Szendrő, Zs

2014-07-01

The objective of the study was to compare production and welfare of rabbit does and their kits housed in various types of cages. Female rabbits were randomly allocated to four groups with the following cage types: CN: common wire-mesh flat-deck cage, without footrest; CF: cage similar to the CN but with plastic footrest; ECWP: enlarged cage with wire-mesh platform; and ECPP: extra enlarged cage with plastic-mesh platform. All does were inseminated on the same day, 11 days after kindlings. Reproductive performance was evaluated during the first five consecutive kindlings. Severity of sore hocks was scored at each insemination. Location preference of the does and the platform usage of their kits were evaluated. Kindling rate, litter size (total born, born alive, alive at 21 and 35 days) and kit mortality were not significantly influenced by the cage types. The litter weight at 21 days was higher in ECWP and ECPP cages than in the CF group (3516, 3576 and 3291 g, respectively; P2.5 cm) and 3 to 4 (3=callus opened, cracks present; 4=wounds) were 58%, 60%, 78% and 48%, and 0%, 5%, 0% and 48% in groups ECPP, ECWP, CF and CN, respectively. Higher number of daily nest visits was observed for CF does than for ECWP does (12.5 v. 5.9; P2/day) was higher in the CF group than in the ECWP group (12.1 v. 3.2%; P<0.01). Within large cages, the does were observed on the platform more frequently in the ECPP cages compared with the ECWP cages (56.9% v. 31.7%; P<0.001). Similarly, 2.7% and 0.2% of kits at 21 days of age, and 33.2% and 5.2% of kits at 28 days of age, were found on the platforms of ECPP and ECWP cages, respectively. In conclusion, cages larger than the conventional ones improved kits' weaning weight, plastic footrests and plastic-mesh platforms in conventional and/or large cages reduced sore hocks' problems, plastic-mesh platforms were more used by both does and kits compared with the wire-mesh platforms.

Stand-alone lumbar cage subsidence: A biomechanical sensitivity study of cage design and placement.

PubMed

Calvo-Echenique, Andrea; Cegoñino, José; Chueca, Raúl; Pérez-Del Palomar, Amaya

2018-08-01

Spinal degeneration and instability are commonly treated with interbody fusion cages either alone or supplemented with posterior instrumentation with the aim to immobilise the segment and restore intervertebral height. The purpose of this work is to establish a tool which may help to understand the effects of intervertebral cage design and placement on the biomechanical response of a patient-specific model to help reducing post-surgical complications such as subsidence and segment instability. A 3D lumbar functional spinal unit (FSU) finite element model was created and a parametric model of an interbody cage was designed and introduced in the FSU. A Drucker-Prager Cap plasticity formulation was used to predict plastic strains and bone failure in the vertebrae. The effect of varying cage size, cross-sectional area, apparent stiffness and positioning was evaluated under 500 N preload followed by 7.5 Nm multidirectional rotation and the results were compared with the intact model. The most influential cage parameters on the FSU were size, curvature congruence with the endplates and cage placement. Segmental stiffness was higher when increasing the cross-sectional cage area in all loading directions and when the cage was anteriorly placed in all directions but extension. In general, the facet joint forces were reduced by increasing segmental stiffness. However, these forces were higher than in the intact model in most of the cases due to the displacement of the instantaneous centre of rotation. The highest plastic deformations took place at the caudal vertebra under flexion and increased for cages with greater stiffness. Thus, wider cages and a more anteriorly placement would increase the volume of failed bone and, therefore, the risk of subsidence. Cage geometry plays a crucial role in the success of lumbar surgery. General considerations such as larger cages may be applied as a guideline, but parameters such as curvature or cage placement should be determined for each specific patient. This model provides a proof-of-concept of a tool for the preoperative evaluation of lumbar surgical outcomes. Copyright © 2018 Elsevier B.V. All rights reserved.

The effect of cage and house design on egg production and egg weight of White Leghorn hens: an epidemiological study.

PubMed

Garner, J P; Kiess, A S; Mench, J A; Newberry, R C; Hester, P Y

2012-07-01

Hen performance can be affected by many interacting variables related to cage design, such as floor area, height, tier arrangement, and feeder and drinker type and placement within the cage. Likewise, features of house design such as waste management and lighting can also affect hen productivity. The influence of these design aspects on hen performance has not been fully assessed. Determining the effects of numerous, interacting variables is impractical in a traditional experiment; therefore, an epidemiological approach, using variability in cage and house design among and within commercial producers, was employed to identify features that affect egg production and egg weight. A universal cage measurement system was created to calculate cage design variables. A database for recording information on cage design, resource location, waste management, environmental conditions, and hen productivity was developed. Production outcomes were assessed from placement to 60 wk of age in White Leghorns (n = 165-168 houses). Using GLM, a statistical model was identified that best described the variance in egg traits. Eggs/hen-housed increased with greater feeder space allocation (P = 0.031); taller cages (P = 0.029); rear (vs. front) drinker location in vertical cages (P = 0.026); and regular removal of manure from the house (P = 0.005). Case weight of eggs was greater in A-frame houses where manure was removed regularly instead of being left in the house (P < 0.001); with increasing cage floor slope (P = 0.001); in cages where drinkers were placed more toward the front or back of the cage as compared with the middle of the cage (P < 0.001); with more space/hen (P = 0.024); and with higher caloric intake (P < 0.001). Perhaps because of its negative correlation with egg production, case weight of eggs increased with less feeder space allocation (P = 0.004) and shorter cage heights (P < 0.001). These results reveal important effects of feeder space, floor space, cage height, drinker position, and waste management on hen productivity.

Experimental evidence supports the abscess theory of development of radicular cysts.

PubMed

Nair, P N R; Sundqvist, Göran; Sjögren, Ulf

2008-08-01

The objective of this study was to experimentally induce inflammatory cysts in an animal model so as to test the hypothesis that radicular cysts develop via the "abscess pathway." Twenty-eight perforated custom-made Teflon cages were surgically implanted into defined locations in the back of 7 Sprague Dawley rats. A week after the implantation of the cages, a known quantity of freshly grown, close allogeneic oral keratinocytes in phosphate buffer solution (PBS) was injected into each cage. One cage per animal was treated as the control that received only epithelial cells. The remaining 3 cages of each animal were trials. Seven days post epithelial cell inoculation; a suspension of 0.2 mL of Fusobacterium nucleatum (10(8) bacteria per mL) was injected into each of the 3 trial cages. Two, 12, and 24 weeks after the inoculation of the bacteria, the cages were taken out, and the tissue contents were fixed and processed by correlative light and transmission electron microscopy. Sixteen of the 21 trial cages could be processed and yielded results. Inoculations of epithelial cells followed 1 week later by F. nucleatum into tissue cages resulted in the development inflammatory cysts in 2 of the 16 cages. The 2 cages contained a total of 4 cystic sites. None of the control cages showed the presence of any cyst-like pathology. Inflammatory cysts were induced by initiating acute inflammatory foci (abscess/necrotic area) by bacterial injection that got enclosed by a proliferating epithelium. This finding provides strong experimental evidence in support of the "abscess theory" of development of radicular cysts.

Bisphenol A is released from used polycarbonate animal cages into water at room temperature

USGS Publications Warehouse

Howdeshell, Kembra L.; Peterman, Paul H.; Judy, Barbara M.; Taylor, Julia A.; Orazio, Carl E.; Ruhlen, Rachel L.; vom Saal, Frederick S.; Welshons, Wade V.

2003-01-01

Bisphenol A (BPA) is a monomer with estrogenic activity that is used in the production of food packaging, dental sealants, polycarbonate plastic, and many other products. The monomer has previously been reported to hydrolyze and leach from these products under high heat and alkaline conditions, and the amount of leaching increases as a function of use. We examined whether new and used polycarbonate animal cages passively release bioactive levels of BPA into water at room temperature and neutral pH. Purified water was incubated at room temperature in new polycarbonate and polysulfone cages and used (discolored) polycarbonate cages, as well as control (glass and used polypropylene) containers. The resulting water samples were characterized with gas chromatography/mass spectrometry (GC/MS) and tested for estrogenic activity using an MCF-7 human breast cancer cell proliferation assay. Significant estrogenic activity, identifiable as BPA by GC/MS (up to 310 micro g/L), was released from used polycarbonate animal cages. Detectable levels of BPA were released from new polycarbonate cages (up to 0.3 micro g/L) as well as new polysulfone cages (1.5 micro g/L), whereas no BPA was detected in water incubated in glass and used polypropylene cages. Finally, BPA exposure as a result of being housed in used polycarbonate cages produced a 16% increase in uterine weight in prepubertal female mice relative to females housed in used polypropylene cages, although the difference was not statistically significant. Our findings suggest that laboratory animals maintained in polycarbonate and polysulfone cages are exposed to BPA via leaching, with exposure reaching the highest levels in old cages.

Molecular marriage through partner preferences in covalent cage formation and cage-to-cage transformation.

PubMed

Acharyya, Koushik; Mukherjee, Sandip; Mukherjee, Partha Sarathi

2013-01-16

Unprecedented self-sorting of three-dimensional purely organic cages driven by dynamic covalent bonds is described. Four different cages were first synthesized by condensation of two triamines and two dialdehydes separately. When a mixture of all the components was allowed to react, only two cages were formed, which suggests a high-fidelity self-recognition. The issue of the preference of one triamine for a particular dialdehyde was further probed by transforming a non-preferred combination to either of the two preferred combinations by reacting it with the appropriate triamine or dialdehyde.

A stochastic frontier analysis of technical efficiency of fish cage culture in Peninsular Malaysia.

PubMed

Islam, Gazi Md Nurul; Tai, Shzee Yew; Kusairi, Mohd Noh

2016-01-01

Cage culture plays an important role in achieving higher output and generating more export earnings in Malaysia. However, the cost of fingerlings, feed and labour have increased substantially for cage culture in the coastal areas in Peninsular Malaysia. This paper uses farm level data gathered from Manjung, Perak and Kota Tinggi, Johor to investigate the technical efficiency of brackish water fish cage culture using the stochastic frontier approach. The technical efficiency was estimated and specifically the factors affecting technical inefficiencies of fish cage culture system in Malaysia was investigated. On average, 37 percent of the sampled fish cage farms are technically efficient. The results suggest very high degrees of technical inefficiency exist among the cage culturists. This implies that great potential exists to increase fish production through improved efficiency in cage culture management in Peninsular Malaysia. The results indicate that farmers obtained grouper fingerlings from other neighboring countries due to scarcity of fingerlings from wild sources. The cost of feeding for grouper (Epinephelus fuscoguttatus) requires relatively higher costs compared to seabass (Lates calcarifer) production in cage farms in the study areas. Initiatives to undertake extension programmes at the farm level are needed to help cage culturists in utilizing their resources more efficiently in order to substantially enhance their fish production.

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.

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.

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

A secure semi-field system for the study of Aedes aegypti.

PubMed

Ritchie, Scott A; Johnson, Petrina H; Freeman, Anthony J; Odell, Robin G; Graham, Neal; Dejong, Paul A; Standfield, Graeme W; Sale, Richard W; O'Neill, Scott L

2011-03-22

New contained semi-field cages are being developed and used to test novel vector control strategies of dengue and malaria vectors. We herein describe a new Quarantine Insectary Level-2 (QIC-2) laboratory and field cages (James Cook University Mosquito Research Facility Semi-Field System; MRF SFS) that are being used to measure the impact of the endosymbiont Wolbachia pipientis on populations of Aedes aegypti in Cairns Australia. The MRF consists of a single QIC-2 laboratory/insectary that connects through a central corridor to two identical QIC-2 semi-field cages. The semi-field cages are constructed of two layers of 0.25 mm stainless steel wire mesh to prevent escape of mosquitoes and ingress of other insects. The cages are covered by an aluminum security mesh to prevent penetration of the cages by branches and other missiles in the advent of a tropical cyclone. Parts of the cage are protected from UV light and rainfall by 90% shade cloth and a vinyl cover. A wooden structure simulating the understory of a Queenslander-style house is also situated at one end of each cage. The remainder of the internal aspect of the cage is covered with mulch and potted plants to emulate a typical yard. An air conditioning system comprised of two external ACs that feed cooled, moistened air into the cage units. The air is released from the central ceiling beam from a long cloth tube that disperses the airflow and also prevents mosquitoes from escaping the cage via the AC system. Sensors located inside and outside the cage monitor ambient temperature and relative humidity, with AC controlled to match ambient conditions. Data loggers set in the cages and outside found a <2 °C temperature difference. Additional security features include air curtains over exit doors, sticky traps to monitor for escaping mosquitoes between layers of the mesh, a lockable vestibule leading from the connecting corridor to the cage and from inside to outside of the insectary, and screened (0.25 mm mesh) drains within the insectary and the cage. A set of standard operating procedures (SOP) has been developed to ensure that security is maintained and for enhanced surveillance for escaping mosquitoes on the JCU campus where the MRF is located. A cohort of male and female Aedes aegypti mosquitoes were released in the cage and sampled every 3-4 days to determine daily survival within the cage; log linear regression from BG-sentinel trapping collections produced an estimated daily survival of 0.93 and 0.78 for females and males, respectively. The MRF SFS allows us to test novel control strategies within a secure, contained environment. The air-conditioning system maintains conditions within the MRF cages comparable to outside ambient conditions. This cage provides a realistic transitional platform between the laboratory and the field in which to test novel control measures on quarantine level insects.

A Secure Semi-Field System for the Study of Aedes aegypti

PubMed Central

Ritchie, Scott A.; Johnson, Petrina H.; Freeman, Anthony J.; Odell, Robin G.; Graham, Neal; DeJong, Paul A.; Standfield, Graeme W.; Sale, Richard W.; O'Neill, Scott L.

2011-01-01

Background New contained semi-field cages are being developed and used to test novel vector control strategies of dengue and malaria vectors. We herein describe a new Quarantine Insectary Level-2 (QIC-2) laboratory and field cages (James Cook University Mosquito Research Facility Semi-Field System; MRF SFS) that are being used to measure the impact of the endosymbiont Wolbachia pipientis on populations of Aedes aegypti in Cairns Australia. Methodology/Principal Findings The MRF consists of a single QIC-2 laboratory/insectary that connects through a central corridor to two identical QIC-2 semi-field cages. The semi-field cages are constructed of two layers of 0.25 mm stainless steel wire mesh to prevent escape of mosquitoes and ingress of other insects. The cages are covered by an aluminum security mesh to prevent penetration of the cages by branches and other missiles in the advent of a tropical cyclone. Parts of the cage are protected from UV light and rainfall by 90% shade cloth and a vinyl cover. A wooden structure simulating the understory of a Queenslander-style house is also situated at one end of each cage. The remainder of the internal aspect of the cage is covered with mulch and potted plants to emulate a typical yard. An air conditioning system comprised of two external ACs that feed cooled, moistened air into the cage units. The air is released from the central ceiling beam from a long cloth tube that disperses the airflow and also prevents mosquitoes from escaping the cage via the AC system. Sensors located inside and outside the cage monitor ambient temperature and relative humidity, with AC controlled to match ambient conditions. Data loggers set in the cages and outside found a <2°C temperature difference. Additional security features include air curtains over exit doors, sticky traps to monitor for escaping mosquitoes between layers of the mesh, a lockable vestibule leading from the connecting corridor to the cage and from inside to outside of the insectary, and screened (0.25 mm mesh) drains within the insectary and the cage. A set of standard operating procedures (SOP) has been developed to ensure that security is maintained and for enhanced surveillance for escaping mosquitoes on the JCU campus where the MRF is located. A cohort of male and female Aedes aegypti mosquitoes were released in the cage and sampled every 3–4 days to determine daily survival within the cage; log linear regression from BG-sentinel trapping collections produced an estimated daily survival of 0.93 and 0.78 for females and males, respectively. Conclusions/Significance The MRF SFS allows us to test novel control strategies within a secure, contained environment. The air-conditioning system maintains conditions within the MRF cages comparable to outside ambient conditions. This cage provides a realistic transitional platform between the laboratory and the field in which to test novel control measures on quarantine level insects. PMID:21445333

Interactions Between Housing Density and Ambient Temperature in the Cage Environment: Effects on Mouse Physiology and Behavior.

PubMed

Toth, Linda A; Trammell, Rita A; Ilsley-Woods, Megan

2015-11-01

To determine how housing density and ambient temperature interact to influence the physiology and behavior of mice, we systematically varied housing density (1 to 5 mice per cage) and ambient temperature (22, 26, or 30 °C) and measured effects on body weight, food intake, diurnal patterns of locomotor activity and core temperature, fecal corticosterone, and serum cytokine and adipokine panels. Temperatures inside cages housing 5 mice were 1 to 2 °C higher than the ambient temperature. As the housing density decreased, in-cage temperatures began to fall at a density of 2 or 3 mice per cage and did not differ from ambient temperature at 1 mouse per cage. Ambient temperature, but not housing density, significantly affected food intake. Although neither ambient temperature nor housing density affected core temperature or activity, hyperthermia and behavioral activation occurred during the 12-h period after cage change. Fecal concentrations of corticosterone metabolites and serum cytokines, chemokines, insulin, and leptin were not influenced by cage density and were only sporadically influenced by ambient temperature. Our data document that the number of mice housed per cage influences the intracage environmental conditions and that ambient temperature influences food intake even when temperatures are within or near recommended or thermoneutral ranges. We conclude that investigators should be cautious when changing the number of mice housed in a cage over the course of a study, because doing so significantly alters the cage environment to which remaining mice are exposed.

Interactions Between Housing Density and Ambient Temperature in the Cage Environment: Effects on Mouse Physiology and Behavior

PubMed Central

Toth, Linda A; Trammell, Rita A; Ilsley-Woods, Megan

2015-01-01

To determine how housing density and ambient temperature interact to influence the physiology and behavior of mice, we systematically varied housing density (1 to 5 mice per cage) and ambient temperature (22, 26, or 30 °C) and measured effects on body weight, food intake, diurnal patterns of locomotor activity and core temperature, fecal corticosterone, and serum cytokine and adipokine panels. Temperatures inside cages housing 5 mice were 1 to 2 °C higher than the ambient temperature. As the housing density decreased, in-cage temperatures began to fall at a density of 2 or 3 mice per cage and did not differ from ambient temperature at 1 mouse per cage. Ambient temperature, but not housing density, significantly affected food intake. Although neither ambient temperature nor housing density affected core temperature or activity, hyperthermia and behavioral activation occurred during the 12-h period after cage change. Fecal concentrations of corticosterone metabolites and serum cytokines, chemokines, insulin, and leptin were not influenced by cage density and were only sporadically influenced by ambient temperature. Our data document that the number of mice housed per cage influences the intracage environmental conditions and that ambient temperature influences food intake even when temperatures are within or near recommended or thermoneutral ranges. We conclude that investigators should be cautious when changing the number of mice housed in a cage over the course of a study, because doing so significantly alters the cage environment to which remaining mice are exposed. PMID:26632780

Effects of single caging and cage size on behavior and stress level of domestic neutered cats housed in an animal shelter.

PubMed

Uetake, Katsuji; Goto, Akihiro; Koyama, Rumi; Kikuchi, Rieko; Tanaka, Toshio

2013-03-01

Cats need a minimum amount of space even in animal shelters. In this study the effects of single caging and cage size on the behavior and stress level of domestic cats were investigated. Six neutered cats (2-15 years old) that had been housed in a group for at least 7 months were moved to three kinds of single cages (small, medium and large) by rotation on a Latin square design. They experienced each cage size for 6 days. Cats could use vertical dimensions when housed in a group room and the large cage. Behavioral observation was conducted for 3 h in the evening, and stress levels were assessed by urine cortisol-to-creatinine ratios. The amounts (estimated proportions) of time spent in locomotion and social/solitary play were lower even in large cages than in group housing (both P < 0.05). Conversely, the amount of time spent resting tended to increase when housed singly (P = 0.104). The urine cortisol-to-creatinine ratios of singly housed cats tended to be higher than that of group-housed cats (P = 0.086). The results indicate that cats become less active when they are housed singly in cages regardless of the cage size. Cats seem to feel no undue stress even in small cages if the stay is short. © 2012 The Authors. Animal Science Journal © 2012 Japanese Society of Animal Science.

Play Caging Benefits the Behavior of Singly Housed Laboratory Rhesus Macaques (Macaca mulatta)

PubMed Central

Griffis, Caroline M; Martin, Allison L; Perlman, Jaine E; Bloomsmith, Mollie A

2013-01-01

This study addresses a recommendation in The Guide for the Care and Use of Laboratory Animals to provide singly housed nonhuman primates with intermittent access to large, enriched (play) caging. Research on the potential benefits of this type of caging is limited. The present study examines the effects of play caging on behavior, activity, and enrichment use. Singly housed, adult male, rhesus macaques (n = 10) underwent a baseline phase in their home cages, a 2-wk treatment phase with housing in play cages, and a posttreatment phase after returning to their home cages. Each subject underwent focal behavioral observations (n = 10; duration 30 min each) during each study phase, for a total of 150 h of data collection. Results showed increases in locomotion and enrichment use and a trend toward decreased abnormal behavior while subjects were in the play cage, with the durations of these behaviors returning to baseline levels after treatment. Anxiety-related behaviors decreased between the treatment and posttreatment phases but not between baseline and treatment, suggesting that outside factors may have influenced the decline. During the treatment phase, subjects spent more time in the upper quadrants of the play caging and preferred a mirror and forage boards as forms of enrichment. The greatest behavioral improvement occurred during the first week in the play cage. This study provides evidence to support the benefits of play caging for singly housed rhesus macaques. PMID:24041207

Particulate matter in animal rooms housing mice in microisolation caging.

PubMed

Langham, Gregory L; Hoyt, Robert F; Johnson, Thomas E

2006-11-01

Reactions to allergens created by laboratory animals are among the most frequently encountered occupational illnesses associated with research animals. Personnel are exposed to these allergens through airborne particulate matter. Although the use of microisolation caging systems can reduce particulate matter concentrations in rooms housing mice, the operating parameters of ventilated caging systems vary extensively. We compared room air in mouse rooms containing 5 different types of caging: 1) individually ventilated caging under positive pressure with filtered intake air and exhaust air returned to the room (VCR+), 2) individually ventilated caging under negative pressure with exhaust air returned to the room (VCR-), 3) individually ventilated caging under positive pressure with exhaust air returned to the heating, ventilation, and air-conditioning (HVAC) system, 4) individually ventilated caging under negative pressure with exhaust air returned to the HVAC system, and 5) static microisolation cages. We found that rooms under VCR conditions had fewer large particles than did those under other conditions, but the numbers of 0.3 microm particles did not differ significantly among systems. Static, positive or negative pressure applied to caging units as well as route of air exhaust were found to have little influence on the total number of particles in the atmosphere. Therefore, considering the heat load, odor, and overall particulate concentration in the room, placing individually ventilated caging under negative pressure with exhaust air returned to the HVAC system appears to be the optimal overall choice when using microisolation housing for rodents.

The effect of pesticide residue on caged mosquito bioassays.

PubMed

Barber, J A S; Greer, Mike; Coughlin, Jamie

2006-09-01

Wind tunnel experiments showed that secondary pickup of insecticide residue by mosquitoes in cage bioassays had a significant effect on mortality. Cage bioassays using adult Ochlerotatus taeniorhynchus (Wiedemann) investigated the effect of exposure time to a contaminated surface. Cages were dosed in a wind tunnel using the LC50 for naled (0.124 mg a.i./ml) and an LC25 (0.0772 mg a.i./ml) for naled. Half of the bioassay mosquitoes were moved directly into clean cages with the other half remaining in the sprayed, hence contaminated, cage. Treatment mortality was assessed at 8, 15, 30, 60, 120, 240, and 1,440 min postapplication. Cage contamination had a significant effect on mosquito mortality for both the LC25 and LC50 between 15 and 30 min postapplication.

[Design and research progress of zero profile cervical Interbody cage].

PubMed

Zhu, Jia; Wang, Song; Liao, Zhenhua; Liu, Weiqiang

2017-02-01

Zero profile cervical interbody cage is an improvement of traditional fusion products and necessary supplement of emerging artificial intervertebral disc products. When applied in Anterior Cervical Decompression Fusion(ACDF), zero profile cervical interbody cage can preserve the advantages of traditional fusion and reduce the incidence of postoperative complications. Moreover, zero profile cervical interbody cage can be applied under the tabu symptoms of Artificial Cervical Disc Replacement(ACDR). This article summarizes zero profile interbody cage products that are commonly recognized and widely used in clinical practice in recent years, and reviews the progress of structure design and material research of zero profile cervical interbody cage products. Based on the latest clinical demands and research progress, this paper also discusses the future development directions of zero profile interbody cage.

Effects of Cage-Change Frequency and Bedding Volume on Mice and Their Microenvironment

PubMed Central

Rosenbaum, Matthew D; VandeWoude, Susan; Johnson, Thomas E

2009-01-01

The frequency at which mouse cages are changed has important implications for the animals, animal care personnel, and facility managers. The objective of this study was to determine how bedding volume and the interval between changes affect microenvironmental conditions, health, and behavior of mice housed in individually ventilated cages (IVC). A total of 15 cages (n = 5 cages per bedding volume) housing ICR female mice (n = 5 animals per cage) were monitored for 17 d. Parameters monitored included clinical evaluation of each animal, appearance of the cage, fecal corticosterone levels, bedding weight, and mouse mass. Atmospheric analysis was performed daily to determine intracage ammonia cage humidity and temperature on a daily basis. Mice were videotaped for 10 min on days 1, 8, and 15, and videos were analyzed for abnormal behaviors. On day 17, 1 mouse from each cage was euthanized, and bronchoalveolar lavage was performed. Statistical differences in parameters were most often noted between low- and high-volume bedding groups. Correlation between visual appearance and actual intracage environmental conditions and mouse health and behavior at specific time points indicated cages that appear dirty to most observers did not have measurably adverse effects on the animals for any of the many parameters evaluated in this study. This study demonstrated that a 2-wk interval between cage changes for ICR female mice housed in IVC caging (with approximately 90 air changes per hour) and aspen chip bedding did not significantly affect measures of animal well-being in this study. This lack of effect occurred despite the appearance of excessive soiling by the 2-wk time point. PMID:19930825

Determinants of acute mortality of Hippodamia convergens (Coleoptera: Coccinellidae) to ultra-low volume permethrin used for mosquito management

PubMed Central

Preftakes, Collin J.; Bodin, Jennifer L.; Brown, Christopher R.; Piccolomini, Alyssa M.; Schleier, Jerome J.

2016-01-01

There are relatively few experimental studies and risk assessments of the effects on non-target insects from ultra-low volume (ULV) insecticides used for management of adult mosquitoes. Therefore, we evaluated factors that may influence the ability of an insect to intercept the insecticide at the time of application by using Hippodamia convergens (Coleoptera: Coccinellidae) in field bioassay experiments in 2011 and 2015. Treatment factors included different distances, two cage heights (ground-level and 1.5 m above ground) to the point of the application, and covered vs. uncovered cage faces (2015 only). Insecticides used included a water-based formulation (Aqua-Reslin®) and an oil-based formulation (Permanone® 30-30) of permethrin. Cage height was highly significant both years, with much less acute (i.e., short-term exposure) mortality at ground-level compared with 1.5 m. In 2011, acute mortality was less at ground-level (mean = 3.2%, median = 0%) compared to 1.5 m (mean = 85.2%, median = 100%). Cage type also was highly significant, with less mortality in covered cages compared to uncovered cages. Mortality by cage height and cage type was as follows: ground level, covered cage (mean = 2.8%, median = 0.1%); ground level, uncovered cage (mean = 41.9%, median = 9.6%); 1.5 m, covered cage (mean = 6.8%, median = 0%); 1.5 m, uncovered cage (mean = 83.7%, median = 100%). Results suggest that acute mortality to non-target insects may vary considerably based on their height and their ability to directly intercept the insecticide as the aerosol passes through the area being sprayed. PMID:27366655

Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C4 cage axes.

PubMed

Theil, Elizabeth C; Turano, Paola; Ghini, Veronica; Allegrozzi, Marco; Bernacchioni, Caterina

2014-06-01

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe2O3·H2O minerals from Fe(2+) for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe(2+) transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe(2+)/O2 catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe(2+) + O2 protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe2O3·H2O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.

Coordinating Subdomains of Ferritin Protein Cages with Catalysis and Biomineralization viewed from the C4 Cage Axes

PubMed Central

Theil, Elizabeth C.; Turano, Paola; Ghini, Veronica; Allegrozzi, Marco; Bernacchioni, Caterina

2014-01-01

Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe2O3•H2O minerals from Fe2+, for metabolic iron concentrates and oxidant protection; biomineral order varies in different ferritin proteins. The conserved 4, 3, 2 geometric symmetry of ferritin protein cages, parallels subunit dimer, trimer and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self- assembling ferritin nanocages have functional relationships to cage symmetry such as Fe2+ transport though ion channels (3-fold symmetry), biomineral nucleation/order (4-fold symmetry) and mineral dissolution (3-fold symmetry) studied in ferritin variants. Cage subunit dimers (2-fold symmetry) influence iron oxidation and mineral dissolution, based on effects of natural or synthetic subunit dimer crosslinks. 2Fe2+/O2 catalysis in ferritin occurs in single subunits, but with cooperativity (n=3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of 3 subunits. Here, we study 2Fe2+ + O2 protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe2O3•H2O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein 2-fold and 3-fold cage axes to show function at ferritin 4-fold cage axes. Here, conserved amino acids facilitate dissolution of ferritin protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage 4-fold symmetry and solid state mineral properties remain largely unexplored. PMID:24504941

Laser photolysis of caged compounds at 405 nm: photochemical advantages, localisation, phototoxicity and methods for calibration.

PubMed

Trigo, Federico F; Corrie, John E T; Ogden, David

2009-05-30

Rapid, localised photolytic release of neurotransmitters from caged precursors at synaptic regions in the extracellular space is greatly hampered at irradiation wavelengths in the near-UV, close to the wavelength of maximum absorption of the caged precursor, because of inner-filtering by strong absorption of light in the cage solution between the objective and cell. For this reason two-photon excitation is commonly used for photolysis, particularly at multiple points distributed over large fields; or, with near-UV, if combined with local perfusion of the cage. These methods each have problems: the small cross-sections of common cages with two-photon excitation require high cage concentrations and light intensities near the phototoxic limit, while local perfusion gives non-uniform cage concentrations over the field of view. Single-photon photolysis at 405 nm, although less efficient than at 330-350 nm, with present cages is more efficient than two-photon photolysis. The reduced light absorption in the bulk cage solution permits efficient wide-field uncaging at non-toxic intensities with uniform cage concentration. Full photolysis of MNI-glutamate with 100 micros pulses required intensities of 2 mW microm(-2) at the preparation, shown to be non-toxic with repeated exposures. Light scattering at 405 nm was estimated as 50% at 18 microm depth in 21-day rat cerebellum. Methods are described for: (1) varying the laser spot size; (2) photolysis calibration in the microscope with the caged fluorophore NPE-HPTS over the wavelength range 347-405 nm; and (3) determining the point-spread function of excitation. Furthermore, DM-Nitrophen photolysis at 405 nm was efficient for intracellular investigations of Ca2+-dependent processes.

Segmental kyphosis after cervical interbody fusion with stand-alone polyetheretherketone (PEEK) cages: a comparative study on 2 different PEEK cages.

PubMed

Kim, Chi Heon; Chung, Chun Kee; Jahng, Tae-Ahn; Park, Sung Bae; Sohn, Seil; Lee, Sungjoon

2015-02-01

Retrospective comparative study. Two polyetheretherketone (PEEK) cages of different designs were compared in terms of the postoperative segmental kyphosis after anterior cervical discectomy and fusion. Segmental kyphosis occasionally occurs after the use of a stand-alone cage for anterior cervical discectomy and fusion. Although PEEK material seems to have less risk of segmental kyphosis compared with other materials, the occurrence of segmental kyphosis for PEEK cages has been reported to be from 0% to 29%. There have been a few reports that addressed the issue of PEEK cage design. A total of 41 consecutive patients who underwent single-level anterior discectomy and fusion with a stand-alone cage were included. Either a round tube-type (Solis; 18 patients, S-group) or a trapezoidal tube-type (MC+; 23 patients, M-group) cage was used. The contact area between the cage and the vertebral body is larger in MC+ than in Solis, and anchoring pins were present in the Solis cage. The effect of the cage type on the segmental angle (SA) (lordosis vs. kyphosis) at postoperative month 24 was analyzed. Preoperatively, segmental lordosis was present in 12/18 S-group and 16/23 M-group patients (P=0.84). The SA was more lordotic than the preoperative angle in both groups just after surgery, with no difference between groups (P=0.39). At 24 months, segmental lordosis was observed in 9/18 S-group and 20/23 M-group patients (P=0.01). The patients in M-group were 7.83 times more likely than patients in S-group (P=0.04; odds ratio, 7.83; 95% confidence interval, 1.09-56.28) not to develop segmental kyphosis. The design of the PEEK cage used may influence the SA, and this association needs to be considered when using stand-alone PEEK cages.

The Impact of Cage Dimensions, Positioning, and Side of Approach in Extreme Lateral Interbody Fusion.

PubMed

Alimi, Marjan; Lang, Gernot; Navarro-Ramirez, Rodrigo; Perrech, Moritz; Berlin, Connor; Hofstetter, Christoph P; Moriguchi, Yu; Elowitz, Eric; Härtl, Roger

2018-02-01

This is a retrospective single-center study. The aim of the study was to evaluate the impact of cage characteristics and position toward clinical and radiographic outcome measures in patients undergoing extreme lateral interbody fusion (ELIF). ELIF is utilized for indirect decompression and minimally invasive surgical treatment for various degenerative spinal disorders. However, evidence regarding the influence of cage characteristics in patient outcome is minimal. Patients undergoing ELIF between 2007 and 2011 were included in a retrospective study. Demographic and perioperative data, as well as cage characteristics and side of approach were extracted. Radiographic parameters including lumbar lordosis, foraminal height, and disc height as well as clinical outcome parameters (Oswestry Disability Index and Visual Analog Scale) were measured preoperatively, postoperatively, and at the latest follow-up examination. Cage dimensions, in situ position, and type were correlated with radiographic and clinical outcome parameters. In total, 84 patients with a total of 145 functional spinal units were analyzed. At the last follow-up of 17.7 months, radiographic and clinical outcome measures revealed significant improvement compared with before surgery with both, 18 and 22 mm cage anterior-posterior diameter subgroups (P≤0.05). Among cage characteristics, 22 mm cages presented superior restoration of foraminal and disc heights compared with 18 mm cages (P≤0.05). Neither position of the cage (anterior vs. posterior), nor the type (parallel vs. lordotic) had a significant impact on restoration of foraminal height and lumbar lordosis. Moreover, the side of surgical approach did not influence the amount of foraminal height increase. Cage anterior-posterior diameter is the determining factor in restoration of foraminal height in ELIF. Cage height, type, positioning, and side of approach do not have a determining role in radiographic outcome in the present study. Sustainable foraminal height restoration is achieved by implantation of wider cages. Level 3.

Effect of 2 Bedding Materials on Ammonia Levels in Individually Ventilated Cages

PubMed Central

Koontz, Jason M; Kumsher, David M; III, Richard Kelly; Stallings, Jonathan D

2016-01-01

This study sought to identify an optimal rodent bedding and cage-change interval to establish standard procedures for the IVC in our rodent vivarium. Disposable cages were prefilled with either corncob or α-cellulose bedding and were used to house 2 adult Sprague–Dawley rats (experimental condition) or contained no animals (control). Rats were observed and intracage ammonia levels measured daily for 21 d. Intracage ammonia accumulation became significant by day 8 in experimental cages containing α-cellulose bedding, whereas experimental cages containing corncob bedding did not reach detectable levels of ammonia until day 14. In all 3 experimental cages containing α-cellulose, ammonia exceeded 100 ppm (our maximum acceptable limit) by day 11. Two experimental corncob cages required changing at days 16 and 17, whereas the remaining cage containing corncob bedding lasted the entire 21 d without reaching the 100-ppm ammonia threshold. These data suggests that corncob bedding provides nearly twice the service life of α-cellulose bedding in the IVC system. PMID:26817976

Effect of 2 Bedding Materials on Ammonia Levels in Individually Ventilated Cages.

PubMed

Koontz, Jason M; Kumsher, David M; Kelly, Richard; Stallings, Jonathan D

2016-01-01

This study sought to identify an optimal rodent bedding and cage-change interval to establish standard procedures for the IVC in our rodent vivarium. Disposable cages were prefilled with either corncob or α-cellulose bedding and were used to house 2 adult Sprague-Dawley rats (experimental condition) or contained no animals (control). Rats were observed and intracage ammonia levels measured daily for 21 d. Intracage ammonia accumulation became significant by day 8 in experimental cages containing α-cellulose bedding, whereas experimental cages containing corncob bedding did not reach detectable levels of ammonia until day 14. In all 3 experimental cages containing α-cellulose, ammonia exceeded 100 ppm (our maximum acceptable limit) by day 11. Two experimental corncob cages required changing at days 16 and 17, whereas the remaining cage containing corncob bedding lasted the entire 21 d without reaching the 100-ppm ammonia threshold. These data suggests that corncob bedding provides nearly twice the service life of α-cellulose bedding in the IVC system.

A Pseudo-Atomic Model of the COPII Cage Obtained from CryoEM and Mass Spectrometry Analyses

PubMed Central

Noble, Alex J.; Zhang, Qian; O’Donnell, Jason; Hariri, Hanaa; Bhattacharya, Nilakshee; Marshall, Alan G.

2012-01-01

COPII vesicles transport proteins from the ER to the Golgi apparatus. Previous cryoEM structures of the COPII cage lacked the resolution necessary to determine the residues of Sec13 and Sec31 that mediate assembly and flexibility of the COPII cage. Here we present a 12Å-resolution structure of the COPII cage, where the tertiary structure of Sec13 and Sec31 is clearly identifiable. We employ this structure and a homology model of the Sec13-Sec31 complex to create a reliable pseudo-atomic model of the COPII cage. We combined this model with hydrogen/deuterium exchange mass spectrometry analysis to characterize four distinct contact regions at the vertices of the COPII cage. Furthermore, we found that the 2-fold symmetry of the Sec31 dimeric region of Sec13-31 is broken on cage formation, and that the resulting hinge is essential to form the proper edge geometry in COPII cages. PMID:23262493

A library of protein cage architectures as nanomaterials.

PubMed

Flenniken, M L; Uchida, M; Liepold, L O; Kang, S; Young, M J; Douglas, T

2009-01-01

Virus capsids and other structurally related cage-like proteins such as ferritins, dps, and heat shock proteins have three distinct surfaces (inside, outside, interface) that can be exploited to generate nanomaterials with multiple functionality by design. Protein cages are biological in origin and each cage exhibits extremely homogeneous size distribution. This homogeneity can be used to attain a high degree of homogeneity of the templated material and its associated property. A series of protein cages exhibiting diversity in size, functionality, and chemical and thermal stabilities can be utilized for materials synthesis under a variety of conditions. Since synthetic approaches to materials science often use harsh temperature and pH, it is an advantage to utilize protein cages from extreme environments. In this chapter, we review recent studies on discovering novel protein cages from harsh natural environments such as the acidic thermal hot springs at Yellowstone National Park (YNP) and on utilizing protein cages as nano-scale platforms for developing nanomaterials with wide range of applications from electronics to biomedicine.

The Evolution of the Cup-Cage Technique for Major Acetabular Defects: Full and Half Cup-Cage Reconstruction.

PubMed

Sculco, Peter K; Ledford, Cameron K; Hanssen, Arlen D; Abdel, Matthew P; Lewallen, David G

2017-07-05

Complex acetabular reconstruction for major bone loss can require advanced methods such as the use of a cup-cage construct. The purpose of this study was to review outcomes after the initial development of the cup-cage technique and the subsequent evolution to the use of a half cup-cage construct. We performed a retrospective, single-center review of 57 patients treated with cup-cage reconstruction for major acetabular bone loss. All patients had major acetabular defects graded as Paprosky Type 2B through 3B, with 34 (60%) having an associated pelvic discontinuity. Thirty patients received a full cup-cage construct and 27, a half cup-cage construct. The mean follow-up was 5 years. Both the full and half cup-cage cohorts demonstrated significantly improved Harris hip score (HHS) values, from 36 to 72 at a minimum of 2 years of follow-up (p < 0.05). Early construct migration occurred in 4 patients, with stabilization prior to 2-year follow-up in all but 1 patient. Incomplete, zone-3, nonprogressive acetabular radiolucencies were observed in 2 (7%) of the full cup-cage constructs and 6 (22%) of the half cup-cage constructs. One patient with a full cup-cage construct underwent re-revision of the acetabular component for progressive migration and aseptic loosening. Short-term survivorship free from re-revision for any cause or reoperation was 89% (83% and 96% for full and half cup-cage cohorts, respectively). Both full and half cup-cage constructs demonstrated successful clinical outcomes and survivorship in the treatment of major acetabular defects and pelvic discontinuity. Each method is utilized on the basis of individual intraoperative findings, including the extent and pattern of bone loss, the quality and location of host bone remaining after preparation, and the presence of pelvic discontinuity. Longer-term follow-up is required to understand the durability of these constructs in treating major acetabular defects and pelvic discontinuity. Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Caged compounds: tools for illuminating neuronal responses and connections.

PubMed

Nerbonne, J M

1996-06-01

A number of new 'caged' intracellular second messengers and neurotransmitters have been developed using the photolabile o-nitrobenzyl group. This chemistry has also recently been exploited in novel ways, including the development of caged enzyme substrates and caged proteins. Although caged compounds continue to be used primarily for mechanistic (kinetic) studies of processes mediated by transmitters or second messengers, the spatial resolution afforded by the use of light to effect changes in transmitter concentrations has now been clearly demonstrated. The increased availability of caged compounds and of the technologies required to exploit them provides neurobiologists with powerful tools for probing neuronal response properties and connectivity patterns.

Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells.

PubMed

Arisaka, Akio; Mogaki, Rina; Okuro, Kou; Aida, Takuzo

2018-02-21

We developed dendritic caged molecular glues ( Caged Glue-R) as tags for nucleus-targeted drug delivery, whose multiple guanidinium ion (Gu + ) pendants are protected by an anionic photocleavable unit (butyrate-substituted nitroveratryloxycarbonyl; BA NVOC). Negatively charged Caged Glue-R hardly binds to anionic biomolecules because of their electrostatic repulsion. However, upon exposure of Caged Glue-R to UV light or near-infrared (NIR) light, the BA NVOC groups of Caged Glue-R are rapidly detached to yield an uncaged molecular glue ( Uncaged Glue-R) that carries multiple Gu + pendants. Because Gu + forms a salt bridge with PO 4 - , Uncaged Glue-R tightly adheres to anionic biomolecules such as DNA and phospholipids in cell membranes by a multivalent salt-bridge formation. When tagged with Caged Glue-R, guests can be taken up into living cells via endocytosis and hide in endosomes. However, when the Caged Glue-R tag is photochemically uncaged to form Uncaged Glue-R, the guests escape from the endosome and migrate into the cytoplasm followed by the cell nucleus. We demonstrated that quantum dots (QDs) tagged with Caged Glue-R can be delivered efficiently to cell nuclei eventually by irradiation with light.

Application of the independent molecule model to elucidate the dynamics of structure I methane hydrate: a third report.

PubMed

Yoshioki, Shuzo

2009-01-01

Two new model systems of methane hydrate, larger than the previous systems, are constructed. One consists of 63 small and large cages with a small cage at the centre. The other has 65 small and large cages with a large cage at the centre. Three different H-bonding network patterns between water are formed, and three random orientations of methane in each cage are chosen. Using the surface water fixed method, we obtained the energy minimum conformations, fitted to the X-ray crystallographic structure. With normal mode analysis, we calculated frequencies of 2915.1 cm(-1) for a small cage, and 2911.0 cm(-1) for a large cage. These frequencies are a little nearer to the Raman spectra than were previous model systems. Treating three force constants of anharmonic potential energy and the strength of H-bonding between methane and water as four parameters, we obtained frequencies of 2913.6 cm(-1) for a small cage, a little lower than the Raman, and 2906.6 cm(-1) for a large cage, a little higher than the Raman. The calculations thus almost reach the Raman spectra.

Virucidal effects of rodent cage-cleaning practices on the viability of adenovirus vectors.

PubMed

Porter, Jacqueline D; Lyons, Russette M

2002-09-01

Human adenoviruses and adenoviral vectors are classified as Risk Group 2 agents and require BSL2 containment and practices. An additional consideration in using adenoviruses and viral vectors in laboratory animal studies is the possible transmission of these agents to other animals and/or personnel as a result of viral shedding in animal urine and feces. When handling BSL2 agents, cage-wash staff are required to wear appropriate personnel protective equipment, including scrubs, Tyvek suit, hair covering, dust mask, shoes covers, and gloves. Current decontamination procedures are to bag and autoclave soiled rodent cages containing bedding prior to washing in the cage washer to prevent possible adenoviral transmission. However, the practice of autoclaving softens the polycarbonate-based rodent cages, allowing damaging agents or conditions to affect the integrity of the plastic and degrade the cages. The objective of this study was to determine whether current rodent cage-cleaning practices produced virucidal effects for use in lieu of or prior to autoclaving the cages. We found that heating an Av3GFP vector in a test tube to a temperature of 74 degrees C (165 degrees F) for 6 min conditions equivalent to those of the cage washer resulted in greater than an 11-log reduction in infectivity of the vector as evaluated by its cytopathic effect on cells. The combination of heating and a liquid, phosphate-free alkaline detergent produced the same reduction in vector infectivity. However, common cage-cleaning solutions alone possessed no virucidal activity. The high temperatures used in cage-washing procedures alone or in combination with a cleaning solution reduced or eliminated the risk of transmission from viral shedding through urine and feces even at vector concentrations far greater than would ever be expected to be present. Autoclaving cages diminishes the stability and integrity of the polycarbonate cages without providing a further reduction in the risk of virus or vector transmission. On the basis of results from this study, new cage-wash recommendations include dumping the contaminated bedding into a HEPA-filtered waste disposal system and autoclaving the bags of bedding before disposal, then cleaning the cages in the rack washer at wash temperatures of 74 degrees C (165 F) and rinse temperatures of 82 degrees C (180 F).

[Detection of alcoholism in the medical office: applicability of the CAGE questionnaire by the practicing physician. Group of Medical Practitioners PMU].

PubMed

Perdrix, A; Decrey, H; Pécoud, A; Burnand, B; Yersin, B

1995-09-23

The general practitioner (GP) plays a very important role in early detection of alcoholism. Clinical evaluation is often the only method used, although it has been suggested that any systematic patient history should include the CAGE test. We compare the effectiveness of these two approaches and attempt to determine the applicability of the CAGE test in a general practitioner's usual practice. 12 GPs took part in this study during 6 months. They looked for possible alcohol abuse in each new patient by a standard patient history and clinical examination. Patients were randomized into 2 groups, one of which was given the CAGE test and the other not. For each patient in the CAGE group the applicability of the test was quantified by the GP. 416 patients were included; 214 were randomized into the "CAGE group" and 202 into the control group. On a clinical basis, 15 patients in the control group and 16 in the "CAGE group" (14 men, 2 women) were suspected of alcohol abuse. The CAGE test was positive in 15 patients (7%); among these, 6 were not suspect on a clinical basis. In patients aged 18-34, the detection rate of alcohol-related problems more than doubled when the CAGE test was used. The age of the patients influenced performance of the CAGE test and clinical evaluation. Only 2% of women had a positive CAGE test. Administration of the CAGE test was considered easy in 112 patients and average to difficult in 50, while the test was inapplicable with 52 patients. The latter proportion was higher than that observed in institutions (hospitals, outpatient departments) of the same region. Applicability was influenced neither by the sex nor the age of the patients, but varied greatly according to the physician (from 38% to 100%). The CAGE test increases the number of patients detected with alcohol problems by 37% and seems to be especially useful when administered to young people. The number of women with alcohol problems is probably underestimated by both clinical evaluation and the CAGE test. Furthermore, physicians in private practice are more reluctant to use the CAGE test systematically than those in a public institution.

A high-precision instrument for analyzing nonlinear dynamic behavior of bearing cage.

PubMed

Yang, Z; Chen, H; Yu, T; Li, B

2016-08-01

The high-precision ball bearing is fundamental to the performance of complex mechanical systems. As the speed increases, the cage behavior becomes a key factor in influencing the bearing performance, especially life and reliability. This paper develops a high-precision instrument for analyzing nonlinear dynamic behavior of the bearing cage. The trajectory of the rotational center and non-repetitive run-out (NRRO) of the cage are used to evaluate the instability of cage motion. This instrument applied an aerostatic spindle to support and spin test the bearing to decrease the influence of system error. Then, a high-speed camera is used to capture images when the bearing works at high speeds. A 3D trajectory tracking software tema Motion is used to track the spot which marked the cage surface. Finally, by developing the matlab program, a Lissajous' figure was used to evaluate the nonlinear dynamic behavior of the cage with different speeds. The trajectory of rotational center and NRRO of the cage with various speeds are analyzed. The results can be used to predict the initial failure and optimize cage structural parameters. In addition, the repeatability precision of instrument is also validated. In the future, the motorized spindle will be applied to increase testing speed and image processing algorithms will be developed to analyze the trajectory of the cage.

A high-precision instrument for analyzing nonlinear dynamic behavior of bearing cage

NASA Astrophysics Data System (ADS)

Yang, Z.; Chen, H.; Yu, T.; Li, B.

2016-08-01

The high-precision ball bearing is fundamental to the performance of complex mechanical systems. As the speed increases, the cage behavior becomes a key factor in influencing the bearing performance, especially life and reliability. This paper develops a high-precision instrument for analyzing nonlinear dynamic behavior of the bearing cage. The trajectory of the rotational center and non-repetitive run-out (NRRO) of the cage are used to evaluate the instability of cage motion. This instrument applied an aerostatic spindle to support and spin test the bearing to decrease the influence of system error. Then, a high-speed camera is used to capture images when the bearing works at high speeds. A 3D trajectory tracking software tema Motion is used to track the spot which marked the cage surface. Finally, by developing the matlab program, a Lissajous' figure was used to evaluate the nonlinear dynamic behavior of the cage with different speeds. The trajectory of rotational center and NRRO of the cage with various speeds are analyzed. The results can be used to predict the initial failure and optimize cage structural parameters. In addition, the repeatability precision of instrument is also validated. In the future, the motorized spindle will be applied to increase testing speed and image processing algorithms will be developed to analyze the trajectory of the cage.

Biomechanical Evaluation of a Novel Apatite-Wollastonite Ceramic Cage Design for Lumbar Interbody Fusion: A Finite Element Model Study

PubMed Central

Şenköylü, Alpaslan; Aktaş, Erdem; Sarıkaya, Baran; Sipahioğlu, Serkan; Gürbüz, Rıza; Timuçin, Muharrem

2018-01-01

Objectives Cage design and material properties play a crucial role in the long-term results, since interbody fusions using intervertebral cages have become one of the basic procedures in spinal surgery. Our aim is to design a novel Apatite-Wollastonite interbody fusion cage and evaluate its biomechanical behavior in silico in a segmental spinal model. Materials and Methods Mechanical properties for the Apatite-Wollastonite bioceramic cages were obtained by fitting finite element results to the experimental compression behavior of a cage prototype. The prototype was made from hydroxyapatite, pseudowollastonite, and frit by sintering. The elastic modulus of the material was found to be 32 GPa. Three intact lumbar vertebral segments were modelled with the ANSYS 12.0.1 software and this model was modified to simulate a Posterior Lumbar Interbody Fusion. Four cage designs in different geometries were analyzed in silico under axial loading, flexion, extension, and lateral bending. Results The K2 design had the best overall biomechanical performance for the loads considered. Maximum cage stress recorded was 36.7 MPa in compression after a flexion load, which was within the biomechanical limits of the cage. Conclusion Biomechanical analyses suggest that K2 bioceramic cage is an optimal design and reveals essential material properties for a stable interbody fusion. PMID:29581974

A high-precision instrument for analyzing nonlinear dynamic behavior of bearing cage

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

Yang, Z., E-mail: zhaohui@nwpu.edu.cn; Yu, T.; Chen, H.

2016-08-15

The high-precision ball bearing is fundamental to the performance of complex mechanical systems. As the speed increases, the cage behavior becomes a key factor in influencing the bearing performance, especially life and reliability. This paper develops a high-precision instrument for analyzing nonlinear dynamic behavior of the bearing cage. The trajectory of the rotational center and non-repetitive run-out (NRRO) of the cage are used to evaluate the instability of cage motion. This instrument applied an aerostatic spindle to support and spin test the bearing to decrease the influence of system error. Then, a high-speed camera is used to capture images whenmore » the bearing works at high speeds. A 3D trajectory tracking software TEMA Motion is used to track the spot which marked the cage surface. Finally, by developing the MATLAB program, a Lissajous’ figure was used to evaluate the nonlinear dynamic behavior of the cage with different speeds. The trajectory of rotational center and NRRO of the cage with various speeds are analyzed. The results can be used to predict the initial failure and optimize cage structural parameters. In addition, the repeatability precision of instrument is also validated. In the future, the motorized spindle will be applied to increase testing speed and image processing algorithms will be developed to analyze the trajectory of the cage.« less

Evaluation of cage micro-environment of mice housed on various types of bedding materials

USGS Publications Warehouse

Smith, E.; Stockwell, J.D.; Schweitzer, I.; Langley, S.H.; Smith, A.L.

2004-01-01

A variety of environmental factors can affect the outcomes of studies using laboratory rodents. One such factor is bedding. Several new bedding materials and processing methods have been introduced to the market in recent years, but there are few reports of their performance. In the studies reported here, we have assessed the cage micro-environment (in-cage ammonia levels, temperature, and humidity) of mice housed on various kinds of bedding and their combinations. We also compared results for bedding supplied as Nestpaks versus loose bedding. We studied C57BL/6J mice (commonly used) and NOD/LtJ mice (heavy soilers) that were maintained, except in one study, in static duplex cages. In general, we observed little effect of bedding type on in-cage temperature or humidity; however, there was considerable variation in ammonia concentrations. The lowest ammonia concentrations occurred in cages housing mice on hardwood bedding or a mixture of corncob and alpha cellulose. In one experiment comparing the micro-environments of NOD/LtJ male mice housed on woodpulp fiber bedding in static versus ventilated caging, we showed a statistically significant decrease in ammonia concentrations in ventilated cages. Therefore, our data show that bedding type affects the micro-environment in static cages and that effects may differ for ventilated cages, which are being used in vivaria with increasing frequency.

Antisense RNA that Affects Rhodopseudomonas palustris Quorum-Sensing Signal Receptor Expression

DTIC Science & Technology

2012-01-01

antisense molecules were produced, we performed a Northern blot analysis with RNA harvested from wild-type and rpaR-mutant R. palustris cells by using...aeruginosa, cells were grown to late-log phase, harvested by cen- trifugation, suspended in SDS/PAGE buffer, and lysed by boiling and sonication. Cell...a selectable DNA fragment. Gene 29:303–313. 17. Egland KA, Greenberg EP (1999) Quorum sensing in Vibrio fischeri: Elements of the luxl promoter. Mol

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

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

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.

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.

Water ordering controls the dynamic equilibrium of micelle-fibre formation in self-assembly of peptide amphiphiles.

PubMed

Deshmukh, Sanket A; Solomon, Lee A; Kamath, Ganesh; Fry, H Christopher; Sankaranarayanan, Subramanian K R S

2016-08-24

Understanding the role of water in governing the kinetics of the self-assembly processes of amphiphilic peptides remains elusive. Here, we use a multistage atomistic-coarse-grained approach, complemented by circular dichroism/infrared spectroscopy and dynamic light scattering experiments to highlight the dual nature of water in driving the self-assembly of peptide amphiphiles (PAs). We show computationally that water cage formation and breakage near the hydrophobic groups control the fusion dynamics and aggregation of PAs in the micellar stage. Simulations also suggest that enhanced structural ordering of vicinal water near the hydrophilic amino acids shifts the equilibrium towards the fibre phase and stimulates structure and order during the PA assembly into nanofibres. Experiments validate our simulation findings; the measured infrared O-H bond stretching frequency is reminiscent of an ice-like bond which suggests that the solvated water becomes increasingly ordered with time in the assembled peptide network, thus shedding light on the role of water in a self-assembly process.

Mice selectively bred for open-field thigmotaxis: life span and stability of the selection trait.

PubMed

Leppänen, Pia K; Ewalds-Kvist, S Béatrice M; Selander, Ritva-Kajsa

2005-04-01

In 2 experiments, the authors examined 69 mice selectively bred for high or low levels of open-field (OF) thigmotactic behavior (high open-field thigmotaxis [HOFT] and low open-field thigmotaxis [LOFT], respectively). They found that the strains differed in defecation during the 60-min exposure to the OF. Furthermore, the strains differed with regard to their life spans: The more thigmotactic HOFT mice lived longer than the LOFT mice. The strains were not differentiated by food intake or excretion. The strain difference in thigmotaxis was not age dependent, and it persisted in the home-cage condition as well. Neither the location (center or wall) of the starting point nor the shape (circular or square) of the OF arena affected the difference in wall-seeking behavior between the two strains. The authors concluded that the difference in thigmotaxis (or emotionality) between the HOFT and LOFT mice is a stable and robust feature of these animals.

Porous titanium-6 aluminum-4 vanadium cage has better osseointegration and less micromotion than a poly-ether-ether-ketone cage in sheep vertebral fusion.

PubMed

Wu, Su-Hua; Li, Yi; Zhang, Yong-Quan; Li, Xiao-Kang; Yuan, Chao-Fan; Hao, Yu-Lin; Zhang, Zhi-Yong; Guo, Zheng

2013-12-01

Interbody fusion cages made of poly-ether-ether-ketone (PEEK) have been widely used in clinics for spinal disorders treatment; however, they do not integrate well with surrounding bone tissue. Ti-6Al-4V (Ti) has demonstrated greater osteoconductivity than PEEK, but the traditional Ti cage is generally limited by its much greater elastic modulus (110 GPa) than natural bone (0.05-30 GPa). In this study, we developed a porous Ti cage using electron beam melting (EBM) technique to reduce its elastic modulus and compared its spinal fusion efficacy with a PEEK cage in a preclinical sheep anterior cervical fusion model. A porous Ti cage possesses a fully interconnected porous structure (porosity: 68 ± 5.3%; pore size: 710 ± 42 μm) and a similar Young's modulus as natural bone (2.5 ± 0.2 GPa). When implanted in vivo, the porous Ti cage promoted fast bone ingrowth, achieving similar bone volume fraction at 6 months as the PEEK cage without autograft transplantation. Moreover, it promoted better osteointegration with higher degree (2-10x) of bone-material binding, demonstrated by histomorphometrical analysis, and significantly higher mechanical stability (P < 0.01), shown by biomechanical testing. The porous Ti cage fabricated by EBM could achieve fast bone ingrowth. In addition, it had better osseointegration and superior mechanical stability than the conventional PEEK cage, demonstrating great potential for clinical application. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.

Influence of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage.

PubMed

Baker, Joseph L; Furbish, Jeffrey; Lindberg, Gerrick E

2015-11-01

We examine the effect of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage and contrast these results with the behavior of Trp-cage in water. We find the ionic liquid has a dramatic effect on Trp-cage, though many similarities with aqueous Trp-cage are observed. We assess Trp-cage folding by monitoring root mean square deviation from the crystallographic structure, radius of gyration, proline cis/trans isomerization state, protein secondary structure, amino acid contact formation and distance, and native and non-native contact formation. Starting from an unfolded configuration, Trp-cage folds in water at 298 K in less than 500 ns of simulation, but has very little mobility in the ionic liquid at the same temperature, which can be ascribed to the higher ionic liquid viscosity. At 365 K, the mobility of the ionic liquid is increased and initial stages of Trp-cage folding are observed, however Trp-cage does not reach the native folded state in 2 μs of simulation in the ionic liquid. Therefore, in addition to conventional molecular dynamics, we also employ scaled molecular dynamics to expedite sampling, and we demonstrate that Trp-cage in the ionic liquid does closely approach the aqueous folded state. Interestingly, while the reduced mobility of the ionic liquid is found to restrict Trp-cage motion, the ionic liquid does facilitate proline cis/trans isomerization events that are not seen in our aqueous simulations. Copyright © 2015 Elsevier Inc. All rights reserved.

50 CFR 648.77 - Cage identification.

Code of Federal Regulations, 2012 CFR

2012-10-01

... Atlantic Surf Clam and Ocean Quahog Fisheries § 648.77 Cage identification. Except as provided in § 648.78... for surfclams and ocean quahogs: (a) Tagging. Before offloading, all cages that contain surfclams or... Administrator. (g) Transfer. See § 648.74(b)(2). (h) Presumptions. Surfclams and ocean quahogs found in cages...

50 CFR 648.77 - Cage identification.

Code of Federal Regulations, 2013 CFR

2013-10-01

... Atlantic Surf Clam and Ocean Quahog Fisheries § 648.77 Cage identification. Except as provided in § 648.78... for surfclams and ocean quahogs: (a) Tagging. Before offloading, all cages that contain surfclams or... Administrator. (g) Transfer. See § 648.74(b)(2). (h) Presumptions. Surfclams and ocean quahogs found in cages...

50 CFR 648.77 - Cage identification.

Code of Federal Regulations, 2014 CFR

2014-10-01

... Atlantic Surf Clam and Ocean Quahog Fisheries § 648.77 Cage identification. Except as provided in § 648.78... for surfclams and ocean quahogs: (a) Tagging. Before offloading, all cages that contain surfclams or... Administrator. (g) Transfer. See § 648.74(b)(2). (h) Presumptions. Surfclams and ocean quahogs found in cages...

Incorporation of organometallic Ru complexes into apo-ferritin cage.

PubMed

Takezawa, Yusuke; Böckmann, Philipp; Sugi, Naoki; Wang, Ziyue; Abe, Satoshi; Murakami, Tatsuya; Hikage, Tatsuo; Erker, Gerhard; Watanabe, Yoshihito; Kitagawa, Susumu; Ueno, Takafumi

2011-03-14

Spherical protein cages such as an iron storage protein, ferritin, have great potential as nanometer-scale capsules to assemble and store metal ions and complexes. We report herein the synthesis of a composite of an apo-ferritin cage and Ru(p-cymene) complexes. Ru complexes were efficiently incorporated into the ferritin cavity without degradation of its cage structure. X-Ray crystallography revealed that the Ru complexes were immobilized on the interior surface of the cage mainly by the coordination of histidine residues.

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.

Effects of an Extended Cage-change Interval on Ammonia Levels and Reproduction in Mongolian Gerbils (Meriones unguiculatus).

PubMed

McCullagh, Elizabeth A; McCullagh, Peter; Klug, Achim; Leszczynski, Jori K; Fong, Derek L

2017-11-01

Prompted by the cage cleanliness of Mongolian gerbils (Meriones unguiculatus), we evaluated a prolonged cage-change interval. We compared the effects of a 2-wk and 6-wk cage-change schedule on ammonia levels, temperature, humidity, and reproductive performance in breeding pairs housed in IVC. We hypothesized that ammonia levels would remain below our threshold for cage changing and that reproductive performance would not be affected. Although ammonia levels increased over time, they remained low (less than 5 ppm) over the 6-wk period. In addition, the 6-wk cage-change interval did not significantly influence reproductive parameters, such as average pup weaning weight, number of litters, and number of pups per litter. We conclude that an extended cage-change interval (6-wk) can be used for gerbils without significant increases in intracage ammonia levels or effects on reproduction.

Testing and Comparative Evaluation of Space Shuttle Main Engine Flowmeter Bearings

NASA Technical Reports Server (NTRS)

Hissam, Andy; Leberman, Mike; McLeroy, Rick

2005-01-01

This paper provides a summary of testing of Space Shuttle Main Engine (SSME) flowmeter bearings and cage material. These tests were con&cM over a several month period in 2004 at the Marshall Space Flight Center. The test program's primary objective was to compare the performance of bearings using the existing cage material and bearings using a proposed replacement cage material. In order to meet the test objectives for this program, a flowmeter test rig was designed and fabricated to measure both breakaway and running torque for a flowmeter assembly. Other test parameters,,such as motor current and shaft speed, were also recorded and provide a means of comparing bearing performance. The flowmeter and bearings were tested in liquid hydrogen to simulate the flowmeter's operating environment as closely as possible. Based on the results from this testing, the bearings with the existing cage material are equivalent to the bearings with the proposed replacement cage material. No major differences exist between the old and new cage materials. Therefore, the new cage material is a suitable replacement for the existing cage material.

Synthesis, structural characterisation and magnetic studies of polymetallic iron phosphonate cages.

PubMed

Khanra, Sumit; Helliwell, Madeleine; Tuna, Floriana; McInnes, Eric J L; Winpenny, Richard E P

2009-08-21

Four new polymetallic iron(III) phosphonate cages have been made and structurally characterised. These are an octanuclear cage [Fe(8)O(3)(OH)(2)(O(2)C(t)Bu)(11)(PhCH(2)PO(3))(3)(py)(3)], a decanuclear cage [Fe(10)O(2)(OH)(8)(O(2)C(t)Bu)(10)(PhCH(2)PO(3))(4)(pip)(2)], a heterometallic cage [Fe(6)Li(5)(mu(3)-O)(2)((t)BuPO(3))(6)(O(2)C(t)Bu)(8)(MeOH)(2)(Py)(4)] and a tridecanuclear cage [Et(3)NH](2)[Fe(13)(mu(3)-O)(3)(mu(2)-OH)(7)((t)BuPO(3))(7)(Me(3)CCO(2))(14)(H(2)O)] (pip = piperidine, py = pyridine). Magnetic studies of the first three compounds show anti-ferromagnetic exchange between the iron(III) centers leading to diamagnetic ground states for the homometallic cages. For the heterometallic cage, the six Fe(III) centers are arranged in two triangles, and each triangle has an S = 1/2 spin ground state.

Intracage ammonia levels in static and individually ventilated cages housing C57BL/6 mice on 4 bedding substrates.

PubMed

Ferrecchia, Christie E; Jensen, Kelly; Van Andel, Roger

2014-03-01

The relationship among ammonia levels, cage-changing frequency, and bedding types is an important and potentially controversial topic in the laboratory animal science community. Some bedding options may not provide sufficient urine absorption and bacterial regulation to minimize ammonia production during the interval between cage changes. High intracage ammonia levels can cause subclinical degeneration and inflammation of nasal passages, rhinitis and olfactory epithelial necrosis in exposed mice. Here we sought to compare the effects of 4 commonly used bedding substrates (1/4-in. irradiated corncob, reclaimed wood pulp, aspen wood chips, and recycled newspaper) on ammonia generation when housing female C57BL/6 mice in static and individually ventilated caging. Intracage ammonia levels were measured daily for 1 wk (static cage experiment) or 2 wk (IVC experiment). The results of this study suggest that the corncob, aspen wood chip, and recycled newspaper beddings that we tested are suitable for once-weekly cage changing for static cages and for changing every 2 wk for IVC. However, ammonia levels were not controlled appropriately in cages containing reclaimed wood pulp bedding, and pathologic changes occurred within 1 wk in the nares of mice housed on this bedding in static cages.

Intracage Ammonia Levels in Static and Individually Ventilated Cages Housing C57BL/6 Mice on 4 Bedding Substrates

PubMed Central

Ferrecchia, Christie E; Jensen, Kelly; Andel, Roger Van

2014-01-01

The relationship among ammonia levels, cage-changing frequency, and bedding types is an important and potentially controversial topic in the laboratory animal science community. Some bedding options may not provide sufficient urine absorption and bacterial regulation to minimize ammonia production during the interval between cage changes. High intracage ammonia levels can cause subclinical degeneration and inflammation of nasal passages, rhinitis and olfactory epithelial necrosis in exposed mice. Here we sought to compare the effects of 4 commonly used bedding substrates (1/4-in. irradiated corncob, reclaimed wood pulp, aspen wood chips, and recycled newspaper) on ammonia generation when housing female C57BL/6 mice in static and individually ventilated caging. Intracage ammonia levels were measured daily for 1 wk (static cage experiment) or 2 wk (IVC experiment). The results of this study suggest that the corncob, aspen wood chip, and recycled newspaper beddings that we tested are suitable for once-weekly cage changing for static cages and for changing every 2 wk for IVC. However, ammonia levels were not controlled appropriately in cages containing reclaimed wood pulp bedding, and pathologic changes occurred within 1 wk in the nares of mice housed on this bedding in static cages. PMID:24602540

Finite element analysis and cadaveric cinematic analysis of fixation options for anteriorly implanted trabecular metal interbody cages.

PubMed

Berjano, Pedro; Blanco, Juan Francisco; Rendon, Diego; Villafañe, Jorge Hugo; Pescador, David; Atienza, Carlos Manuel

2015-11-01

To assess, with finite element analysis and an in vitro biomechanical study in cadaver, whether the implementation of an anterior interbody cage made of hedrocel with nitinol shape memory staples in compression increases the stiffness of the stand-alone interbody cage and to compare these constructs' stiffness to other constructs common in clinical practice. A biomechanical study with a finite element analysis and cadaveric testing assessed the stiffness of different fixation modes for the L4-L5 functional spinal unit: intact spine, destabilized spine with discectomy, posterior pedicle-screw fixation, anterior stand-alone interbody cage, anterior interbody cage with bilateral pedicle screws and anterior interbody cage with two shape memory staples in compression. These modalities of vertebral fixation were compared in four loading modes (flexion, extension, lateral bending, and axial rotation). The L4-L5 spinal unit with an anterior interbody cage and two staples was stiffer than the stand-alone cage. The construct stiffness was similar to that of a model of posterior pedicular stabilization. The stiffness was lower than that of the anterior cage plus bilateral pedicle-screw fixation. The use of an anterior interbody implant with shape memory staples in compression may be an alternative to isolated posterior fixation and to anterior isolated implants, with increased stiffness.

Investigation of instability, dynamic forces, and effect of dynamic loading on strength of cages for the bearings in the high pressure oxygen turbopumps for the space shuttle main engine

NASA Technical Reports Server (NTRS)

Dufrane, K. F.; Kannel, J. W.; Merriman, T. L.; Rosenfield, A. R.

1985-01-01

Experiments were performed to determine the effect of cyclic loading on bearing cage strength. A long term working tensile load of approximately 1300 N (300 lbs) was found to be the likely maximum. Higher loads caused a decrease in cage tensile strength after the 125,000 cycle testing period. Poisson's ratio in compression was found to be highly dependent upon the direction of the fiberglass plies. At room temperature the value was 0.15 with the plies and 0.68 across the plies. At -196 C (-321 F), the value with the plies was 0.20. The results of the analyses conducted have again demonstrated the critical need for improved lubrication in the high pressure oxygen turbopump bearings. Lubricant films with low shear strength and low friction coefficients promote cage stability and decrease ball/cage forces during marginal operating conditions. The analysis of the effect of combined bearing loads on ball/cage loads has identified a radial load of 3600 N (800 lbs) as the maximum for the current clearance of the balls and cage pockets. Liquid oxygen impinging on the cage in the direction of rotation was found to enhance cage stability.

Improved Synthesis of Caged Glutamate and Caging Each Functional Group.

PubMed

Guruge, Charitha; Ouedraogo, Yannick P; Comitz, Richard L; Ma, Jingxuan; Losonczy, Attila; Nesnas, Nasri

2018-05-25

Glutamate is an excitatory neurotransmitter that controls numerous pathways in the brain. Neuroscientists make use of photoremovable protecting groups, also known as cages, to release glutamate with precise spatial and temporal control. Various cage designs have been developed and among the most effective has been the nitroindolinyl caging of glutamate. We, hereby, report an improved synthesis of one of the current leading molecules of caged glutamate, 4-carboxymethoxy-5,7-dinitroindolinyl glutamate (CDNI-Glu), which possesses efficiencies with the highest reported quantum yield of at least 0.5. We present the shortest route, to date, for the synthesis of CDNI-Glu in 4 steps, with a total reaction time of 40 h and an overall yield of 20%. We also caged glutamate at the other two functional groups, thereby, introducing two new cage designs: α-CDNI-Glu and N-CDNI-Glu. We included a study of their photocleavage properties using UV-vis, NMR, as well as a physiology experiment of a two-photon uncaging of CDNI-Glu in acute hippocampal brain slices. The newly introduced cage designs may have the potential to minimize the interference that CDNI-Glu has with the GABA A receptor. We are broadly disseminating this to enable neuroscientists to use these photoactivatable tools.

Impact of Room Ventilation Rates on Mouse Cage Ventilation and Microenvironment.

PubMed

Reeb, Carolyn K.; Jones, Robert B.; Bearg, David W.; Bedigian, Hendrick; Paigen, Beverly

1997-01-01

To assess the impact of room ventilation on animal cage microenvironment, intracage ventilation rate, temperature, humidity, and concentrations of carbon dioxide and ammonia were monitored in nonpressurized, bonnet-topped mouse cages. Cages on the top, middle, and bottom rows of a mouse rack were monitored at room ventilation rates of 0, 5, 10, and 20 air changes/h (ACH). Ventilation inside the animal cage increased somewhat from 12.8 to 18.9 ACH as room ventilation rate in- creased from 0 to 20 ACH, but the differences were not statistically significant, and most of the increase occurred in cages in the top row nearest to the fresh air supply. Cages containing mice had ventilation rate between 10 and 15 ACH even when room ventilation was reduced to 0 ACH; this ventilation is a result of the thermal heat load of the mice. After 6 days of soiled bedding, intracage ammonia concentration was c 3 ppm at all room ventilation rates and was not affected by increasing room ventilation. Temperature inside cages did not change with increasing ventilation. Humidity inside cages significantly decreased with increasing ventilation, from 55% relative humidity at 5 ACH to 36% relative humidity at 20 ACH. Carbon dioxide concentration decreased from 2,500 ppm to 1,900 ppm when ventilation rate increased from 5 ACH to 10 ACH, but no further significant decrease was observed at 20 ACH. In conclusion, increasing the room ventilation rate higher than 5 ACH did not result in significant improvements in the cage microenvironment.

Effect of nest design, passages, and hybrid on use of nest and production performance of layers in furnished cages.

PubMed

Wall, H; Tauson, R; Elwinger, K

2002-03-01

Production performance, including egg quality, and proportion of eggs laid in nests were studied in furnished experimental cages incorporating nests, litter baths, and perches. The study comprised a total of 972 hens of two genotypes: Lohmann Selected Leghorn (LSL) and Hy-Line White. The birds were studied from 20 to 80 wk of age, and conventional four-hen cages were included as a reference. In furnished cages for six hens, the effects of 30 or 50% vs. 100% nest bottom lining (Astro turf) were studied with LSL hens. Nest bottom lining had no significant effect on egg production or proportions of cracked or dirty eggs, but the use of nests was significantly higher in cages incorporating nests with 100% lining, compared with 50 or 30%. The two hybrids were compared when housed in large, group-furnished cages for 14 or 16 hens of two designs; with a rear partition with two pop holes or fully open, i.e., no rear partition. LSL birds produced significantly better and had a significantly lower proportion of cracked eggs. There was no difference between H- and O-cages, either in production or in egg quality. LSL birds laid a significantly lower proportion of eggs in the nests, especially in O-cages, implying a significant hybrid x cage interaction. When housed in conventional cages, the hybrids did not differ in proportion of cracked eggs but differed in production traits. It was concluded that with the present nest design, the proportion of nest bottom lining cannot be reduced without affecting birds' use of nests, but the proportion did not affect exterior egg quality. The effect of genotype should be considered in the further development of furnished cages.

Effect of cage design, supplemental posterior instrumentation and approach on primary stability of a lumbar interbody fusion - A biomechanical in vitro study.

PubMed

Schmoelz, Werner; Sandriesser, Sabrina; Loebl, Oded; Bauer, Marlies; Krappinger, Dietmar

2017-10-01

There are various techniques and approaches for lumbar interbody fusion differing in access, cage type and type of supplemental posterior instrumentation. While a transforaminal access usually includes a hemifacetectomy, the facet joint can be preserved with a more lateral extraforaminal access. The supplemental posterior instrumentation required for both fusion techniques is still debated. The purpose of the present study was to compare primary stability of the two accesses for two different cage types with none, unilateral and bilateral supplemental posterior instrumentation. Six monosegmental lumbar functional spinal units (FSUs) were included in each of the two groups, and subjected to a flexibility test. As cages, a newly designed cage was compared to a standard cage in the following states: (a) native, (b) stand-alone cage, (c) bilateral internal fixator, (d) unilateral internal fixator, (e) unilateral facetectomy+bilateral internal fixator, (f) unilateral facetectomy+unilateral internal fixator and (g) unilateral facetectomy with stand-alone cage. For comparison the range of motion was normalized to the native state and the effects of the facetectomy, cage type, and supplemental instrumentation was compared. Within the subject comparison showed a significantly higher flexibility for the unilateral facetectomy in all motion directions (p<0.001). In between subject comparison showed a significant effect of cage type on flexibility in flexion/extension (p=0.002) and lateral bending (p=0.028) but not in axial rotation (p=0.322). The type of supplemental posterior fixation had a significant effect on the flexibility in all motion directions (stand-alone>unilateral fixator>bilateral fixator). Cage design and approach type are affecting the primary stability of lumbar interbody fusion procedures while the type of posterior instrumentation is the most influencing factor. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tissue engineering rib with the incorporation of biodegradable polymer cage and BMSCs/decalcified bone: an experimental study in a canine model.

PubMed

Tang, Hua; Wu, Bin; Qin, Xiong; Zhang, Lu; Kretlow, Jim; Xu, Zhifei

2013-05-20

The reconstruction of large bone defects, including rib defects, remains a challenge for surgeons. In this study, we used biodegradable polydioxanone (PDO) cages to tissue engineer ribs for the reconstruction of 4cm-long costal defects. PDO sutures were used to weave 6cm long and 1cm diameter cages. Demineralized bone matrix (DBM) which is a xenograft was molded into cuboids and seeded with second passage bone marrow mesenchymal stem cells (BMSCs) that had been osteogenically induced. Two DBM cuboids seeded with BMSCs were put into the PDO cage and used to reconstruct the costal defects. Radiographic examination including 3D reconstruction, histologic examination and mechanical test was performed after 24 postoperative weeks. All the experimental subjects survived. In all groups, the PDO cage had completely degraded after 24 weeks and been replaced by fibrous tissue. Better shape and radian were achieved in PDO cages filled with DBM and BMSCs than in the other two groups (cages alone, or cages filled with acellular DBM cuboids). When the repaired ribs were subjected to an outer force, the ribs in the PDO cage/DBMs/BMSCs group kept their original shape while ribs in the other two groups deformed. In the PDO cage/DBMs/BMSCs groups, we also observed bony union at all the construct interfaces while there was no bony union observed in the other two groups. This result was also confirmed by radiographic and histologic examination. This study demonstrates that biodegradable PDO cage in combination with two short BMSCs/DBM cuboids can repair large rib defects. The satisfactory repair rate suggests that this might be a feasible approach for large bone repair.

Tissue engineering rib with the incorporation of biodegradable polymer cage and BMSCs/decalcified bone: an experimental study in a canine model

PubMed Central

2013-01-01

Background The reconstruction of large bone defects, including rib defects, remains a challenge for surgeons. In this study, we used biodegradable polydioxanone (PDO) cages to tissue engineer ribs for the reconstruction of 4cm-long costal defects. Methods PDO sutures were used to weave 6cm long and 1cm diameter cages. Demineralized bone matrix (DBM) which is a xenograft was molded into cuboids and seeded with second passage bone marrow mesenchymal stem cells (BMSCs) that had been osteogenically induced. Two DBM cuboids seeded with BMSCs were put into the PDO cage and used to reconstruct the costal defects. Radiographic examination including 3D reconstruction, histologic examination and mechanical test was performed after 24 postoperative weeks. Results All the experimental subjects survived. In all groups, the PDO cage had completely degraded after 24 weeks and been replaced by fibrous tissue. Better shape and radian were achieved in PDO cages filled with DBM and BMSCs than in the other two groups (cages alone, or cages filled with acellular DBM cuboids). When the repaired ribs were subjected to an outer force, the ribs in the PDO cage/DBMs/BMSCs group kept their original shape while ribs in the other two groups deformed. In the PDO cage/DBMs/BMSCs groups, we also observed bony union at all the construct interfaces while there was no bony union observed in the other two groups. This result was also confirmed by radiographic and histologic examination. Conclusions This study demonstrates that biodegradable PDO cage in combination with two short BMSCs/DBM cuboids can repair large rib defects. The satisfactory repair rate suggests that this might be a feasible approach for large bone repair. PMID:23688344

Dyspnea, chest wall hyperinflation, and rib cage distortion in exercising patients with chronic obstructive pulmonary disease.

PubMed

Bruni, Giulia Innocenti; Gigliotti, Francesco; Binazzi, Barbara; Romagnoli, Isabella; Duranti, Roberto; Scano, Giorgio

2012-06-01

Whether dyspnea, chest wall dynamic hyperinflation, and abnormalities of rib cage motion are interrelated phenomena has not been systematically evaluated in patients with chronic obstructive pulmonary disease (COPD). Our hypothesis that they are not interrelated was based on the following observations: (i) externally imposed expiratory flow limitation is associated with no rib cage distortion during strenuous incremental exercise, with indexes of hyperinflation not being correlated with dyspnea, and (ii) end-expiratory chest wall volume may either increase or decrease during exercise in patients with COPD, with those who hyperinflate being as breathless as those who do not. Sixteen patients breathed either room air or 50% supplemental O2 at 75% of peak exercise in randomized order. We evaluated the volume of chest wall (V(cw)) and its compartments: the upper rib cage (V(rcp)), lower rib cage (V(rca)), and abdomen (V(ab)) using optoelectronic plethysmography; rib cage distortion was assessed by measuring the phase angle shift between V(rcp) and V(rca). Ten patients increased end-expiratory V(cw) (V(cw,ee)) on air. In seven hyperinflators and three non-hyperinflators, the lower rib cage paradoxed inward during inspiration with a phase angle of 63.4° ± 30.7° compared with a normal phase angle of 16.1° ± 2.3° recorded in patients without rib cage distortion. Dyspnea (by Borg scale) averaged 8.2 and 9 at the end of exercise on air in patients with and without rib cage distortion, respectively. At iso-time during exercise with oxygen, decreased dyspnea was associated with a decrease in ventilation regardless of whether patients distorted the rib cage, dynamically hyperinflated, or deflated the chest wall. Dyspnea, chest wall dynamic hyperinflation, and rib cage distortion are not interrelated phenomena.

The Impact of Environmental Light Intensity on Experimental Tumor Growth.

PubMed

Suckow, Mark A; Wolter, William R; Duffield, Giles E

2017-09-01

Cancer research requires for consistent models that minimize environmental variables. Within the typical laboratory animal housing facility, animals may be exposed to varying intensities of light as a result of cage type, cage position, light source, and other factors; however, studies evaluating the differential effect of light intensity during the light phase on tumor growth are lacking. The effect of cage face light intensity, as determined by cage rack position was evaluated with two tumor models using the C57Bl/6NHsd mouse and transplantable B16F10 melanoma cells or Lewis lung carcinoma (LLC) cells. Animals were housed in individually-ventilated cages placed at the top, middle, or bottom of the rack in a diagonal pattern so that the top cage was closest to the ceiling light source, and cage face light intensity was measured. Following a two-week acclimation period at the assigned cage position, animals were subcutaneously administered either 1.3×10 6 B16F10 melanoma cells or 2.5×10 5 Lewis lung carcinoma cells. Weights of excised tumors were measured following euthanasia 18 days (melanoma) or 21 days (LCC) after tumor cell administration. Cage face light intensity was significantly different depending on the location of the cage, with cages closest to the light source have the greatest intensity. Mean tumor weights were significantly less (p<0.001 for melanoma; p≤0.01 for LCC) in middle light intensity mice compared to high and low light intensity mice. The environmental light intensity to which experimental animals are exposed may vary markedly with cage location and can significantly influence experimental tumor growth, thus supporting the idea that light intensity should be controlled as an experimental variable for animals used in cancer research. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The role of acoustic nonlinearity in tissue heating behind a rib cage using a high-intensity focused ultrasound phased array

NASA Astrophysics Data System (ADS)

Yuldashev, Petr V.; Shmeleva, Svetlana M.; Ilyin, Sergey A.; Sapozhnikov, Oleg A.; Gavrilov, Leonid R.; Khokhlova, Vera A.

2013-04-01

The goal of this study was to investigate theoretically the effects of nonlinear propagation in a high-intensity focused ultrasound (HIFU) field produced by a therapeutic phased array and the resultant heating of tissue behind a rib cage. Three configurations of focusing were simulated: in water, in water with ribs in the beam path and in water with ribs backed by a layer of soft tissue. The Westervelt equation was used to model the nonlinear HIFU field, and a 1 MHz phased array consisting of 254 circular elements was used as a boundary condition to the model. The temperature rise in tissue was modelled using the bioheat equation, and thermally necrosed volumes were calculated using the thermal dose formulation. The shapes of lesions predicted by the modelling were compared with those previously obtained in in vitro experiments at low-power sonications. Intensity levels at the face of the array elements that corresponded to the formation of high-amplitude shock fronts in the focal region were determined as 10 W cm-2 in the free field in water and 40 W cm-2 in the presence of ribs. It was shown that exposures with shocks provided a substantial increase in tissue heating, and its better spatial localization in the main focal region only. The relative effects of overheating ribs and splitting of the focus due to the periodic structure of the ribs were therefore reduced. These results suggest that utilizing nonlinear propagation and shock formation effects can be beneficial for inducing confined HIFU lesions when irradiating through obstructions such as ribs. Design of compact therapeutic arrays to provide maximum power outputs with lower intensity levels at the elements is necessary to achieve shock wave regimes for clinically relevant sonication depths in tissue.

The role of acoustic nonlinearity in tissue heating behind the rib cage using high intensity focused ultrasound phased array

PubMed Central

Yuldashev, Petr V.; Shmeleva, Svetlana M.; Ilyin, Sergey A.; Sapozhnikov, Oleg A.; Gavrilov, Leonid R.; Khokhlova, Vera A.

2013-01-01

The goal of this study was to investigate theoretically the effects of nonlinear propagation in a high intensity focused ultrasound (HIFU) field produced by a therapeutic phased array and the resultant heating of tissue behind a rib cage. Three configurations of focusing were simulated: in water, in water with ribs in the beam path, and in water with ribs backed by a layer of soft tissue. The Westervelt equation was used to model the nonlinear HIFU field and a 1 MHz phased array consisting of 254 circular elements was used as a boundary condition to the model. The temperature rise in tissue was modelled using the bioheat equation, and thermally necrosed volumes were calculated using the thermal dose formulation. The shapes of lesions predicted by the modelling were compared with those previously obtained in in vitro experiments at low power sonications. Intensity levels at the face of the array elements that corresponded to formation of high amplitude shock fronts in the focal region were determined as 10 W·cm−2 in the free field in water and 40 W·cm−2 in the presence of ribs. It was shown that exposures with shocks provided a substantial increase in tissue heating, and its better spatial localization in the main focal region only. The relative effects of overheating ribs and splitting of the focus due to the periodic structure of the ribs were therefore reduced. These results suggest that utilizing nonlinear propagation and shock formation effects can be beneficial for inducing confined HIFU lesions when irradiating through obstructions such as ribs. Design of compact therapeutic arrays to provide maximum power outputs with lower intensity levels at the elements is necessary to achieve shock wave regimes for clinically relevant sonication depths in tissue. PMID:23528338

Maternal antibody transfer to broiler progeny varies among strains and is affected by grain source and cage density.

PubMed

Leandro, N M; Ali, R; Koci, M; Moraes, V; Eusebio-Balcazar, P E; Jornigan, J; Malheiros, R D; Wineland, M J; Brake, J; Oviedo-Rondón, E O

2011-12-01

Two experiments were conducted to examine the effects of broiler breeder dietary grain source and cage density on maternal antibody (MatAb) transfer to progeny in 2 genetic strains (A and B). Broiler breeders were assigned to 16 litter floor pens and fed either corn- or wheat-based diets. Breeders were administered 4 live vaccines against Newcastle disease virus (NDV). At 23 wk of age, pullets and cocks, which reflected the full BW distribution from each treatment, were moved to a cage breeder house and placed at 1 or 2 hens/cage. Breeders were artificially inseminated at 44 wk (experiment 1) and 52 wk of age (experiment 2). Eggs were collected for 8 d, incubated, and placed in individual pedigree bags at d 19 of incubation. Blood samples from 5 chicks per treatment combination were collected at hatch in both experiments. Spleen and bursa were collected from the same chicks for histomorphometry analyses in experiment 2. In the second experiment, 12 chicks per treatment were placed in cages. Progeny were provided diets based on the same grain (corn or wheat) as their parents. Serum samples were collected at 5, 9, and 13 d of age and analyzed for anti-NDV MatAb. Data were analyzed as a 2 × 2 × 2 factorial design considering strain, dietary grain source, and cage density as main factors. Interaction effects were observed in breeders and progeny. Experiment 1 showed that strain A chicks had lower levels of MatAb when hens were housed at 2 hens/cage rather than 1 hen/cage. The MatAb levels of strain B chickens were not affected by cage density in either experiment. Experiment 2 demonstrated similar effects of cage density on MatAb levels and the area of bursa follicles for both strains. Progeny of breeders fed corn-based diets had smaller spleen white pulp only when hens were housed at 2 hens/cage compared with 1 hen/cage. The results of these experiments suggest that breeder strain and cage-density conditions affected MatAb transfer to progeny and embryo development of spleen and bursa.

Vertebral body fracture after anterior cervical discectomy and fusion with zero-profile anchored cages in adjacent levels: a cautionary tale.

PubMed

Mattei, Tobias A; Teles, Alisson R; Dinh, Dzung H

2016-01-05

Zero-profile (also called self-locking, anchored or stand-alone cages) have been recently proposed as an interesting alternative for anterior cervical discectomy and fusion (ACDF), as they are supposed to reduce the rates of post-operative cage extrusion without necessarily incurring in the additional surgical time and increased rates of dysphagia associated with plating. Nevertheless, the exact indications of zero-profile anchored cages have not yet been established in the literature. To report the first case of a vertebral body fracture between the blades of zero-profile anchored cages after ACDFs in adjacent levels and to review the available literature on hardware-related complications after multi-level ACDFs with zero-profile anchored cages. Case report and systematic literature review. The authors report the first case of a vertebral body fracture between the blades of zero-profile anchored cages after ACDFs in adjacent levels. The patient presented with refractory mechanical neck pain at the 1-month post-operative follow-up, ultimately requiring a posterior instrumented fusion. A comprehensive systematic literature review on the available data regarding the safety, complications as well as radiological and clinical outcomes of zero-profile anchored cages is also performed. In the reported case, the use of zero-profile anchored cages in adjacent levels on the cervical spine led to a fracture of the vertebral body between the cages at the 1-month follow-up, with anterior avulsion of the part of the vertebral body where the blades from the two cages converged. According to the systematic literature review which included 409 patients from 10 different clinical series (with a total cumulative follow-up of approximately 535 patients-year), there were only two reported hardware-related complications after ACDF with zero-profile anchored cages, none of them involving fracture at the level of convergence of blades or screws. Although hardware-related complications after the use of zero-profile anchored cages seem to be rare events, future biomechanical and clinical studies are warranted in order to evaluate the safety of employing such devices for the treatment of multilevel degenerative disc disease in the cervical spine.

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

Biomechanics of lateral lumbar interbody fusion constructs with lateral and posterior plate fixation: laboratory investigation.

PubMed

Fogel, Guy R; Parikh, Rachit D; Ryu, Stephen I; Turner, Alexander W L

2014-03-01

Lumbar interbody fusion is indicated in the treatment of degenerative conditions. Laterally inserted interbody cages significantly decrease range of motion (ROM) compared with other cages. Supplemental fixation options such as lateral plates or spinous process plates have been shown to provide stability and to reduce morbidity. The authors of the current study investigate the in vitro stability of the interbody cage with a combination of lateral and spinous process plate fixation and compare this method to the established bilateral pedicle screw fixation technique. Ten L1-5 specimens were evaluated using multidirectional nondestructive moments (± 7.5 N · m), with a custom 6 degrees-of-freedom spine simulator. Intervertebral motions (ROM) were measured optoelectronically. Each spine was evaluated under the following conditions at the L3-4 level: intact; interbody cage alone (stand-alone); cage supplemented with lateral plate; cage supplemented with ipsilateral pedicle screws; cage supplemented with bilateral pedicle screws; cage supplemented with spinous process plate; and cage supplemented with a combination of lateral plate and spinous process plate. Intervertebral rotations were calculated, and ROM data were normalized to the intact ROM data. The stand-alone laterally inserted interbody cage significantly reduced ROM with respect to the intact state in flexion-extension (31.6% intact ROM, p < 0.001), lateral bending (32.5%, p < 0.001), and axial rotation (69.4%, p = 0.002). Compared with the stand-alone condition, addition of a lateral plate to the interbody cage did not significantly alter the ROM in flexion-extension (p = 0.904); however, it was significantly decreased in lateral bending and axial rotation (p < 0.001). The cage supplemented with a lateral plate was not statistically different from bilateral pedicle screws in lateral bending (p = 0.579). Supplemental fixation using a spinous process plate was not significantly different from bilateral pedicle screws in flexion-extension (p = 0.476). The combination of lateral plate and spinous process plate was not statistically different from the cage supplemented with bilateral pedicle screws in all the loading modes (p ≥ 0.365). A combination of lateral and spinous process plate fixation to supplement a laterally inserted interbody cage helps achieve rigidity in all motion planes similar to that achieved with bilateral pedicle screws.

Protein cages and synthetic polymers: a fruitful symbiosis for drug delivery applications, bionanotechnology and materials science.

PubMed

Rother, Martin; Nussbaumer, Martin G; Renggli, Kasper; Bruns, Nico

2016-11-07

Protein cages are hollow protein nanoparticles, such as viral capsids, virus-like particles, ferritin, heat-shock proteins and chaperonins. They have well-defined capsule-like structures with a monodisperse size. Their protein subunits can be modified by genetic engineering at predetermined positions, allowing for example site-selective introduction of attachment points for functional groups, catalysts or targeting ligands on their outer surface, in their interior and between subunits. Therefore, protein cages have been extensively explored as functional entities in bionanotechnology, as drug-delivery or gene-delivery vehicles, as nanoreactors or as templates for the synthesis of organic and inorganic nanomaterials. The scope of functionalities and applications of protein cages can be significantly broadened if they are combined with synthetic polymers on their surface or within their interior. For example, PEGylation reduces the immunogenicity of protein cage-based delivery systems and active targeting ligands can be attached via polymer chains to favour their accumulation in diseased tissue. Polymers within protein cages offer the possibility of increasing the loading density of drug molecules, nucleic acids, magnetic resonance imaging contrast agents or catalysts. Moreover, the interaction of protein cages and polymers can be used to modulate the size and shape of some viral capsids to generate structures that do not occur with native viruses. Another possibility is to use the interior of polymer cages as a confined reaction space for polymerization reactions such as atom transfer radical polymerization or rhodium-catalysed polymerization of phenylacetylene. The protein nanoreactors facilitate a higher degree of control over polymer synthesis. This review will summarize the hybrid structures that have been synthesized by polymerizing from protein cage-bound initiators, by conjugating polymers to protein cages, by embedding protein cages into bulk polymeric materials, by forming two- and three-dimensional crystals of protein cages and dendrimers, by adsorbing proteins to the surface of materials, by layer-by-layer deposition of proteins and polyelectrolytes and by encapsulating polymers into protein cages. The application of these hybrid materials in the biomedical context or as tools and building blocks for bionanotechnology, biosensing, memory devices and the synthesis of materials will be highlighted. The review aims to showcase recent developments in this field and to suggest possible future directions and opportunities for the symbiosis of protein cages and polymers.

Lordosis restoration after anterior longitudinal ligament release and placement of lateral hyperlordotic interbody cages during the minimally invasive lateral transpsoas approach: a radiographic study in cadavers.

PubMed

Uribe, Juan S; Smith, Donald A; Dakwar, Elias; Baaj, Ali A; Mundis, Gregory M; Turner, Alexander W L; Cornwall, G Bryan; Akbarnia, Behrooz A

2012-11-01

In the surgical treatment of spinal deformities, the importance of restoring lumbar lordosis is well recognized. Smith-Petersen osteotomies (SPOs) yield approximately 10° of lordosis per level, whereas pedicle subtraction osteotomies result in as much as 30° increased lumbar lordosis. Recently, selective release of the anterior longitudinal ligament (ALL) and placement of lordotic interbody grafts using the minimally invasive lateral retroperitoneal transpsoas approach (XLIF) has been performed as an attempt to increase lumbar lordosis while avoiding the morbidity of osteotomy. The objective of the present study was to measure the effect of the selective release of the ALL and varying degrees of lordotic implants placed using the XLIF approach on segmental lumbar lordosis in cadaveric specimens between L-1 and L-5. Nine adult fresh-frozen cadaveric specimens were placed in the lateral decubitus position. Lateral radiographs were obtained at baseline and after 4 interventions at each level as follows: 1) placement of a standard 10° lordotic cage, 2) ALL release and placement of a 10° lordotic cage, 3) ALL release and placement of a 20° lordotic cage, and 4) ALL release and placement of a 30° lordotic cage. All four cages were implanted sequentially at each interbody level between L-1 and L-5. Before and after each intervention, segmental lumbar lordosis was measured in all specimens at each interbody level between L-1 and L-5 using the Cobb method on lateral radiography. The mean baseline segmental lordotic angles at L1-2, L2-3, L3-4, and L4-5 were -3.8°, 3.8°, 7.8°, and 22.6°, respectively. The mean lumbar lordosis was 29.4°. Compared with baseline, the mean postimplantation increase in segmental lordosis in all levels combined was 0.9° in Intervention 1 (10° cage without ALL release); 4.1° in Intervention 2 (ALL release with 10° cage); 9.5° in Intervention 3 (ALL release with 20° cage); and 11.6° in Intervention 4 (ALL release with 30° cage). Foraminal height in the same sequence of conditions increased by 6.3%, 4.6%, 8.8% and 10.4%, respectively, while central disc height increased by 16.1%, 22.3%, 52.0% and 66.7%, respectively. Following ALL release and placement of lordotic cages at all 4 lumbar levels, the average global lumbar lordosis increase from preoperative lordosis was 3.2° using 10° cages, 12.0° using 20° cages, and 20.3° using 30° cages. Global lumbar lordosis with the cages at 4 levels exhibited a negative correlation with preoperative global lordosis (10°, R = -0.756; 20°, -0.730; and 30°, R = -0.437). Combined ALL release and placement of increasingly lordotic lateral interbody cages leads to progressive gains in segmental lordosis in the lumbar spine. Mean global lumbar lordosis similarly increased with increasingly lordotic cages, although the effect with a single cage could not be evaluated. Greater global lordosis was achieved with smaller preoperative lordosis. The mean maximum increase in segmental lordosis of 11.6° followed ALL release and placement of the 30° cage.

G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing.

PubMed

Verma, Shiv Prakash; Das, Parimal

2018-03-01

Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing. Copyright © 2017 Elsevier B.V. All rights reserved.

Ball to separator contact forces in angular contact ball bearings under thrust and radial loads

NASA Technical Reports Server (NTRS)

Nypan, L. J.

1977-01-01

Experimental data is reported on ball to cage contact forces in a 110 mm bore ball bearing operating at speeds to 12000 rpm under radial and thrust loads. Information is also reported on cage to inner race land contact force, cage to inner race land clearance, and cage to shaft speed ratios.

Cage drying and the application of a dry treatment (absorbent cornstarch powder) as a means to diminish Campylobacter on transport cages.

USDA-ARS?s Scientific Manuscript database

Broiler transport cages soiled with Campylobacter-positive feces have been shown to facilitate cross contamination of broilers. Campylobacter organisms are sensitive to dry stress. Allowing feces left on transport cage flooring to dry during extended periods of nonuse would be an effective method ...

Sensitivity of BN nano-cages to caffeine and nicotine molecules

NASA Astrophysics Data System (ADS)

Soltani, Alireza; Baei, Mohammad T.; Tazikeh Lemeski, E.; Shahini, Malihe

2014-12-01

Adsorption of caffeine and nicotine molecules over B12N12 and B16N16 nano-cages were investigated by using first-principles calculations to define whether BN nano-cages are applicable for filtering or sensing caffeine and nicotine molecules. The chemisorption energy of nicotine molecule on BN nano-cages is very stronger than caffeine molecule. Upon the adsorption of caffeine and nicotine molecules, the electronic properties of the BN nano-cages can be significantly changed, being too much sensitized on the caffeine and nicotine adsorptions.

Lowest-energy cage structures of medium-sized ( ZnO )n clusters with n = 15 - 24

NASA Astrophysics Data System (ADS)

Tang, Lingli; Sai, Linwei; Zhao, Jijun; Qiu, Ruifeng

2015-01-01

Fullerene-like cage structures of medium-sized ( ZnO )n clusters with n = 15 - 24 were generated by spiral algorithm and optimized using density functional theory calculations. Most of these lowest-energy cage structures contain only four-membered and six-membered rings, whereas eight-membered rings were found in the lowest-energy cages of ( ZnO )n (n = 19, 20, 23, 24). Our best cage configurations either reproduce or prevail the previously reported ones. The size-dependent electronic properties were also discussed.

Lowest-energy cage structures of medium-sized (ZnO){sub n} clusters with n = 15 − 24

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

Tang, Lingli; Sai, Linwei; Zhao, Jijun, E-mail: zhaojj@dlut.edu.cn

2015-01-22

Fullerene-like cage structures of medium-sized (ZnO){sub n} clusters with n = 15 − 24 were generated by spiral algorithm and optimized using density functional theory calculations. Most of these lowest-energy cage structures contain only four-membered and six-membered rings, whereas eight-membered rings were found in the lowest-energy cages of (ZnO){sub n} (n = 19, 20, 23, 24). Our best cage configurations either reproduce or prevail the previously reported ones. The size-dependent electronic properties were also discussed.

Two zeolite-type frameworks in one metal-organic framework with Zn24 @Zn104 cube-in-sodalite architecture.

PubMed

Bu, Fei; Lin, Qipu; Zhai, Quanguo; Wang, Le; Wu, Tao; Zheng, Shou-Tian; Bu, Xianhui; Feng, Pingyun

2012-08-20

Two in one: A metal-organic framework obtained from three different inorganic building blocks (tetrameric Zn(4) O, trimeric Zn(3) OH, and monomeric Zn) posseses a nested cage-in-cage and framework-in-framework architecture. 24 Zn(4) O tetramers and eight Zn monomers form a sodalite cage into which a cubic cage made from eight Zn(3) (OH) trimers is nestled. Eight monomeric Zn(2+) centers interconnect these two cages. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

'Snorkel' lice barrier technology reduced two co- occurring parasites, the salmon louse (Lepeophtheirus salmonis) and the amoebic gill disease causing agent (Neoparamoeba perurans), in commercial salmon sea-cages.

PubMed

Wright, D W; Stien, L H; Dempster, T; Vågseth, T; Nola, V; Fosseidengen, J-E; Oppedal, F

2017-05-01

Diverse chemical-free parasite controls are gaining status in Atlantic salmon sea-cage farming. Yet, the intricacies of their use at commercial scale, including effects on co-occurring parasites, are seldom reported. A new salmon lice prevention method involves installing a deep net roof and 'snorkel' lice barrier in cages to shelter salmon from free-living infective larvae which concentrate at shallow depths, and allows salmon to jump and re- inflate their buoyancy-regulating swim bladder by swallowing air. We document use of snorkel cages (10m deep barrier) in commercial farms, where their effects on salmon lice levels, amoebic gill disease (AGD)-related gill scores, the cage environment, fish welfare and farm management practices were compared to standard cages. During an autumn-winter study involving only snorkel cages, high AGD-related gill scores were observed to decline when freshwater was pumped into snorkels, creating a freshwater surface layer for salmon to enter for self-treatment. In a spring-summer study incorporating snorkel and standard cages, snorkel cages were found to reduce new lice infestations by 84%. The deployment of snorkels and intermittent oxygen depletion detected within them in the spring-summer study did not alter fish welfare parameters. Overall, the results suggest snorkel technology has a place in the toolkit of commercial salmon sea-cage farmers co-managing salmon lice and amoebic gill disease outbreaks - two principal parasite issues facing the industry. Copyright © 2017 Elsevier B.V. All rights reserved.

Finite element analysis of lordosis restoration with anterior longitudinal ligament release and lateral hyperlordotic cage placement.

PubMed

Uribe, Juan S; Harris, Jeffrey E; Beckman, J M; Turner, Alexander W L; Mundis, Gregory M; Akbarnia, Behrooz A

2015-04-01

Restoring sagittal alignment is an important factor in the treatment of spinal deformities. Recent investigations have determined that releasing the anterior longitudinal ligament (ALL) and placing hyperlordotic cages can increase lordosis, while minimizing need for 3 column osteotomies. The influences of parameters such as cage height and angle have not been determined. Finite element analysis was employed to assess the extent of lordosis achievable after placement of different sized lordotic cages. A 3-dimensional model of a L3-4 segment was used. Disc distraction was simulated by inserting interbody cages mid-body in the disc space. Analyses were performed in the following conditions: (1) intact, (2) ALL release, (3) ALL release + facetectomy, and (4) ALL release + posterior column osteotomy. Changes in segmental lordosis, disc height, foraminal height, and foraminal area were measured. After ALL resection and insertion of hyperlordotic cages, lordosis was increased in all cases. The lordosis achieved by the shorter cages was less due to posterior disc height maintained by the facet joints. A facetectomy increased segmental lordosis, but led to contact between the spinous processes. For some configurations, a posterior column osteotomy was required if the end goal was to match cage angle to intradiscal angle. Increased segmental lumbar lordosis is achievable with hyperlordotic cages after ALL resection. Increased cage height tended to increase the amount of lordosis achieved, although in some cases additional posterior bone resection was required to maximize lordosis. Further studies are needed to evaluate the impact on regional lumbar lordosis.

Caging, but not air deprivation, slows tadpole growth and development in the amphibian Xenopus laevis.

PubMed

Rose, Christopher S

2014-08-01

Xenopus laevis tadpoles raised in submerged cages in normoxic water develop more slowly than tadpoles raised with access to air. This study distinguishes between the effects of being caged and being deprived access to air on development and growth. Tadpoles were raised in high and low density control tanks and in cages in the same tank that were either completely submerged or with the top exposed to air. Experiments were repeated with the cages in different positions relative to the air stones and with and without the water flow from air stones supplemented with a pump. Whereas caging tadpoles has a large effect on their development and growth, additionally depriving them of air has a small effect and this effect can be removed by optimizing water flow through the cage. The effect of caging, though significant in this study, is small compared to the variation in growth and developmental rates that is commonly encountered within and among controls in lab studies. Caging effects can also be diminished by optimizing rearing conditions and/or having exceptionally vigorous tadpoles. The effects of air deprivation and caging thus pose less of a problem for experimenting on air-deprived (AD) and air-restored Xenopus tadpoles than their inherent variability in growth and developmental rates and their susceptibility to growth and developmental arrest. Further, the effect of air deprivation in this air-breathing amphibian does not pose a conflict with evolutionary hypotheses for lung loss involving lengthening of the larval period and delay in the onset of air breathing. © 2014 Wiley Periodicals, Inc.

Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments

PubMed Central

2017-01-01

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor–acceptor pairs during solution-based smFRET. We use this “caged FRET” methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules. PMID:28362086

A New Vertebral Body Replacement Strategy Using Expandable Polymeric Cages

PubMed Central

Liu, Xifeng; Paulsen, Alex; Giambini, Hugo; Guo, Ji; Miller, A. Lee; Lin, Po-Chun; Yaszemski, Michael J.

2017-01-01

We have developed a novel polymeric expandable cage that can be delivered via a posterior-only surgical approach for the treatment of noncontained vertebral defects. This approach is less invasive than an anterior-only or combined approach and much more cost-effective than currently used expandable metal cages. The polymeric expandable cage is composed of oligo poly(ethylene glycol) fumarate (OPF), a hydrogel that has been previously shown to have excellent nerve and bone tissue biocompatibility. OPF hydrogel cages can expand to twice their original diameter and length within a surgical time frame following hydration. Modulation of parameters such as polymeric network crosslink density or the introduction of charge to the network allowed for precise expansion kinetics. To meet specific requirements due to size variations in patient vertebral bodies, we fabricated a series of molds with varied diameters and explored the expansion kinetics of the OPF cages. Results showed a stable expansion ratio of approximately twofold to the original size within 20 min, regardless of the absolute value of the cage size. Following implantation of a dried OPF cage into a noncontained vertebral defect and its in situ expansion with normal saline, other augmentation biomaterials, such as poly(propylene fumarate) (PPF), can be injected to the lumen of the OPF cage and allowed to crosslink in situ. The OPF/PPF composite scaffold can provide the necessary rigidity and stability to the augmented spine. PMID:27835935

Effect of lipopolysaccharide on sickness behaviour in hens kept in cage and free range environments.

PubMed

Gregory, N G; Payne, S R; Devine, C D; Cook, C J

2009-08-01

The aim of this study was to assess whether environmental enrichment and environmental conditions can influence the expression of sickness behaviour. The behaviour in response to injection of lipopolysaccharide or saline was examined in a total of 96 62-weeks old hatchmate hens kept in a free range or cage environment. There were eight experimental treatments, each with 12 birds. Half the birds were sourced from a commercial cage layer unit (C/-) and half from a commercial free range unit (FR/-). After intraperitoneal injection with either lipopolysaccharide or saline (as a control), the hens were placed in either a cage (-/C) or free range (-/FR) environment. Lipopolysaccharide caused greater suppression of activity in free range (FR/FR) than in caged hens, including less walking (53% reduction), roosting (-86%) and preening (-60%) (p<0.05). Those responses were not observed in caged birds released into free range, nor in free range birds introduced to cages, suggesting that both the presence of and the familiarity with an environment affected sickness behaviour patterns. Increased sleeping was the most consistent response (+147%; p<0.001), and it was least influenced by environment. It was concluded that free range layer hens can express a greater range of sickness behaviours than caged hens, and this may make it more difficult to recognise disease expression in the caged environment.

78 FR 32374 - Magnuson-Stevens Act Provisions; General Provisions for Domestic Fisheries; Application for...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-30

...) 281-9135. FOR FURTHER INFORMATION CONTACT: Jason Berthiaume, Fishery Management Specialist, 978-281...-standard cages and 32-bu (1,703.68 L) cage tags. The traditional cages do not fit on smaller day-boats and... system, with each tag representing 32 bu (1,703.68 L) of allocation, or one cage. In addition to managing...

Eggshell bacterial levels of non-washed and washed eggs from caged and cage-free hens

USDA-ARS?s Scientific Manuscript database

The bacteria levels of non-washed and washed eggs obtained from caged and cage-free laying hens housed on either all shavings or all wire slat floors were determined. On eight sample days (from 22 to 52 weeks at 4 week intervals), 20 eggs were collected from each pen (n=120/sample day). Ten eggs p...

Ball to separator contact forces in angular contact ball bearings under thrust and radial loads

NASA Technical Reports Server (NTRS)

Nypan, L. J.

1978-01-01

Experimental data are reported on ball to cage contact forces in a 110 mm bore ball bearing operating at speeds to 12,000 rpm under radial and thrust loads. Information is also reported on cage to inner race land contact force, cage to inner race land clearance, and cage to shaft speed ratios.

Hen genetic strain and extended cold storage implications on physical egg quality from cage-free aviary housing system

USDA-ARS?s Scientific Manuscript database

In the US there is a sudden increase in the need for cage-free eggs in retail and food manufacturing sectors. As the US industry adapts existing and builds new cage-free housing structures, there is a need to understand the impact of cage-free systems and the corresponding management on egg quality...

Individually ventilated cages cause chronic low-grade hypoxia impacting mice hematologically and behaviorally

PubMed Central

York, Jason M.; McDaniel, Allison W.; Blevins, Neil A.; Guillet, Riley R.; Allison, Sarah O.; Cengel, Keith A.; Freund, Gregory G.

2012-01-01

Use of individually ventilated caging (IVC) systems for mouse-based laboratory investigation has dramatically increased. We found that without mice present, intra-cage oxygen concentration was comparable (21%) between IVC housing and ambient environment caging (AEC) that used wire top lids. However, when mice were housed 4-to-a-cage for 1 week, intra-cage oxygen dropped to 20.5% in IVC housing as compared to 21% for AEC housing. IVC intra-cage humidity was also elevated relative to AEC housing. Mice raised in IVC housing as compared to mice raised in AEC housing had higher RBC mass, hematocrit and hemoglobin concentrations. They also had elevated platelet counts but lower white blood cell counts. IVC mice relative to AEC mice had increased saccharin preference and increased fluid consumption but similar locomotion, food intake, social exploration and novel object recognition when tested in an AEC environment. Taken together, these data indicate that ventilated caging systems can have a 0.5% reduction from ambient oxygen concentration that is coupled to mouse red blood cell indices indicative of chronic exposure to a hypoxia. Importantly, IVC housing can impact behavioral testing for depressive-like behavior. PMID:22561683

CAGEd-oPOSSUM: motif enrichment analysis from CAGE-derived TSSs.

PubMed

Arenillas, David J; Forrest, Alistair R R; Kawaji, Hideya; Lassmann, Timo; Wasserman, Wyeth W; Mathelier, Anthony

2016-09-15

With the emergence of large-scale Cap Analysis of Gene Expression (CAGE) datasets from individual labs and the FANTOM consortium, one can now analyze the cis-regulatory regions associated with gene transcription at an unprecedented level of refinement. By coupling transcription factor binding site (TFBS) enrichment analysis with CAGE-derived genomic regions, CAGEd-oPOSSUM can identify TFs that act as key regulators of genes involved in specific mammalian cell and tissue types. The webtool allows for the analysis of CAGE-derived transcription start sites (TSSs) either provided by the user or selected from ∼1300 mammalian samples from the FANTOM5 project with pre-computed TFBS predicted with JASPAR TF binding profiles. The tool helps power insights into the regulation of genes through the study of the specific usage of TSSs within specific cell types and/or under specific conditions. The CAGEd-oPOSUM web tool is implemented in Perl, MySQL and Apache and is available at http://cagedop.cmmt.ubc.ca/CAGEd_oPOSSUM CONTACTS: anthony.mathelier@ncmm.uio.no or wyeth@cmmt.ubc.ca Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Automated home cage observations as a tool to measure the effects of wheel running on cage floor locomotion.

PubMed

de Visser, Leonie; van den Bos, Ruud; Spruijt, Berry M

2005-05-28

This paper introduces automated observations in a modular home cage system as a tool to measure the effects of wheel running on the time distribution and daily organization of cage floor locomotor activity in female C57BL/6 mice. Mice (n = 16) were placed in the home cage system for 6 consecutive days. Fifty percent of the subjects had free access to a running wheel that was integrated in the home cage. Overall activity levels in terms of duration of movement were increased by wheel running, while time spent inside a sheltering box was decreased. Wheel running affected the hourly pattern of movement during the animals' active period of the day. Mice without a running wheel, in contrast to mice with a running wheel, showed a clear differentiation between novelty-induced and baseline levels of locomotion as reflected by a decrease after the first day of introduction to the home cage. The results are discussed in the light of the use of running wheels as a tool to measure general activity and as an object for environmental enrichment. Furthermore, the possibilities of using automated home cage observations for e.g. behavioural phenotyping are discussed.

Polarization response of clathrate hydrates capsulated with guest molecules

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

Zeng, Qun; Li, Jinshan, E-mail: ljs915@263.net, E-mail: myang@scu.edu.cn; Huang, Hui

2016-05-28

Clathrate hydrates are characterized by their water cages encapsulating various guest atoms or molecules. The polarization effect of these guest-cage complexes was studied with combined density functional theory and finite-field calculations. An addition rule was noted for these systems whose total polarizability is approximately equal to the polarizability sum of the guest and the cage. However, their distributional polarizability computed with Hirshfeld partitioning scheme indicates that the guest–cage interaction has considerable influence on their polarization response. The polarization of encapsulated guest is reduced while the polarization of water cage is enhanced. The counteraction of these two opposite effects leads tomore » the almost unchanged total polarizability. Further analysis reveals that the reduced polarizability of encapsulated guest results from the shielding effect of water cage against the external field and the enhanced polarizability of water cage from the enhanced bonding of hydrogen bonds among water molecules. Although the charge transfer through the hydrogen bonds is rather small in the water cage, the polarization response of clathrate hydrates is sensitive to the changes of hydrogen bonding strength. The guest encapsulation strengthens the hydrogen bonding network and leads to enhanced polarizability.« less

CAGEd-oPOSSUM: motif enrichment analysis from CAGE-derived TSSs

PubMed Central

Arenillas, David J.; Forrest, Alistair R. R.; Kawaji, Hideya; Lassmann, Timo; Wasserman, Wyeth W.; Mathelier, Anthony

2016-01-01

With the emergence of large-scale Cap Analysis of Gene Expression (CAGE) datasets from individual labs and the FANTOM consortium, one can now analyze the cis-regulatory regions associated with gene transcription at an unprecedented level of refinement. By coupling transcription factor binding site (TFBS) enrichment analysis with CAGE-derived genomic regions, CAGEd-oPOSSUM can identify TFs that act as key regulators of genes involved in specific mammalian cell and tissue types. The webtool allows for the analysis of CAGE-derived transcription start sites (TSSs) either provided by the user or selected from ∼1300 mammalian samples from the FANTOM5 project with pre-computed TFBS predicted with JASPAR TF binding profiles. The tool helps power insights into the regulation of genes through the study of the specific usage of TSSs within specific cell types and/or under specific conditions. Availability and Implementation: The CAGEd-oPOSUM web tool is implemented in Perl, MySQL and Apache and is available at http://cagedop.cmmt.ubc.ca/CAGEd_oPOSSUM. Contacts: anthony.mathelier@ncmm.uio.no or wyeth@cmmt.ubc.ca Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27334471

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.

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

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

Homogenized boundary conditions and resonance effects in Faraday cages

PubMed Central

Hewitt, I. J.

2016-01-01

We present a mathematical study of two-dimensional electrostatic and electromagnetic shielding by a cage of conducting wires (the so-called ‘Faraday cage effect’). Taking the limit as the number of wires in the cage tends to infinity, we use the asymptotic method of multiple scales to derive continuum models for the shielding, involving homogenized boundary conditions on an effective cage boundary. We show how the resulting models depend on key cage parameters such as the size and shape of the wires, and, in the electromagnetic case, on the frequency and polarization of the incident field. In the electromagnetic case, there are resonance effects, whereby at frequencies close to the natural frequencies of the equivalent solid shell, the presence of the cage actually amplifies the incident field, rather than shielding it. By appropriately modifying the continuum model, we calculate the modified resonant frequencies, and their associated peak amplitudes. We discuss applications to radiation containment in microwave ovens and acoustic scattering by perforated shells. PMID:27279775

Homogenized boundary conditions and resonance effects in Faraday cages

NASA Astrophysics Data System (ADS)

Hewett, D. P.; Hewitt, I. J.

2016-05-01

We present a mathematical study of two-dimensional electrostatic and electromagnetic shielding by a cage of conducting wires (the so-called `Faraday cage effect'). Taking the limit as the number of wires in the cage tends to infinity, we use the asymptotic method of multiple scales to derive continuum models for the shielding, involving homogenized boundary conditions on an effective cage boundary. We show how the resulting models depend on key cage parameters such as the size and shape of the wires, and, in the electromagnetic case, on the frequency and polarization of the incident field. In the electromagnetic case, there are resonance effects, whereby at frequencies close to the natural frequencies of the equivalent solid shell, the presence of the cage actually amplifies the incident field, rather than shielding it. By appropriately modifying the continuum model, we calculate the modified resonant frequencies, and their associated peak amplitudes. We discuss applications to radiation containment in microwave ovens and acoustic scattering by perforated shells.

Homogenized boundary conditions and resonance effects in Faraday cages.

PubMed

Hewett, D P; Hewitt, I J

2016-05-01

We present a mathematical study of two-dimensional electrostatic and electromagnetic shielding by a cage of conducting wires (the so-called 'Faraday cage effect'). Taking the limit as the number of wires in the cage tends to infinity, we use the asymptotic method of multiple scales to derive continuum models for the shielding, involving homogenized boundary conditions on an effective cage boundary. We show how the resulting models depend on key cage parameters such as the size and shape of the wires, and, in the electromagnetic case, on the frequency and polarization of the incident field. In the electromagnetic case, there are resonance effects, whereby at frequencies close to the natural frequencies of the equivalent solid shell, the presence of the cage actually amplifies the incident field, rather than shielding it. By appropriately modifying the continuum model, we calculate the modified resonant frequencies, and their associated peak amplitudes. We discuss applications to radiation containment in microwave ovens and acoustic scattering by perforated shells.

Mitochondria mediate septin cage assembly to promote autophagy of Shigella.

PubMed

Sirianni, Andrea; Krokowski, Sina; Lobato-Márquez, Damián; Buranyi, Stephen; Pfanzelter, Julia; Galea, Dieter; Willis, Alexandra; Culley, Siân; Henriques, Ricardo; Larrouy-Maumus, Gerald; Hollinshead, Michael; Sancho-Shimizu, Vanessa; Way, Michael; Mostowy, Serge

2016-07-01

Septins, cytoskeletal proteins with well-characterised roles in cytokinesis, form cage-like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single-cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri-infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin-related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin-polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti-Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

PubMed

Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

2007-05-15

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

Photo-dependent protein biosynthesis using a caged aminoacyl-tRNA.

PubMed

Akahoshi, Akiya; Doi, Yoshio; Sisido, Masahiko; Watanabe, Kazunori; Ohtsuki, Takashi

2014-12-01

Translation systems with four-base codons provide a powerful strategy for protein engineering and protein studies because they enable site-specific incorporation of non-natural amino acids into proteins. In this study, a caged aminoacyl-tRNA with a four-base anticodon was synthesized. The caged aminoacyl-tRNA contains a photocleavable nitroveratryloxycarbonyl (NVOC) group. This study showed that the caged aminoacyl-tRNA was not deacylated, did not bind to EF-Tu, and was activated by light. Photo-dependent translation of an mRNA containing the four-base codon was demonstrated using the caged aminoacyl-tRNA.

A new germfree chicken cage--characteristics and use.

PubMed

MILLER, H T; LUCKEY, T D

1962-01-01

A new germfree chicken cage for rearing chicks up to 3 or 4 weeks of age has been designed and is in use at the University of Missouri. The cage and the accessory parts, the small magnet and hinged door, the "Bactytector," the built-in air filter assembly, and the glass top with connecting air outlet filter have been described in detail. The complete operating procedure for sterilizing the cage and diet and the method of adding sterile embryonated eggs have been outlined. Data on the effectiveness of the cage as a physical barrier to microbes have been presented.

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.

Establishment of a medium-scale mosquito facility: tests on mass production cages for Aedes albopictus (Diptera: Culicidae).

PubMed

Zhang, Dongjing; Li, Yongjun; Sun, Qiang; Zheng, Xiaoying; Gilles, Jeremie R L; Yamada, Hanano; Wu, Zhongdao; Xi, Zhiyong; Wu, Yu

2018-03-19

Mass egg production is an important component of Aedes albopictus mosquito control programs, such as the sterile insect technique and incompatible insect technique, which requires the releases of large number of sterile males. Developing standard operating procedures and optimized cages for adult maintenance of Ae. albopictus can improve the mass rearing efficiency. Three different sex ratios of females to males with a total number of 4,000 mosquitoes were tested by evaluating the insemination rate, egg production (total number of eggs per cage), female fecundity and egg hatch rate in small cage (30 × 30 × 30 cm). Blood meals with adenosine triphosphate (ATP, 0.05 g/ml), cage structures (Big cage A: 90 × 30 × 30 cm; Big cage B: 90 × 30 × 50 cm or 90 × 50 × 30 cm) and rearing densities (12,000, 16,000 and 20,000 mosquitoes, corresponding to 0.9 cm 2 /mosquito, 0.675 cm 2 /mosquito and 0.54 cm 2 /mosquito, respectively) were also tested and evaluated on the basis of egg production, female fecundity and egg hatch rate. An adult rearing unit holding 15 of Big cage A with optimal egg production was designed to produce 10 million eggs per rearing cycle in a 1.8 m 2 space. Female to male ratios at 3:1 in small cages resulted in higher egg production but did not affect insemination rate, female fecundity and egg hatch rate. A concentration of 0.05 g/ml of ATP added to blood meals improved the blood-feeding frequency and thus increased the overall egg production per cage. Cage structures affected the egg production per cage, but not egg hatch rate. A medium rearing density at 0.675 cm 2 /mosquito (16,000 mosquitoes) resulted in higher egg production compared to both low and high densities. An adult rearing unit for Ae. albopictus on the basis of Big cage A has been developed with the capacity of producing 10 million eggs within 15 days. Our results have indicated that the adult rearing methods and adult maintenance unit are recommended for Ae. albopictus mass rearing in support of the establishment of a medium-sized mosquito factory.

Interbody fusion cage design using integrated global layout and local microstructure topology optimization.

PubMed

Lin, Chia-Ying; Hsiao, Chun-Ching; Chen, Po-Quan; Hollister, Scott J

2004-08-15

An approach combining global layout and local microstructure topology optimization was used to create a new interbody fusion cage design that concurrently enhanced stability, biofactor delivery, and mechanical tissue stimulation for improved arthrodesis. To develop a new interbody fusion cage design by topology optimization with porous internal architecture. To compare the performance of this new design to conventional threaded cage designs regarding early stability and long-term stress shielding effects on ingrown bone. Conventional interbody cage designs mainly fall into categories of cylindrical or rectangular shell shapes. The designs contribute to rigid stability and maintain disc height for successful arthrodesis but may also suffer mechanically mediated failures of dislocation or subsidence, as well as the possibility of bone resorption. The new optimization approach created a cage having designed microstructure that achieved desired mechanical performance while providing interconnected channels for biofactor delivery. The topology optimization algorithm determines the material layout under desirable volume fraction (50%) and displacement constraints favorable to bone formation. A local microstructural topology optimization method was used to generate periodic microstructures for porous isotropic materials. Final topology was generated by the integration of the two-scaled structures according to segmented regions and the corresponding material density. Image-base finite element analysis was used to compare the mechanical performance of the topology-optimized cage and conventional threaded cage. The final design can be fabricated by a variety of Solid Free-Form systems directly from the image output. The new design exhibited a narrower, more uniform displacement range than the threaded cage design and lower stress at the cage-vertebra interface, suggesting a reduced risk of subsidence. Strain energy density analysis also indicated that a higher portion of total strain energy density was transferred into the new bone region inside the new designed cage, indicating a reduced risk of stress shielding. The new design approach using integrated topology optimization demonstrated comparable or better stability by limited displacement and reduced localized deformation related to the risk of subsidence. Less shielding of newly formed bone was predicted inside the new designed cage. Using the present approach, it is also possible to tailor cage design for specific materials, either titanium or polymer, that can attain the desired balance between stability, reduced stress shielding, and porosity for biofactor delivery.

Room environment influence on eggshell bacterial levels of non-washed and washed eggs from caged and cage-free laying hens

USDA-ARS?s Scientific Manuscript database

The bacteria levels of non-washed and washed eggs obtained from caged and cage-free hens housed in either wire slats or shaving-covered pens were determined. On eight days (from 22 to 52 wk), 20 eggs were collected from each pen. Ten eggs/pen were washed with a commercial egg washing solution, whi...

Study of rolling element dynamic interactions with separators and raceway paths: Roller to separator contact forces and cage to shaft speed ratios in roller bearings

NASA Technical Reports Server (NTRS)

Nypan, L. J.

1978-01-01

Cage to roller force measurements, cage to shaft forces, and cage to shaft speed ratios are reported for 115 and 118mm bore roller bearings operating at speeds of 4,000, 8,000, and 12,000 rpm under loads ranging from 360 to 6670 N (80 to 1500 lb).

Furnished Cage System and Hen Well-Being: Comparative Effects of Furnished and Battery Cages on Egg Production and Physiological Parameters of White Leghorn Hens

USDA-ARS?s Scientific Manuscript database

Laboratory animal well-being can be improved by housing the animals in a species-special “natural” or “near to natural” environment. This study was to examine if housing environment, furnished cages vs. battery cages, causes a similar impact on well-being in laying hens. One hundred seventy-two, on...

Anterior cervical corpectomy: review and comparison of results using titanium mesh cages and carbon fibre reinforced polymer cages.

PubMed

Kabir, Syed M R; Alabi, J; Rezajooi, Kia; Casey, Adrian T H

2010-10-01

Different types of cages have recently become available for reconstruction following anterior cervical corpectomy. We review the results using titanium mesh cages (TMC) and stackable CFRP (carbon fibre reinforced polymer) cages. Forty-two patients who underwent anterior cervical corpectomy between November 2001 and September 2008 were retrospectively reviewed. Pathologies included cervical spondylotic myelopathy (CSM), cervical radiculopathy, OPLL (ossified posterior longitudinal ligament), metastasis/primary bone tumour, rheumatoid arthritis and deformity correction. All patients were evaluated clinically and radiologically. Outcome was assessed on the basis of the Odom's criteria, neck disability index (NDI) and myelopathy disability index (MDI). Mean age was 60 years and mean follow-up was 1½ years. Majority of the patients had single-level corpectomy. Twenty-three patients had TMC cages while 19 patients had CFRP cages. The mean subsidence noted with TMC cage was 1.91 mm, while with the stackable CFRP cage it was 0.5 mm. This difference was statistically significant (p < 0.05). However, there was no statistically significant correlation noted between subsidence and clinical outcome (p > 0.05) or between subsidence and post-operative sagittal alignment (p > 0.05) in either of the groups. Three patients had significant subsidence (> 3 mm), one of whom was symptomatic. There were no hardware-related complications. On the basis of the Odom's criterion, 9 patients (21.4%) had an excellent outcome, 14 patients (33.3%) had a good outcome, 9 patients (21.4%) had a fair outcome and 5 patients (11.9%) had a poor outcome, i.e. symptoms and signs unchanged or exacerbated. Mean post-operative NDI was 26.27% and mean post-operative MDI was 19.31%. Fusion was noted in all 42 cases. Both TMC and stackable CFRP cages provide solid anterior column reconstruction with good outcome following anterior cervical corpectomy. However, more subsidence is noted with TMC cages though this might not significantly alter the clinical outcome unless the subsidence is significant (>3 mm).

PMMA versus titanium cage after anterior cervical discectomy - a prospective randomized trial.

PubMed

Schröder, J; Grosse-Dresselhaus, F; Schul, C; Wassmann, H

2007-02-01

Nonautologous interbody fusion materials are utilised in increasing numbers after anterior cervical disc surgery to overcome the problem of donor site morbidity of autologous bone grafts. This study investigates the performance of two nonautologous materials, the bone cement Polymethylmethacrylate (PMMA) and titanium cages. This prospective randomised trial, with assessment of the results by an independent observer, evaluates whether a Polymethylmethacrylate (PMMA) spacer or a titanium cage provides a better fusion rate around the implant and a better clinical outcome. Between 2000 and 2002, 115 patients with monoradicular cervical nerve root compression syndrome caused by soft cervical disc herniation were eligible for this study. Myelopathy, excessive osteophyte formation, and adjacent level degeneration were exclusion criteria. A block-restricted randomisation was applied. The 2-year clinical outcome served as the primary endpoint of the study. Clinical outcome was assessed according to the Odom scale by an independent observer at the follow-up examination. Preoperative, postoperative, and follow-up radiographs were taken. The study was completed by 107 patients (53 with PMMA and 54 with titanium cage). No significant difference between the two groups could be established with respect to the clinical outcome. In each group, 26 patients scored excellent. Good results were found in 19 PMMA patients and 16 titanium cage patients; satisfactory results were found in 8 PMMA patients and 9 titanium cage patients; bad results were found in 3 titanium cage patients. In 47 titanium cage cases (87%), fusion occurred radiologically as bony bridging around the implant. The fusion rate was significantly lower (p=0.011) in the PMMA group, with 35 cases (66%) united at follow-up. The radiological result of the titanium cage is superior to that of PMMA with respect to the fusion rate. Although the titanium cage achieves a better fusion rate, there is no difference between titanium cages and PMMA with respect to the clinical outcome.

Reinventing the wheel: comparison of two wheel cage styles for assessing mouse voluntary running activity.

PubMed

Seward, T; Harfmann, B D; Esser, K A; Schroder, E A

2018-04-01

Voluntary wheel cage assessment of mouse activity is commonly employed in exercise and behavioral research. Currently, no standardization for wheel cages exists resulting in an inability to compare results among data from different laboratories. The purpose of this study was to determine whether the distance run or average speed data differ depending on the use of two commonly used commercially available wheel cage systems. Two different wheel cages with structurally similar but functionally different wheels (electromechanical switch vs. magnetic switch) were compared side-by-side to measure wheel running data differences. Other variables, including enrichment and cage location, were also tested to assess potential impacts on the running wheel data. We found that cages with the electromechanical switch had greater inherent wheel resistance and consistently led to greater running distance per day and higher average running speed. Mice rapidly, within 1-2 days, adapted their running behavior to the type of experimental switch used, suggesting these running differences are more behavioral than due to intrinsic musculoskeletal, cardiovascular, or metabolic limits. The presence of enrichment or location of the cage had no detectable impact on voluntary wheel running. These results demonstrate that mice run differing amounts depending on the type of cage and switch mechanism used and thus investigators need to report wheel cage type/wheel resistance and use caution when interpreting distance/speed run across studies. NEW & NOTEWORTHY The results of this study highlight that mice will run different distances per day and average speed based on the inherent resistance present in the switch mechanism used to record data. Rapid changes in running behavior for the same mouse in the different cages demonstrate that a strong behavioral factor contributes to classic exercise outcomes in mice. Caution needs to be taken when interpreting mouse voluntary wheel running activity to include potential behavioral input and physiological parameters.

Persistence of fecal shedding of Salmonella Enteritidis by experimentally infected laying hens housed in conventional or enriched cages.

PubMed

Gast, Richard K; Guraya, Rupa; Jones, Deana R; Anderson, Kenneth E

2015-07-01

Salmonella Enteritidis can be deposited inside eggs laid by infected hens, so the prevalence of this pathogen in commercial egg-producing flocks is an important risk factor for human illness. Opportunities for the introduction, transmission, and persistence of salmonellae in poultry are potentially influenced by flock housing and management systems. Animal welfare concerns have spurred the development of alternatives to traditional cage-based housing. However, the consequences of poultry housing systems for food safety have not been fully resolved by prior research. The present study assessed the effects of two different housing systems (conventional cages and colony cages enriched with perching and nesting areas) on the persistence of fecal shedding of Salmonella Enteritidis by groups of experimentally infected laying hens. In each of two trials, 136 hens were distributed among cages of both housing systems and orally inoculated with doses of 10(8) cfu of Salmonella Enteritidis (phage type 13a in one trial and phage type 4 in the other). At weekly intervals, samples of voided feces were collected from beneath each cage and cultured to detect Salmonella Enteritidis. Fecal shedding of Salmonella Enteritidis was detected for up to 8 wk post-inoculation by hens housed in enriched colony cages and 10 wk by hens housed in conventional cages. For both trials combined, the frequency of positive fecal cultures was significantly (P < 0.05) greater for conventional cages than for enriched colony cages at 1 wk (84.7 vs. 71.5%), 2 wk (54.2 vs. 31.3%), 3 wk (21.5 vs. 7.6%), and 4 wk (9.7 vs. 2.8%) post-inoculation. These results demonstrate that the susceptibility of hens to intestinal colonization by Salmonella Enteritidis can differ between conventional and enriched cage-based production systems, although this effect does not necessarily translate into a corresponding difference in the longer-term persistence of fecal shedding. © 2015 Poultry Science Association Inc.

Group selection for adaptation to multiple-hen cages: humoral immune response.

PubMed

Hester, P Y; Muir, W M; Craig, J V

1996-11-01

A selected line of White Leghorns, which has shown improved survivability and reduced feather loss in large multiple-hen cages, was evaluated for humoral immune response to SRBC under both stressed and unstressed conditions. Three lines of chickens (selected, control, and commercial) were housed in either single- (1 hen) or multiple-hen cages (12 hens, social competition) and subjected to a cold ambient temperature (0 C) at 33 wk of age and to two heating episodes (38 C) at 44 wk of age. Each hen was challenged intravenously with 1 mL of a 7% saline suspension of SRBC at the time that cold exposure was initiated. Hens subjected to high ambient temperatures had been exposed previously to a cold temperature, but were not challenged with SRBC until 16 to 18 h following the end of the second heating episode. Exposure to cold caused immunosuppression in single-caged hens, but not in hens in colony cages. Single- vs colony-caged hens of the control environment challenged with SRBC at 33 wk of age had similar primary hemagglutinin responses to SRBC. Hens subjected to heat experienced immunosuppression at 9 and 12 d following challenge to SRBC when compared to the controls. Hens of multiple-bird cages challenged with antigen at 44 wk of age had a significantly lower hemagglutinin response to SRBC than those reared in single-bird cages. The three lines of genetic stock had similar primary hemagglutinin responses to SRBC; the interactions of genetic stock with cage size or environmental temperature were not significant. It was concluded that genetically selecting hens for survival in multiple-hen cages did not affect their humoral immune response to SRBC.

The Vertical Dynamics of Larval Chironomids on Artificial Substrates in Lake Lido (Bogor, Indonesia)

PubMed Central

Wardiatno, Yusli; Krisanti, Majariana

2013-01-01

The composition and abundance of chironomid larval communities was studied on artificial substrates in Lido Lake, located in Bogor, West Java, Indonesia. The lake is organically enriched as a result of fish farming activity. Seventy two artificial substrates were deployed at three depths (2.0, 3.5 and 5.0 m) at two sites: a cage culture site and a non-cage culture site (control). Larval chironomid larvae were collected 7, 14, 28 and 56 days after the artificial substrates were deployed. In addition, selected physical and chemical parameters of the water were simultaneously measured. Three chironomid subfamilies, the Chironominae, Tanypodinae and Orthocladiinae, were found at both sites. At the cage culture site, both diversity and total abundance were significantly higher at the 2.0 and 3.5 m depths than at the 5.0 m depth, but this was not the case at the non-cage culture site. Based on pooling of the data from all depths, a Mann-Whitney U test showed that the non-cage culture site had a significantly higher diversity and total abundance than the cage culture site. Dissolved oxygen (DO) and turbidity showed significant differences between the 2.0 m depth and the 2 greater depths at the cage culture site, whereas none of the environmental parameters showed significant differences among the three depths at the non-cage culture site. A comparison of the environmental parameters at the same depth at the two sites showed significant differences in turbidity, pH and DO. A Spearman rank correlation analysis at the cage culture site showed that abundance and DO were positively correlated, whereas abundance and turbidity were negatively correlated. However, only pH was negatively correlated with abundance at the non-cage culture site. PMID:24575246

Dynamic free energy surfaces for sodium diffusion in type II silicon clathrates.

PubMed

Slingsby, J G; Rorrer, N A; Krishna, L; Toberer, E S; Koh, C A; Maupin, C M

2016-02-21

Earth abundant semiconducting type II Si clathrates have attracted attention as photovoltaic materials due to their wide band gaps. To realize the semiconducting properties of these materials, guest species that arise during the synthesis process must be completely evacuated from the host cage structure post synthesis. A common guest species utilized in the synthesis of Si clathrates is Na (metal), which templates the clathrate cage formation. Previous experimental investigations have identified that it is possible to evacuate Na from type II clathrates to an occupancy of less than 1 Na per unit cell. This work investigates the energetics, kinetics, and resulting mechanism of Na diffusion through type II Si clathrates by means of biased molecular dynamics and kinetic Monte Carlo simulations. Well-tempered metadynamics has been used to determine the potential of mean force for Na moving between clathrate cages, from which the thermodynamic preferences and transition barrier heights have been obtained. Kinetic Monte Carlo simulations based on the metadynamics results have identified the mechanism of Na diffusion in type II Si clathrates. The overall mechanism consists of a coupled diffusive process linked via electrostatic guest-guest interactions. The large occupied hexakaidechedral cages initially empty their Na guests to adjacent empty large cages, thereby changing the local electrostatic environment around the occupied small pentagonal dodecahedral cages and increasing the probability of Na guests to leave the small cages. This coupled process continues through the cross-over point that is identified as the point where large and small cages are equally occupied by Na guests. Further Na removal results in the majority of guests residing in the large cages as opposed to the small cages, in agreement with experiments, and ultimately a Na free structure.

Stand-alone anchored cage versus cage with plating for single-level anterior cervical discectomy and fusion: a prospective, randomized, controlled study with a 2-year follow-up.

PubMed

Nemoto, Osamu; Kitada, Akira; Naitou, Satoko; Tachibana, Atsuko; Ito, Yuya; Fujikawa, Akira

2015-07-01

To avoid complications associated with plating in anterior cervical discectomy and fusion (ACDF), stand-alone anchored PEEK cage was developed and favourable outcomes with a low rate of dysphasia have been described. The objective of this study was to compare the clinical and radiological outcomes of ACDF using a standalone anchored PEEK cage (PREVAIL; Medtronic Sofamor Danek, Memphis, TN) with those of a PEEK cage with plating in a prospective randomized manner. Fifty patients with single-level cervical radiculopathy were randomly assigned to a PREVAIL or a PEEK cage with plating. Following 3, 6, 12, and 24 months, clinical and radiological outcomes were assessed. The mean surgical time for the patients with a PREVAIL was significantly shorter than that for those with a PEEK cage with plating. The clinical outcomes evaluated by visual analogue scale for pain and the Odom's criteria were comparable between both the groups. Both the groups demonstrated the high fusion rate (92% in PREVAIL; 96% in PEEK cage with plating). The subsidence rate and the improvement of cervical alignment were comparable between both the groups. The incidence of adjacent-level ossification was significantly lower for patients with a PREVAIL than that for those with a PEEK cage with plating. The rate of dysphasia graded by the method of Bazaz and measurement of prevertebral soft tissue swelling indicated no significant differences between both the groups. Our prospective randomized study confirmed that stand-alone anchored PEEK cage is a valid alternative to plating in ACDF with a low rate of adjacent-level ossification. However, the potential to reduce the incidence of dysphasia was not confirmed.

Transition-Metal-Catalyzed Selective Cage B-H Functionalization of o-Carboranes.

PubMed

Quan, Yangjian; Qiu, Zaozao; Xie, Zuowei

2018-02-26

Carboranes are a class of carbon-boron molecular clusters with unusual thermal and chemical stabilities. They have been proved as very useful building blocks in supramolecular design, optoelectronics, nanomaterials, boron neutron capture therapy agents and organometallic/coordination chemistry. Thus, the functionalization of o-carboranes has received growing interests. Over the past decades, most of the works in this area have been focused on cage carbon functionalization as the weakly acidic cage C-H proton can be readily deprotonated by strong bases. In sharp contrast, selective cage B-H activation/functionalization among chemically very similar ten B-H vertices is very challenging. Considering the differences in electron density of ten cage B-H bonds in o-carborane and the nature of transition metal complexes, we have tackled this selectivity issue by means of organometallic chemistry. Our strategy is as follows: using electron-rich transition metal catalysts for the functionalization of the most electron-deficient B(3,6)-H vertices (bonded to both cage CH vertices); using electron-deficient transition-metal catalysts for the functionalization of relatively electron-rich B(8,9,10,12)-H vertices (with no bonding to both cage CH vertices); and using the combination of directing groups and electrophilic transition metal catalysts for the functionalization of B(4,5,7,11)-H vertices (bonded to only one cage CH vertex). Successful applications of such a strategy result in the preparation of a large variety of cage B-functionalized carboranes in a regioselective and catalytic manner, which are inaccessible by other means. It is believed that as this field progresses, other cage B-functionalized carboranes are expected to be synthesized, and the results detailed in this concept article will further these efforts. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Crystal Structure of a Maxi/Mini-Ferritin Chimera Reveals Guiding Principles for the Assembly of Protein Cages

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

Cornell, Thomas A.; Srivastava, Yogesh; Jauch, Ralf

Cage proteins assemble into nanoscale structures with large central cavities. They play roles, including those as virus capsids and chaperones, and have been applied to drug delivery and nanomaterials. Furthermore, protein cages have been used as model systems to understand and design protein quaternary structure. Ferritins are ubiquitous protein cages that manage iron homeostasis and oxidative damage. Two ferritin subfamilies have strongly similar tertiary structure yet distinct quaternary structure: maxi-ferritins normally assemble into 24-meric, octahedral cages with C-terminal E-helices centered around 4-fold symmetry axes, and mini-ferritins are 12-meric, tetrahedral cages with 3-fold axes defined by C-termini lacking E-domains. To understandmore » the role E-domains play in ferritin quaternary structure, we previously designed a chimera of a maxi-ferritin E-domain fused to the C-terminus of a mini-ferritin. The chimera is a 12-mer cage midway in size between those of the maxi- and mini-ferritin. The research described herein sets out to understand (a) whether the increase in size over a typical mini-ferritin is due to a frozen state where the E-domain is flipped out of the cage and (b) whether the symmetrical preference of the E-domain in the maxi-ferritin (4-fold axis) overrules the C-terminal preference in the mini-ferritin (3-fold axis). With a 1.99 Å resolution crystal structure, we determined that the chimera assembles into a tetrahedral cage that can be nearly superimposed with the parent mini-ferritin, and that the E-domains are flipped external to the cage at the 3-fold symmetry axes.« less

A Geometric Principle May Guide Self-Assembly of Fullerene Cages from Clathrin Triskelia and from Carbon Atoms☆

PubMed Central

Schein, Stan; Sands-Kidner, Michelle

2008-01-01

Abstract Clathrin triskelia and carbon atoms alike self-assemble into a limited selection of fullerene cages (with n three connected vertices, 3n/2 edges, 12 pentagonal faces, and (n−20)/2 hexagonal faces). We show that a geometric constraint—exclusion of head-to-tail dihedral angle discrepancies (DADs)—explains this limited selection as well as successful assembly into such closed cages in the first place. An edge running from a pentagon to a hexagon has a DAD, since the dihedral angles about the edge broaden from its pentagon (tail) end to its hexagon (head) end. Of the 21 configurations of a central face and surrounding faces, six have such DAD vectors arranged head-to-tail. Of the 5770 mathematically possible fullerene cages for n ≤ 60, excluding those with any of the six configurations leaves just 15 cages plus buckminsterfullerene (n = 60), among them the known clathrin cages. Of the 216,739 mathematically possible cages for 60 < n ≤ 84, just the 50 that obey the isolated-pentagon rule, among them known carbon cages, pass. The absence of likely fullerenes for some n (30,34,46,48,52–58,62–68) explains the abundance of certain cages, including buckminsterfullerene. These principles also suggest a “probable roads” path to self-assembly in place of pentagon-road and fullerene-road hypotheses. PMID:17921209

Single transverse-orientation cage via MIS-TLIF approach for the treatment of degenerative lumbar disease: a technical note.

PubMed

Wang, Shan-Jin; Han, Ying-Chao; Pan, Fu-Min; Ma, Bin; Tan, Jun

2015-01-01

Single transverse cage placed in the anterior vertebral column can better maintain lumbar lordosis and sagittal alignment and is frequently used via the lateral transpsoas approach. However, there is no clear description in the literature of the steps required to place the single transverse cage during the instrumented transforaminal lumbar interbody fusion (TLIF) procedure for the treatment of degenerative lumbar disease. The objective of this study is to describe the technique using single transverse-orientation cage when performing TLIF procedures. We present 18 illustrative cases in which single transverse-orientation cage was placed according to a step-by-step technique that can be used during the TLIF procedure. Information acquired included procedure time, intraoperative blood loss and postoperative complications. The preoperative and postoperative Oswestry Disability Index (ODI) and the visual analogue scale (VAS) scores were recorded. Changes in disc height and segmental lordosis were measured at radiographs. The single transverse-orientation cage was successfully placed in 18 patients in a stepwise technique to achieve lumbar fusion. Using this technique, the patients significantly improved clinically and radiographically at postoperative visits. This is the first report demonstrating the safety and efficacy of instrumented TLIF with single transverse-orientation cage for the treatment of degenerative lumbar disease. Single transverse-orientation cage via MIS-TLIF approach can maintain greater lumbar lordosis and avoid the unique complications of lateral transpsoas approach. Understanding the options for cage placement is important for surgeons considering the use of this technique.

Solid-State Gas Adsorption Studies with Discrete Palladium(II) [Pd2 (L)4 ]4+ Cages.

PubMed

Preston, Dan; White, Keith F; Lewis, James E M; Vasdev, Roan A S; Abrahams, Brendan F; Crowley, James D

2017-08-04

The need for effective CO 2 capture systems remains high, and due to their tunability, metallosupramolecular architectures are an attractive option for gas sorption. While the use of extended metal organic frameworks for gas adsorption has been extensively explored, the exploitation of discrete metallocage architectures to bind gases remains in its infancy. Herein the solid state gas adsorption properties of a series of [Pd 2 (L) 4 ] 4+ lantern shaped coordination cages (L = variants of 2,6-bis(pyridin-3-ylethynyl)pyridine), which had solvent accessible internal cavities suitable for gas binding, have been investigated. The cages showed little interaction with dinitrogen gas but were able to take up CO 2 . The best performing cage reversibly sorbed 1.4 mol CO 2 per mol cage at 298 K, and 2.3 mol CO 2 per mol cage at 258 K (1 bar). The enthalpy of binding was calculated to be 25-35 kJ mol -1 , across the number of equivalents bound, while DFT calculations on the CO 2 binding in the cage gave ΔE for the cage-CO 2 interaction of 23-28 kJ mol -1 , across the same range. DFT modelling suggested that the binding mode is a hydrogen bond between the carbonyl oxygen of CO 2 and the internally directed hydrogen atoms of the cage. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Plastic cages to protect Douglas-fir seedlings from animal damage in western Oregon.

Treesearch

Glen C. Crouch

1980-01-01

Effects of plastic mesh cages designed to protect Douglas-fir seedlings from animals were evaluated in western Oregon. In two tests over 5-year periods, caging increased survival by 0 and 13 percent and increased height growth by 0.8 and 1.2 feet compared with uncaged trees. Benefits from caging might have been greater if damage had been more prevalent during the tests...

Final Report for DUSEL R&D: BetaCage: A Screener of Ultra-Low-Level Radioactive Surface Contamination

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

Golwala, Sunil R.

2013-12-20

The eventual full-size, radiopure BetaCage will be a low-background, atmospheric-pressure neon drift chamber with unprecedented sensitivity to emitters of low-energy electrons and alpha particles. We expect that the prototype BetaCage already developed will be an excellent screener of alpha particles. Both the prototype and final BetaCage will provide new infrastructure for rare-event science.

Extended cage adjustable speed electric motors and drive packages

DOEpatents

Hsu, John S.

1999-01-01

The rotor cage of a motor is extended, a second stator is coupled to this extended rotor cage, and the windings have the same number of poles. The motor torque and speed can be controlled by either injecting energy into or extracting energy out from the rotor cage. The motor produces less harmonics than existing doubly-fed motors. Consequently, a new type of low cost, high efficiency drive is produced.

Acute neurological symptoms during hypobaric exposure: consider cerebral air embolism.

PubMed

Weenink, Robert P; Hollmann, Markus W; van Hulst, Robert A

2012-11-01

Cerebral arterial gas embolism (CAGE) is well known as a complication of invasive medical procedures and as a risk in diving and submarine escape. In the underwater environment, CAGE is caused by trapped air, which expands and leads to lung vessel rupture when ambient pressure decreases during ascent. Pressure decrease also occurs during hypobaric activities such as flying and, therefore, CAGE may theoretically be a risk in hypobaric exposure. We reviewed the available literature on this subject. Identified were 12 cases of CAGE due to hypobaric exposure. Based on these cases, we discuss pathophysiology, diagnosis, and treatment of CAGE due to hypobaric exposure. The low and slow pressure decrease during most hypobaric activities (as opposed to diving) account for the low incidence of CAGE during these exposures and suggest that severe air trapping must be present to cause barotrauma. This is also suggested by the large prevalence of air filled cysts in the case reports reviewed. We recommend considering CAGE in all patients presenting with acute central neurological injury during or shortly after pressure decrease such as flying. A CT scan of head and chest should be performed in these patients. Treatment with hyperbaric oxygen therapy should be initiated as soon as possible in cases of proven or probable CAGE.

Validation of an automatic system (DoubleCage) for detecting the location of animals during preference tests.

PubMed

Tsai, P P; Nagelschmidt, N; Kirchner, J; Stelzer, H D; Hackbarth, H

2012-01-01

Preference tests have often been performed for collecting information about animals' acceptance of environmental refinement objects. In numerous published studies animals were individually tested during preference experiments, as it is difficult to observe group-housed animals with an automatic system. Thus, videotaping is still the most favoured method for observing preferences of socially-housed animals. To reduce the observation workload and to be able to carry out preference testing of socially-housed animals, an automatic recording system (DoubleCage) was developed for determining the location of group-housed animals in a preference test set-up. This system is able to distinguish the transition of individual animals between two cages and to record up to 16 animals at the same time (four animals per cage). The present study evaluated the reliability of the DoubleCage system. The data recorded by the DoubleCage program and the data obtained by human observation were compared. The measurements of the DoubleCage system and manual observation of the videotapes are comparable and significantly correlated (P < 0.0001) with good agreement. Using the DoubleCage system enables precise and reliable recording of the preferences of group-housed animals and a considerable reduction of animal observation time.

Zero-profile anchored cage reduces risk of postoperative dysphagia compared with cage with plate fixation after anterior cervical discectomy and fusion.

PubMed

Xiao, ShanWen; Liang, ZhuDe; Wei, Wu; Ning, JinPei

2017-04-01

To compare the rate of postoperative dysphagia between zero-profile anchored cage fixation (ZPC group) and cage with plate fixation (CP group) after anterior cervical discectomy and fusion (ACDF). A meta-analysis of cohort studies between zero-profile anchored cage and conventional cage with plate fixation after ACDF for the treatment of cervical diseases from 2008 to May 2016. An extensive search of studies was performed in PubMed, Medline, Embase, Cochrane library and Google Scholar. Dysphagia rate was extracted. Data analysis was conducted with RevMan 5.2. Sixteen trials involving 1066 patients were included in this meta-analysis. The results suggested that the ZPC group were associated with lower incidences of dysphagia than the CP group at postoperative immediately, 2 weeks, 2, 3, 6 and 12 months. In subgroup analysis, although significant differences were only found in the mild dysphagia at 3 and 6 months postoperatively and in the moderate dysphagia at 2 weeks after surgery; the ZPC group had a lower rate of postoperative dysphagia than the CCP group in short, medium and long term follow-up periods. Zero-profile anchored cage had a lower risk of postoperative dysphagia than cage with plate.

Effect of curcumin caged silver nanoparticle on collagen stabilization for biomedical applications.

PubMed

Srivatsan, Kunnavakkam Vinjimur; Duraipandy, N; Begum, Shajitha; Lakra, Rachita; Ramamurthy, Usha; Korrapati, Purna Sai; Kiran, Manikantan Syamala

2015-04-01

The current study aims at understanding the influence of curcumin caged silver nanoparticle (CCSNP) on stability of collagen. The results indicated that curcumin caged silver nanoparticles efficiently stabilize collagen, indicated by enhanced tensile strength, fibril formation and viscosity. The tensile strength of curcumin caged silver nanoparticle cross-linked collagen and elongation at break was also found to be higher than glutaraldehyde cross-linked collagen. The physicochemical characteristics of curcumin caged nanoparticle cross-linked collagen exhibited enhanced strength. The thermal properties were also good with both thermal degradation temperature and hydrothermal stability higher than native collagen. CD analysis showed no structural disparity in spite of superior physicochemical properties suggesting the significance of curcumin caged nanoparticle mediated cross-linking. The additional enhancement in the stabilization of collagen could be attributed to multiple sites for interaction with collagen molecule provided by curcumin caged silver nanoparticles. The results of cell proliferation and anti-microbial activity assays indicated that curcumin caged silver nanoparticles promoted cell proliferation and inhibited microbial growth making it an excellent biomaterial for wound dressing application. The study opens scope for nano-biotechnological strategies for the development of alternate non-toxic cross-linking agents facilitating multiple site interaction thereby improving therapeutic values to the collagen for biomedical application. Copyright © 2015 Elsevier B.V. All rights reserved.

High pressure oxygen turbopump bearing cage stability analyses. [space shuttle main engine

NASA Technical Reports Server (NTRS)

Merriman, T. L.; Kannel, J. W.

1984-01-01

The low service life of the high pressure oxygen turbopump (HPOTP) bearings used in the space shuttle main engine was examined by use of the Battelle "BASDAP' bearing computer stability model. The dynamic instability of the bearing cage resulted in excessive wear and eventual failure of the unit. By maintaining a cage/race clearance of no more than 0.25 millimeters (0.010 inches), ball/pocket clearance of no less than 0.54 millimeters (0.025 inches), dynamic balancing of the cages, and maintaining adequate lubricant films between the balls and races, cage instability and subsequent bearing degradation can be reduced.

Omni-directional and holonomic rolling platform with decoupled rotational and translational degrees of freedom

DOEpatents

Pin, F.G.; Killough, S.M.

1994-12-20

A wheel assembly includes a support, a cage rotatably mounted on the support and having a longitudinal rotation axis, a first ball wheel rotatably mounted in the cage and having a rotation axis orthogonal to the rotation axis of the cage, and a second ball wheel rotatably mounted in the cage and having a rotation axis orthogonal to the rotation axis or the cage and to the rotation axis of the first ball wheel. A control circuit includes a photodetector signal which indicates ground contact for each ball wheel, and a tachometer which indicates actual drive shaft velocity. 6 figures.

Omni-directional and holonomic rolling platform with decoupled rotational and translational degrees of freedom

DOEpatents

Pin, Francois G.; Killough, Stephen M.

1994-01-01

A wheel assembly includes a support, a cage rotatably mounted on the support and having a longitudinal rotation axis, a first ball wheel rotatably mounted in the cage and having a rotation axis orthogonal to the rotation axis of the cage, and a second ball wheel rotatably mounted in the cage and having a rotation axis orthogonal to the rotation axis or the cage and to the rotation axis of the first ball wheel. A control circuit includes a photodetector signal which indicates ground contact for each ball wheel, and a tachometer which indicates actual drive shaft velocity.

Octa- and hexametallic iron(III)-potassium phosphonate cages.

PubMed

Gopal, Kandasamy; Tuna, Floriana; Winpenny, Richard E P

2011-12-07

Two new iron(III)-potassium phosphonate cage complexes with {K(2)Fe(6)} and {K(2)Fe(4)} cores are reported. Magnetic studies reveal antiferromagnetic interactions between the Fe(III) centres occur in these cages.

Provision Of Carbon Nanotube Bucky Paper Cages For Immune Shielding Of Cells, Tissues, and Medical Devices

NASA Technical Reports Server (NTRS)

Loftus, David J. (Inventor)

2006-01-01

System and method for enclosing cells and/or tissue, for purposes of growth, cell differentiation, suppression of cell differentiation, biological processing and/or transplantation of cells and tissues (biological inserts), and for secretion, sensing and monitoring of selected chemical substances and activation of gene expression of biological inserts implanted into a human body. Selected cells and/or tissue are enveloped in a "cage" that is primarily carbon nanotube Bucky paper, with a selected thickness and porosity. Optionally, selected functional groups, proteins and/or peptides are attached to the carbon nanotube cage, or included within the cage, to enhance the growth and/or differentiation of the cells and/or tissue, to select for certain cellular sub-populations, to optimize certain functions of the cells and/or tissue and/or to optimize the passage of chemicals across the cage surface(s). A cage system is also used as an immuns shield and to control operation of a nano-device or macroscopic device, located within the cage, to provide or transform a selected chemical and/or a selected signal.

Thermodynamics and kinetics of gas storage in porous liquids

DOE PAGES

Zhang, Fei; Yang, Fengchang; Huang, Jingsong; ...

2016-07-05

The recent synthesis of organic molecular liquids with permanent porosity (Giri et al., Nature, 2015, 527, 216) opens up exciting new avenues for gas capture, storage, and separation. Using molecular dynamics simulations, we study the thermodynamics and kinetics for the storage of CH 4, CO 2, and N 2 molecules in porous liquids consisting of crown-ether substituted cage molecules in a 15-crown-5 solvent. It is found that the gas storage capacity per cage molecule follows the order of CH 4 > CO 2 > N 2, which does not correlate simply with the size of gas molecules. Different gas moleculesmore » are stored inside the cage differently, e.g., CO 2 molecules prefer the cage s core while CH 4 molecules favor both the core and the branch regions. All gas molecules considered can enter the cage essentially without energy barriers, and their dynamics inside the cage are only slightly hindered by the nanoscale confinement. In addition, all gas molecules can leave the cage on nanosecond time scale by overcoming a modest energy penalty. The molecular mechanisms of these observations are clarified.« less

Thermodynamics and kinetics of gas storage in porous liquids

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

Zhang, Fei; Yang, Fengchang; Huang, Jingsong

The recent synthesis of organic molecular liquids with permanent porosity (Giri et al., Nature, 2015, 527, 216) opens up exciting new avenues for gas capture, storage, and separation. Using molecular dynamics simulations, we study the thermodynamics and kinetics for the storage of CH 4, CO 2, and N 2 molecules in porous liquids consisting of crown-ether substituted cage molecules in a 15-crown-5 solvent. It is found that the gas storage capacity per cage molecule follows the order of CH 4 > CO 2 > N 2, which does not correlate simply with the size of gas molecules. Different gas moleculesmore » are stored inside the cage differently, e.g., CO 2 molecules prefer the cage s core while CH 4 molecules favor both the core and the branch regions. All gas molecules considered can enter the cage essentially without energy barriers, and their dynamics inside the cage are only slightly hindered by the nanoscale confinement. In addition, all gas molecules can leave the cage on nanosecond time scale by overcoming a modest energy penalty. The molecular mechanisms of these observations are clarified.« less

Consumption of particulate wastes derived from cage fish farming by aggregated wild fish. An experimental approach.

PubMed

Ballester-Moltó, M; Sanchez-Jerez, P; Aguado-Giménez, F

2017-09-01

Particulate wastes derived from cage fish farming are a trophic resource used by wild fish. This study assesses waste consumption by wild fish and the impact on the final balance of wastes. Consumption was determined according to the difference between the particulate matter exiting the cages and that reaching 5 m away at three different depths, in the presence and absence of wild fish. Wild fish around the experimental cages were counted during feeding and non-feeding periods. A weighted abundance of 1057 fish 1000 m -3 consumed 17.75% of the particulate wastes exiting the cages, on average. Consumption was higher below the cages, where waste outflow was greater. However, waste removal by wild fish was noteworthy along the shallow and deep sides of the cages. Wild fish diminished the net particulate wastes by about 14%, transforming them into more easily dispersible and less harmful wastes. This study demonstrates the mitigating potential of wild fish in reducing environmental impact. Copyright © 2017 Elsevier Ltd. All rights reserved.

Can an Endplate-conformed Cervical Cage Provide a Better Biomechanical Environment than a Typical Non-conformed Cage?: A Finite Element Model and Cadaver Study.

PubMed

Zhang, Fan; Xu, Hao-Cheng; Yin, Bo; Xia, Xin-Lei; Ma, Xiao-Sheng; Wang, Hong-Li; Yin, Jun; Shao, Ming-Hao; Lyu, Fei-Zhou; Jiang, Jian-Yuan

2016-08-01

To evaluate the biomechanical characteristics of endplate-conformed cervical cages by finite element method (FEM) analysis and cadaver study. Twelve specimens (C2 -C7 ) and a finite element model (C3 -C7 ) were subjected to biomechanical evaluations. In the cadaver study, specimens were randomly assigned to intact (I), endplate-conformed (C) and non-conformed (N) groups with C4-5 discs as the treated segments. The morphologies of the endplate-conformed cages were individualized according to CT images of group C and the cages fabricated with a 3-D printer. The non-conformed cages were wedge-shaped and similar to commercially available grafts. Axial pre-compression loads of 73.6 N and moment of 1.8 Nm were used to simulate flexion (FLE), extension (EXT), lateral bending (LB) and axial rotation (AR). Range of motion (ROM) at C4-5 of each specimen was recorded and film sensors fixed between the cages and C5 superior endplates were used to detect interface stress. A finite element model was built based on the CT data of a healthy male volunteer. The morphologies of the endplate-conformed and wedge-shaped, non-conformed cervical cages were both simulated by a reverse engineering technique and implanted at the segment of C4-5 in the finite element model for biomechanical evaluation. Force loading and grouping were similar to those applied in the cadaver study. ROM of C4-5 in group I were recorded to validate the finite element model. Additionally, maximum cage-endplate interface stresses, stress distribution contours on adjoining endplates, intra-disc stresses and facet loadings at adjacent segments were measured and compared between groups. In the cadaver study, Group C showed a much lower interface stress in all directions of motion (all P < 0.05) and the ROM of C4-5 was smaller in FLE-EXT (P = 0.001) but larger in AR (P = 0.017). FEM analysis produced similar results: the model implanted with an endplate-conformed cage presented a lower interface stress with a more uniform stress distribution than that implanted with a non-conformed cage. Additionally, intra-disc stress and facet loading at the adjacent segments were obviously increased in both groups C and N, especially those at the supra-jacent segments. However, stress increase was milder in group C than in group N for all directions of motion. Endplate-conformed cages can decrease cage-endplate interface stress in all directions of motion and increase cervical stability in FLE-EXT. Additionally, adjacent segments are possibly protected because intra-disc stress and facet loading are smaller after endplate-conformed cage implantation. However, axial stability was reduced in group C, indicating that endplate-conformed cage should not be used alone and an anterior plate system is still important in anterior cervical discectomy and fusion. © 2016 Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.

Extended cage adjustable speed electric motors and drive packages

DOEpatents

Hsu, J.S.

1999-03-23

The rotor cage of a motor is extended, a second stator is coupled to this extended rotor cage, and the windings have the same number of poles. The motor torque and speed can be controlled by either injecting energy into or extracting energy out from the rotor cage. The motor produces less harmonics than existing doubly-fed motors. Consequently, a new type of low cost, high efficiency drive is produced. 12 figs.

Use of Li.sub.2[B.sub.12H.sub.12] salt to absorb water into polymers

DOEpatents

Eastwood, Eric A.; Bowen, III, Daniel E.

2016-08-30

Methods of adjusting the properties of a composition are provided. The compositions comprise a polymer-containing matrix and a filler comprising a hygroscopic salt. Preferred such salts comprise a cage compound selected from the group consisting of borane cage compounds, carborane cage compounds, metal complexes thereof, residues thereof, mixtures thereof, and/or agglomerations thereof, where the cage compound is not covalently bound to the matrix polymer.

Smallest fullerene-like silicon cage stabilized by a V(2) unit.

PubMed

Xu, Hong-Guang; Kong, Xiang-Yu; Deng, Xiao-Jiao; Zhang, Zeng-Guang; Zheng, Wei-Jun

2014-01-14

We conducted a combined anion photoelectron spectroscopy and density functional theory study on V2Si20 cluster. Our results show that the V2Si20 cluster has an elongated dodecahedron cage structure with a V2 unit encapsulated inside the cage. It is the smallest fullerene-like silicon cage and can be used as building block to make cluster-assembled materials, such as pearl-chain style nanowires.

Smallest fullerene-like silicon cage stabilized by a V2 unit

NASA Astrophysics Data System (ADS)

Xu, Hong-Guang; Kong, Xiang-Yu; Deng, Xiao-Jiao; Zhang, Zeng-Guang; Zheng, Wei-Jun

2014-01-01

We conducted a combined anion photoelectron spectroscopy and density functional theory study on V2Si20 cluster. Our results show that the V2Si20 cluster has an elongated dodecahedron cage structure with a V2 unit encapsulated inside the cage. It is the smallest fullerene-like silicon cage and can be used as building block to make cluster-assembled materials, such as pearl-chain style nanowires.

In silico design of supramolecules from their precursors: odd-even effects in cage-forming reactions.

PubMed

Jelfs, Kim E; Eden, Edward G B; Culshaw, Jamie L; Shakespeare, Stephen; Pyzer-Knapp, Edward O; Thompson, Hugh P G; Bacsa, John; Day, Graeme M; Adams, Dave J; Cooper, Andrew I

2013-06-26

We synthesize a series of imine cage molecules where increasing the chain length of the alkanediamine precursor results in an odd-even alternation between [2 + 3] and [4 + 6] cage macrocycles. A computational procedure is developed to predict the thermodynamically preferred product and the lowest energy conformer, hence rationalizing the observed alternation and the 3D cage structures, based on knowledge of the precursors alone.

In silico Design of Supramolecules from Their Precursors: Odd–Even Effects in Cage-Forming Reactions

PubMed Central

2013-01-01

We synthesize a series of imine cage molecules where increasing the chain length of the alkanediamine precursor results in an odd–even alternation between [2 + 3] and [4 + 6] cage macrocycles. A computational procedure is developed to predict the thermodynamically preferred product and the lowest energy conformer, hence rationalizing the observed alternation and the 3D cage structures, based on knowledge of the precursors alone. PMID:23745577

Electronic structure, stability and magnetic properties of small M1-2Cr (M = Fe, Co, and Ni) alloy encapsulated inside a (BN)48 cage

NASA Astrophysics Data System (ADS)

Liang, Wenjuan; Jia, Jianfeng; Lv, Jin; Wu, Haishun

2015-09-01

The geometrical structure and magnetic properties of M1-2Cr (M = Fe, Co, and Ni) alloy clusters inside a (BN)48 cage were calculated at the BPW91/LANL2DZ level of theory. The doping with Cr significantly changed the magnetic properties of the transition-metal clusters. When M1-2Cr alloys were placed inside a (BN)48 cage, the alloy clusters interacted strongly with the cage, and the M1-2Cr@(BN)48 clusters showed high stability. Moreover, Cr-doped magnetic metal clusters preferably occupied positions off-center and near the hexagonal rings of (BN)48 cages. Thus, the (BN)48 cages can be used to increase the stability of M1-2Cr alloys, and retain their magnetic nature, except for CoCr and Ni2Cr clusters.

Optimization of mosquito egg production under mass rearing setting: effects of cage volume, blood meal source and adult population density for the malaria vector, Anopheles arabiensis.

PubMed

Mamai, Wadaka; Bimbile-Somda, Nanwintoum S; Maiga, Hamidou; Juarez, José Guillermo; Muosa, Zaynab A I; Ali, Adel Barakat; Lees, Rosemary Susan; Gilles, Jeremie R L

2017-01-24

Anopheles arabiensis is one of the major malaria vectors that put millions of people in endemic countries at risk. Mass-rearing of this mosquito is crucial for strategies that use sterile insect technique to suppress vector populations. The sterile insect technique (SIT) package for this mosquito species is being developed by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. To improve mass-rearing outcomes for An. arabiensis, the question of whether the egg production by females would be affected by the size of the adult holding cages, the source of the blood meal and the total number of pupae that could be loaded into the cages was addressed and finally the impact of adding additional pupae to the cage daily to maintain adult numbers on egg productivity assessed. Mass production cages of two different volumes, two different sources of blood meal (bovine and porcine) and two different population densities (cages originally loaded with either 15,000 or 20,000 pupae) were tested and evaluated on the basis of eggs produced/cage or per female. Males and females pupae with a ratio of 1:1 were added to the cages at day 1 and 2 of pupation. The emerging adults had constant access to 5% sugar solution and blood fed via the Hemotek membrane feeding system. Eggs were collected either twice a week or daily. A generalized linear model was used to identify factors which gave significantly higher egg production. Neither cage volume nor blood meal source affected egg production per cage or per female. However, increasing population density to 20,000 pupae had a negative effect on eggs produced per cage and per female. Although high density negatively impacted egg production, adding 1000 daily additional pupae compensating for daily mortality resulted in a substantial increase in egg production. Moreover, in all tests the first and the third egg batches collected were significantly higher than others eggs batches. With the equipment and protocols described here and routinely used at the Insect Pest Control Laboratory (IPCL), it was possible to produce up to 120,000 eggs/cage/day. These results demonstrated that 15,000 is the optimal number of pupae to be loaded into the Anopheles Mass production cages. Under this condition, an average of 40 eggs per female was obtained for five gonotrophic cycles. However, an improvement in egg production can be achieved by daily addition, to the original 15,000 pupae, of one thousand pupae a day. Interestingly, feeding females with bovine or porcine blood using both large and small versions of the mass production cage did not affect egg productivity.

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.

Comparison of behaviour, performance and mortality in restricted and ad libitum-fed growing rabbits.

PubMed

Dalmau, A; Abdel-Khalek, A M; Ramon, J; Piles, M; Sanchez, J P; Velarde, A; Rafel, O

2015-07-01

The objective of this study was to determine whether rabbits fed in a restricted regimen (75%) showed increased competition for feeding, drinking and use of specific areas of the cages as compared with those provided feed ad libitum. This evaluation was carried out by measuring their space utilisation in the cage, the incidence of agonistic behaviour and rates of mortality. In total, 504 rabbits between 31 and 66 days of age were used in this study. A total of 200 heavy-weight rabbits and 56 light-weight rabbits were randomly housed in 32 cages, each cage containing eight rabbits: 25 cages housing heavy rabbits and seven cages housing the light-weight ones. They were all fed ad libitum (AD). In addition, a total of 208 heavy-weight rabbits and 40 light-weight rabbits were randomly housed in 31 cages, each of them containing eight rabbits: 26 cages housing heavy weight rabbits and five cages housing light-weight ones. They were all fed a restricted diet (R) regimen. The restriction was calculated to be 75% of the feed consumed by the AD group. The total space available in the cage was 3252 cm(2), with a stocking density of 24.6 animals/m(2). Animals between 32 and 60 days of age from 20 different cages were observed nine times per week (morning or afternoon) by means of scan and focal sampling by one observer. During each period, cages were assessed for 5 min, registering every minute the position of all the animals in relation to Area A (feeder), Area B (central part) or Area C (back and drinker area). The incidence of agonistic behaviour such as displacement, biting and jumping on each other was also assessed. Performance variables such as daily gain and feed conversion ratio, in addition to general health status and mortality rates, were recorded for all rabbits. When the rabbits were under restricted feeding, the competition for feed and drink increased with clear signs of agonistic behaviour such as biting, displacement and animals jumping on top of each other. Although this competition was maintained during the entire growing period, the BW homogeneity between animals in the same cage was similar in both cases, suggesting that all animals could consume similar quantities of feed. The possible advantages of a restricted diet, such as better feed conversion ratio, were observed in this study only in the last few weeks of the growing period.

Frequency and Duration of Fecal Shedding of Salmonella Enteritidis by Experimentally Infected Laying Hens Housed in Enriched Colony Cages at Different Stocking Densities

PubMed Central

Gast, Richard K.; Guraya, Rupa; Jones, Deana R.; Anderson, Kenneth E.; Karcher, Darrin M.

2017-01-01

Human infections with Salmonella Enteritidis are often attributed to the consumption of contaminated eggs, so the prevalence of this pathogen in egg-laying poultry is an important public health risk factor. Numerous and complex environmental influences on Salmonella persistence and transmission are exerted by management practices and housing facilities used in commercial egg production. In recent years, the animal welfare implications of poultry housing systems have guided the development of alternatives to traditional cage-based housing, but their food safety consequences are not yet fully understood. The present study assessed the effects of different bird stocking densities on the frequency and duration of fecal shedding of S. Enteritidis in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In two trials, groups of laying hens were distributed at two stocking densities (648 and 973 cm2/bird) into enriched colony cages and (along with a group housed in conventional cages at 648 cm2/bird) orally inoculated with doses of 1.0 × 108 cfu of S. Enteritidis. At 10 weekly postinoculation intervals, samples of voided feces were collected from beneath each cage and cultured to detect S. Enteritidis. Fecal shedding of S. Enteritidis was detected for up to 10 weeks postinoculation by hens in all three housing treatment groups. The overall frequency of positive fecal cultures was significantly (P < 0.05) greater from conventional cages than from enriched colony cages (at the lower stocking density) for the total of all sampling dates (45.0 vs. 33.3%) and also for samples collected at 4–9 weeks postinfection. Likewise, the frequency of S. Enteritidis isolation from feces from conventional cages was significantly greater than from enriched colony cages (at the higher hen stocking density) for the sum of all samples (45.0 vs. 36.7%) and at 6 weeks postinoculation. Moreover, the frequency of S. Enteritidis fecal recovery from enriched colony cages at the higher hen stocking was significantly greater than from similar cages at the lower stocking density for all 10 sampling dates combined (39.4 vs. 33.3%). These results suggest that stocking density can affect S. Enteritidis intestinal colonization and fecal shedding in laying hens, but some other difference between conventional and enriched colony cage systems appears to exert an additional influence. PMID:28443289

Mating competitiveness of male Anopheles arabiensis mosquitoes irradiated with a partially or fully sterilizing dose in small and large laboratory cages.

PubMed

Helinski, M E H; Knols, B G J

2008-07-01

Male mating competitiveness is a crucial parameter in many genetic control programs including the sterile insect technique (SIT). We evaluated competitiveness of male Anopheles arabiensis Patton as a function of three experimental variables: (1) small or large cages for mating, (2) the effects of either a partially sterilizing (70 Gy) or fully sterilizing (120 Gy) dose, and (3) pupal or adult irradiation. Irradiated males competed for females with an equal number of unirradiated males. Competitiveness was determined by measuring hatch rates of individually laid egg batches. In small cages, pupal irradiation with the high dose resulted in the lowest competitiveness, whereas adult irradiation with the low dose gave the highest, with the latter males being equal in competitiveness to unirradiated males. In the large cage, reduced competitiveness of males irradiated in the pupal stage was more pronounced compared with the small cage; the males irradiated as adults at both doses performed similarly to unirradiated males. Unexpectedly, males irradiated with the high dose performed better in a large cage than in a small one. A high proportion of intermediate hatch rates was observed for eggs collected in the large cage experiments with males irradiated at the pupal stage. It is concluded that irradiation of adult An. arabiensis with the partially sterilizing dose results in the highest competitiveness for both cage designs. Cage size affected competitiveness for some treatments; therefore, competitiveness determined in laboratory experiments must be confirmed by releases into simulated field conditions. The protocols described are readily transferable to evaluate male competitiveness for other genetic control techniques.

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.

Does impaction of titanium-coated interbody fusion cages into the disc space cause wear debris or delamination?

PubMed

Kienle, Annette; Graf, Nicolas; Wilke, Hans-Joachim

2016-02-01

A large number of interbody fusion cages are made of polyetheretherketone (PEEK). To improve bone on-growth, some are coated with a thin layer of titanium. This coating may fail when subjected to shear loading. The purpose of this testing was to investigate whether impaction of titanium-coated PEEK cages into the disc space can result in wear or delamination of the coating, and whether titanium cages with subtractive surface etching (no coating) are less susceptible to such failure. A biomechanical study was carried out to simulate the impaction process in clinical practice and to evaluate if wear or delamination may result from impaction. Two groups of posterior lumbar interbody fusion cages with a similar geometry were tested: n=6 titanium-coated PEEK and n=6 surface-etched titanium cages. The cages were impacted into the space in between two vertebral body substitutes (polyurethane foam blocks). The two vertebral body substitutes were fixed in a device, through which a standardized axial preload of 390 N was applied. The anterior tip of the cage was positioned at the posterior border of the space between the two vertebral body substitutes. The cages were then inserted using a drop weight with a mass representative of a surgical hammer. The drop weight impacted the insertion instrument at a maximum speed of about 2.6 m/s, which is in the range of the impaction speed in vivo. This was repeated until the cages were fully inserted. The wear particles were captured and analyzed according to the pertinent standards. The surface-etched titanium cages did not show any signs of wear debris or surface damage. In contrast, the titanium-coated PEEK cages resulted in detached wear particles of different sizes (1-191 µm). Over 50% of these particles had a size <10 µm. In median, on 26% of the implants' teeth, the coating was abraded. Full delamination was not observed. In contrast to the surface-etched implants, the titanium-coated PEEK implants lost some coating material. This was visible to the naked eye. More than half of all particles were of a size range that allows phagocytosis. This study shows that titanium-coated implants are susceptible to impaction-related wear debris. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Ncm, a Photolabile Group for Preparation of Caged Molecules: Synthesis and Biological Application

PubMed Central

Muralidharan, Sukumaran; Dirda, Nathaniel D. A.; Katz, Elizabeth J.; Tang, Cha-Min; Bandyopadhyay, Sharba; Kanold, Patrick O.

2016-01-01

Ncm, 6-nitrocoumarin-7-ylmethyl, is a photolabile protective group useful for making “caged” molecules. Ncm marries the reliable photochemistry of 2-nitrobenzyl systems with the excellent stability and spectroscopic properties of the coumarin chromophore. From simple, commercially available starting materials, preparation of Ncm and its caged derivatives is both quick and easy. Photorelease of Ncm-caged molecules occurs on the microsecond time scale, with quantum efficiencies of 0.05–0.08. We report the synthesis and physical properties of Ncm and its caged derivatives. The utility of Ncm-caged glutamate for neuronal photostimulation is demonstrated in cultured hippocampal neurons and in brain slice preparations. PMID:27695074

A Cobalt Supramolecular Triple-Stranded Helicate-based Discrete Molecular Cage

PubMed Central

Mai, Hien Duy; Kang, Philjae; Kim, Jin Kyung; Yoo, Hyojong

2017-01-01

We report a strategy to achieve a discrete cage molecule featuring a high level of structural hierarchy through a multiple-assembly process. A cobalt (Co) supramolecular triple-stranded helicate (Co-TSH)-based discrete molecular cage (1) is successfully synthesized and fully characterized. The solid-state structure of 1 shows that it is composed of six triple-stranded helicates interconnected by four linking cobalt species. This is an unusual example of a highly symmetric cage architecture resulting from the coordination-driven assembly of metallosupramolecular modules. The molecular cage 1 shows much higher CO2 uptake properties and selectivity compared with the separate supramolecular modules (Co-TSH, complex 2) and other molecular platforms. PMID:28262690

Comparing polychlorinated biphenyl concentrations and patterns in the Saginaw River using sediment, caged fish, and semipermeable membrane devices

USGS Publications Warehouse

Echols, K.R.; Gale, R.W.; Schwartz, T.R.; Huckins, J.N.; Williams, L.L.; Meadows, J.C.; Morse, D.; Petty, J.D.; Orazio, C.E.; Tillitt, D.E.

2000-01-01

Three techniques of assessing bioavailable polychlorinated biphenyls (PCBs) in the Saginaw River, MI, were compared: sediments, caged fish, and semipermeable membrane devices (SPMDs). SPMDs and caged fish were placed in the river for 28 days at five sites where sediments were also sampled. The samples were analyzed for PCB congeners to determine concentrations and patterns. Total PCB concentrations ranged from 33 to 280 ng/g (dry weight) in sediments, 46 to 290 ng/g (wet weight) in caged fish, and 77 to 790 ng/g in SPMDs. Previously reported rates of PCB accumulation by SPMDs were used to estimate aqueous concentrations from the PCB concentrations detected in the SPMDs. Sediment-water partition coefficients were used to estimate aqueous PCB concentrations from sediment. Steady-state bioconcentration factors and depuration rate constants were used to estimate dissolved PCB concentrations from caged channel catfish. Relative PCB patterns from the SPMDs, caged fish, and sediment were compared using principal components analysis. SPMD and sediment samples provide complementary information. Sediments reflect long-term accumulation and weathering, while SPMDs integrate water concentrations only during the sampling period. Because of higher water solubilities of lower-chlorinated PCBs these predominate in the SPMDs as compared to in the fish and sediments. Contaminant profile differences between caged fish and SPMDs are likely due to metabolism and depuration of certain PCB congeners by fish.Three techniques of assessing bioavailable polychlorinated biphenyls (PCBs) in the Saginaw River, Ml, were compared: sediments, caged fish, and semipermeable membrane devices (SPMDs). SPMDs and caged fish were placed in the river for 28 days at five sites where sediments were also sampled. The samples were analyzed for PCB congeners to determine concentrations and patterns. Total PCB concentrations ranged from 33 to 280 ng/g (dry weight) in sediments, 46 to 290 ng/g (wet weight) in caged fish, and 77 to 790 ng/g in SPMDs. Previously reported rates of PCB accumulation by SPMDs were used to estimate aqueous concentrations from the PCB concentrations detected in the SPMDs. Sediment-water partition coefficients were used to estimate aqueous PCB concentrations from sediment. Steady-state bioconcentration factors and depuration rate constants were used to estimate dissolved PCB concentrations from caged channel catfish. Relative PCB patterns from the SPMDs, caged fish, and sediment were compared using principal components analysis. SPMD and sediment samples provide complementary information. Sediments reflect long-term accumulation and weathering, while SPMDs integrate water concentrations only during the sampling period. Because of higher water solubilities of lower-chlorinated PCBs these predominate in the SPMDs as compared to in the fish and sediments. Contaminant profile differences between caged fish and SPMDs are likely due to metabolism and depuration of certain PCB congeners by fish.At five sites in the Saginaw River, MI, PCB concentrations were determined in the summer of 1993 using three methods: sediment analysis, concentrations in caged fish, and concentrations in semipermeable membrane devices (SPMDs). On average, total PCB concentrations in the SPMDs were twice those found in caged fish, and the SPMD-to-fish concentration ratios of di-, tri-, tetra-, and pentaCB homologues were 10.0, 3.0, 2.5, and 1.4, respectively. Average concentrations in the sediments were approximately half those in the SPMDs, and the caged fish showed a greater preponderance of higher log octanol-water partition coefficient PCBs similar to the sediment pattern. On average, the water PCB concentrations estimated from sediment concentrations were five times higher than those calculated from SPMDs and three times higher than those estimated from caged fish. The total PCB concentrations in sediment, caged fish, and SPMDs ranged 33-280, 46-290, and 77-790 ng/g, respectiv

Smallest fullerene-like silicon cage stabilized by a V{sub 2} unit

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

Xu, Hong-Guang, E-mail: xuhong@iccas.ac.cn, E-mail: zhengwj@iccas.ac.cn; Kong, Xiang-Yu; Deng, Xiao-Jiao

We conducted a combined anion photoelectron spectroscopy and density functional theory study on V{sub 2}Si{sub 20} cluster. Our results show that the V{sub 2}Si{sub 20} cluster has an elongated dodecahedron cage structure with a V{sub 2} unit encapsulated inside the cage. It is the smallest fullerene-like silicon cage and can be used as building block to make cluster-assembled materials, such as pearl-chain style nanowires.

Computational design and fabrication of a novel bioresorbable cage for tibial tuberosity advancement application.

PubMed

Castilho, Miguel; Rodrigues, Jorge; Vorndran, Elke; Gbureck, Uwe; Quental, Carlos; Folgado, João; Fernandes, Paulo R

2017-01-01

Tibial tuberosity advancement (TTA) is a promising method for the treatment of cruciate ligament rupture in dogs that usually implies the implantation of a titanium cage as bone implant. This cage is non-biodegradable and fails in providing adequate implant-bone tissue integration. The objective of this work is to propose a new process chain for designing and manufacturing an alternative biodegradable cage that can fulfill specific patient requirements. A three-dimensional finite element model (3D FEM) of the TTA system was first created to evaluate the mechanical environment at cage domain during different stages of the dog walk. The cage microstructure was then optimized using a topology optimization tool, which addresses the accessed local mechanical requirements, and at same time ensures the maximum permeability to allow nutrient and oxygen supply to the implant core. The designed cage was then biofabricated by a 3D powder printing of tricalcium phosphate cement. This work demonstrates that the combination of a 3D FEM with a topology optimization approach enabled the design of a novel cage for TTA application with tailored permeability and mechanical properties, that can be successfully 3D printed in a biodegradable bioceramic material. These results support the potential of the design optimization strategy and fabrication method to the development of customized and bioresorbable implants for bone repair. Copyright © 2016 Elsevier Ltd. All rights reserved.

Common experience modifies the reinforcing properties of methamphetamine-injected cage mates but not morphine-injected cage mates in C57 mice.

PubMed

Watanabe, Shigeru

2015-10-01

The aim of this study was to determine whether previous exposure to a drug affects the social facilitation of conditioned place preference (CPP) for a drug-injected cage mate. Twenty-two male C57/BL6J mice received drug injections (methamphetamine or morphine) and 22 male C57/BL6J mice received saline injections. All 44 mice then received CPP training, during which one compartment of a conventional CPP apparatus was associated with a drug-injected cage mate (stimulus mouse) and the other compartment was associated with a saline-injected cage mate (stimulus mouse). The subject mice did not receive any drug injection during this CPP training. Time spent in the compartment associated with the drug-injected cage mate was measured before and after training. Subject mice that had previously received methamphetamine injections showed an increase in the time spent in the compartment associated with the methamphetamine-injected cage mate after CPP training. This effect was not observed in subject mice that had previously received saline injections. Subject mice did not show an increase in the time spent in the compartment associated with the morphine-injected cage mate irrespective of whether they had previously received morphine or saline injections. Therefore, in agreement with previous reports, common experience with methamphetamine induced reinforcing properties, but that with morphine did not.

In situ experiments to assess effects of constraints linked to caging on ecotoxicity biomarkers of the three-spined stickleback (Gasterosteus aculeatus L.).

PubMed

Le Guernic, Antoine; Sanchez, Wilfried; Palluel, Olivier; Bado-Nilles, Anne; Turies, Cyril; Chadili, Edith; Cavalié, Isabelle; Adam-Guillermin, Christelle; Porcher, Jean-Marc; Geffard, Alain; Betoulle, Stéphane; Gagnaire, Béatrice

2016-04-01

The aim of this study was to evaluate the effects of caging constraints on multiple fish biomarkers used during ecotoxicological studies (biometric data, immune and antioxidant systems, and energetic status). Two of these constraints were linked to caging: starvation and fish density in cages, and one in relation to the post-caging handling: a short transport. Three in situ experiments were conducted with three-spined sticklebacks (Gasterosteus aculeatus). The first experiment compared the effects of three densities (low, medium, and high). The second experiment compared effects of starvation in fish fed every two days with fish that were not fed. Finally comparisons between sticklebacks which have suffered a short car transport after caging and sticklebacks killed without preliminary transport were made. The lack of food had no effect on fish energetic reserves but negatively affected their condition index and their immune system. Transport and high density induced oxidative stress, defined as an overproduction of reactive oxygen species and a stimulation of the antioxidant system. These two constraints also harmed the leucocyte viability. In order not to have any impact on ecotoxicity biomarkers during in situ experiments, it is preferable to decrease fish density in cages, prevent transport before dissections, and feed fish when the caging lasts more than two weeks.

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.

Development of perching behavior in 3 strains of pullets reared in furnished cages.

PubMed

Habinski, A M; Caston, L J; Casey-Trott, T M; Hunniford, M E; Widowski, T M

2017-03-01

Furnished rearing cages are becoming more widely available to replace conventional systems for pullets. To date, there is little information on how pullets develop perching behavior in furnished cages or how this varies among strains. The objective of this study was to evaluate the use of perches and a platform in a commercial furnished rearing "Combi-Cage" system by 3 pure-bred heritage strains of pullets (Rhode Island Red, Columbian Rock, and White Leghorn). Each cage had 4 elevated locations: one platform and 3 perches of differing heights and positions in the cage. The length of each cage was visually divided into 4 sections for observation. The number of birds using each section in each location was counted by one observer d per wk at 1200 h (d) from one to 14 wk of age and at 1600 h (night) right after lights were turned off from 4 to 14 wk of age. Mixed model repeated analyses were used to test effects of strain, age, and their interaction on the use of the platform, the 3 perches, and all 4 locations combined (vertical space use) at both time periods. GLM were used to compare overall use of the sections and locations. On average, pullets used vertical space more during the d than at night. There were also effects of age (P < 0.05) and strain (P < 0.05) on the use of the 4 locations in the cage at both time periods. Generally, the Columbian Rocks used the perches and platform the most, and Rhode Island Reds the least. The highest perch in the cage was rarely used and birds showed a preference for perching in sections that were closest to cage walls (P < 0.05). Differences across strains and low use of some perch locations indicate that the design of furnished rearing cages may still require improvements in order to ensure the furnishings are used by pullets as intended. © 2016 Poultry Science Association Inc.

Comparison among perfect-C®, zero-P®, and plates with a cage in single-level cervical degenerative disc disease.

PubMed

Noh, Sung Hyun; Zhang, Ho Yeol

2018-01-25

We intended to analyze the efficacy of a new integrated cage and plate device called Perfect-C for anterior cervical discectomy and fusion (ACDF) to cure single-level cervical degenerative disc disease. We enrolled 148 patients who were subjected to single-level ACDF with one of the following three surgical devices: a Perfect-C implant (41 patients), a Zero-P implant (36 patients), or a titanium plate with a polyetheretherketone (PEEK) cage (71 patients). We conducted a retrospective study to compare the clinical and radiological results among the three groups. The length of the operation, intraoperative blood loss, and duration of hospitalization were significantly lower in the Perfect-C group than in the Zero-P and plate-with-cage groups (P < 0.05). At the last follow-up visit, heterotopic ossification (HO) was not observed in any cases (0%) in the Perfect-C and Zero-P groups but was noted in 21 cases (30%) in the plate-with-cage group. The cephalad and caudal plate-to-disc distance (PDD) and the cephalad and caudal PDD/anterior body height (ABH) were significantly greater in the Perfect-C and Zero-P groups than in the plate-with-cage group (P < 0.05). Subsidence occurred in five cases (14%) in the Perfect-C group, in nine cases (25%) in the Zero-P group, and in 15 cases (21%) in the plate-with-cage group. Fusion occurred in 37 cases (90%) in the Perfect-C group, in 31 cases (86%) in the Zero-P group, and in 68 cases (95%) in the plate-with-cage group. The Perfect-C, Zero-P, and plate-with-cage devices are effective for treating single-level cervical degenerative disc disease. However, the Perfect-C implant has many advantages over both the Zero-P implant and conventional plate-cage treatments. The Perfect-C implant was associated with shorter operation times and hospitalization durations, less blood loss, and lower subsidence rates compared with the Zero-P implant or the titanium plate with a PEEK cage.

Evaluation of anterior cervical reconstruction with titanium mesh cages versus nano-hydroxyapatite/polyamide66 cages after 1- or 2-level corpectomy for multilevel cervical spondylotic myelopathy: a retrospective study of 117 patients.

PubMed

Zhang, Yuan; Quan, Zhengxue; Zhao, Zenghui; Luo, Xiaoji; Tang, Ke; Li, Jie; Zhou, Xu; Jiang, Dianming

2014-01-01

To retrospectively compare the efficacy of the titanium mesh cage (TMC) and the nano-hydroxyapatite/polyamide66 cage (n-HA/PA66 cage) for 1- or 2-level anterior cervical corpectomy and fusion (ACCF) to treat multilevel cervical spondylotic myelopathy (MCSM). A total of 117 consecutive patients with MCSM who underwent 1- or 2-level ACCF using a TMC or an n-HA/PA66 cage were studied retrospectively at a mean follow-up of 45.28 ± 12.83 months. The patients were divided into four groups according to the level of corpectomy (1- or 2-level corpectomy) and cage type used (TMC or n-HA/PA66 cage). Clinical and radiological parameters were used to evaluate outcomes. At the one-year follow-up, the fusion rate in the n-HA/PA66 group was higher, albeit non-significantly, than that in the TMC group for both 1- and 2-level ACCF, but the fusion rates of the procedures were almost equal at the final follow-up. The incidence of cage subsidence at the final follow-up was significantly higher in the TMC group than in the n-HA/PA66 group for the 1-level ACCF (24% vs. 4%, p = 0.01), and the difference was greater for the 2-level ACCF between the TMC group and the n-HA/PA66 group (38% vs. 5%, p = 0.01). Meanwhile, a much greater loss of fused height was observed in the TMC group compared with the n-HA/PA66 group for both the 1- and 2-level ACCF. All four groups demonstrated increases in C2-C7 Cobb angle and JOA scores and decreases in VAS at the final follow-up compared with preoperative values. The lower incidence of cage subsidence, better maintenance of the height of the fused segment and similar excellent bony fusion indicate that the n-HA/PA66 cage may be a superior alternative to the TMC for cervical reconstruction after cervical corpectomy, in particular for 2-level ACCF.

Biomechanical analysis of an expandable lateral cage and a static transforaminal lumbar interbody fusion cage with posterior instrumentation in an in vitro spondylolisthesis model.

PubMed

Mantell, Matthew; Cyriac, Mathew; Haines, Colin M; Gudipally, Manasa; O'Brien, Joseph R

2016-01-01

Insufficient biomechanical data exist from comparisons of the stability of expandable lateral cages with that of static transforaminal lumbar interbody fusion (TLIF) cages. The purpose of this biomechanical study was to compare the relative rigidity of L4-5 expandable lateral interbody constructs with or without additive pedicle screw fixation with that of L4-5 static TLIF cages in a novel cadaveric spondylolisthesis model. Eight human cadaver spines were used in this study. A spondylolisthesis model was created at the L4-5 level by creating 2 injuries. First, in each cadaver, a nucleotomy from 2 channels through the anterior side was created. Second, the cartilage of the facet joint was burred down to create a gap of 4 mm. Light-emitting-diode tracking markers were placed at L-3, L-4, L-5, and S-1. Specimens were tested in the following scenarios: intact model, bilateral pedicle screws, expandable lateral 18-mm-wide cage (alone, with unilateral pedicle screws [UPSs], and with bilateral pedicle screws [BPSs]), expandable lateral 22-mm-wide cage (alone, with UPSs, and with BPSs), and TLIF (alone, with UPSs, and with BPSs). Four of the spines were tested with the expandable lateral cages (18-mm cage followed by the 22-mm cage), and 4 of the spines were tested with the TLIF construct. All these constructs were tested in flexion-extension, axial rotation, and lateral bending. The TLIF-alone construct was significantly less stable than the 18- and 22-mm-wide lateral lumbar interbody fusion (LLIF) constructs and the TLIF constructs with either UPSs or BPSs. The LLIF constructs alone were significantly less stable than the TLIF construct with BPSs. However, there was no significant difference between the 18-mm LLIF construct with UPSs and the TLIF construct with BPSs in any of the loading modes. Expandable lateral cages with UPSs provide stability equivalent to that of a TLIF construct with BPSs in a degenerative spondylolisthesis model.

Pop hole passages and welfare in furnished cages for laying hens.

PubMed

Wall, H; Tauson, R; Elwinger, K

2004-02-01

1. This study included two designs of furnished cages for 16 hens; H-cages divided into two apartments by a partition with pop holes in the middle of the cage, and fully open O-cages, without a partition. The hypothesis was that in this rather large group of birds the pop hole partition would benefit the birds by allowing them to avoid or escape from potential cannibals, feather-peckers or aggressive hens. All cages had two nests, two perches and one litter box. 2. A total of 10 cages (5 H and 5 O) were stocked with Lohmann Selected Leghorn (LSL) and 8 cages (4 H and 4 O) with Hy-Line W36. No birds were beak-trimmed. 3. Heterophil/lymphocyte (H/L) ratios, duration of tonic immobility (TI) and exterior appearance (scoring of plumage condition and wounds at comb or around cloaca) were used as indicators of well-being. Total mortality and deaths due to cannibalism were also recorded. 4. Visits to nests and passages through partition pop holes were studied in samples of 35 and 21 birds, respectively, using a technique based on passive integrated transponder (PIT) tags. 5. Cage design (H- vs O-cage) had no effect on the welfare traits chosen. 6. Hy-Line birds showed higher H/L ratios, longer duration of TI and better plumage condition than LSL birds. These differences are discussed in terms of stress thresholds and copying strategies. 7. On days when a hen made visits to nests, the visiting frequency was 1.4 and the total time in the nest was 41 min on average. Hens made use of the pop hole passages between 1 and 8 times per hen and day. 8. Overall low levels of aggression, lack of injuries or deaths due to cannibalism, and plumage condition indicating moderate feather pecking, together imply a low need to escape. The pop holes were used frequently and birds distributed well between compartments showing that the system worked well. However, at this group size there was no evidence in the measured traits that H-cages provided a better housing environment.

Opinion of Belgian Egg Farmers on Hen Welfare and Its Relationship with Housing Type

PubMed Central

Stadig, Lisanne M.; Ampe, Bart A.; Van Gansbeke, Suzy; Van den Bogaert, Tom; D’Haenens, Evelien; Heerkens, Jasper L.T.; Tuyttens, Frank A.M.

2015-01-01

Simple Summary Until 2012, laying hens in the EU were often housed in conventional cages that offered limited space and few opportunities to perform highly motivated behaviors. Conventional cages are now banned in the EU in order to improve animal welfare. In this study, egg farmers were surveyed (winter 2013–2014) to assess whether they perceived any changes in animal welfare since changing housing systems, what role hen welfare played in choosing a new housing system, and which aspects of hen welfare they find most important. The data show that the answers differ depending on which housing system the farmers currently use and whether they had used conventional cages in the past. Abstract As of 2012, the EU has banned the use of conventional cages (CC) for laying hens, causing a shift in housing systems. This study’s aim was to gain insight into farmers’ opinions on hen health and welfare in their current housing systems. A survey was sent to 218 Belgian egg farmers, of which 127 (58.3%) responded, with 84 still active as egg farmer. Hen welfare tended to be less important in choosing the housing system for farmers with cage than with non-cage systems. Respondents currently using cage systems were more satisfied with hen health than respondents with non-cage systems. Reported mortality increased with farm size and was higher in furnished cages than in floor housing. Feather pecking, cannibalism, smothering and mortality were perceived to be higher in current housing systems than in CC, but only by respondents who shifted to non-cage systems from previously having had CC. Health- and production-related parameters were scored to be more important for hen welfare as compared to behavior-related parameters. Those without CC in the past rated factors relating to natural behavior to be more important for welfare than those with CC. This difference in opinion based on farmer backgrounds should be taken into account in future research. PMID:26703742

The effect of early life experience, environment, and genetic factors on spontaneous home-cage aggression-related wounding in male C57BL/6 mice.

PubMed

Gaskill, Brianna N; Stottler, Aurora M; Garner, Joseph P; Winnicker, Christina W; Mulder, Guy B; Pritchett-Corning, Kathleen R

2017-03-22

Aggression is a major welfare issue in mice, particularly when mice unfamiliar to each other are first placed in cages, as happens on receipt from a vendor, and following cage cleaning. Injuries from aggression are the second leading cause of unplanned euthanasia in mice, following ulcerative dermatitis. Commonly employed strategies for reducing aggression-related injury are largely anecdotal, and may even be counterproductive. Here we report a series of experiments testing potential explanations and interventions for post-shipping aggression-related injuries in C57BL/6 mice. First, we examined the effects of weaning: testing whether manipulating weaning age reduced aggression-related injuries, and if repeated mixing of weaned mice before shipping increased these injuries. Contrary to our predictions, repeated mixing did not increase post-shipping injurious aggression, and early weaning reduced aggression-related injuries. Second, we examined potential post-shipping interventions: testing whether lavender essential oil applied to the cage reduced aggression-related injuries, and whether a variety of enrichments decreased injurious aggression. Again, contrary to predictions, lavender increased wounding, and none of the enrichments reduced it. However, consistent with the effects of weaning age in the first experiment, cages with higher mean body weight showed elevated levels of aggression-related wounding. Finally, we tested whether C57BL/6 substrains and identification methods affected levels of intra-cage wounding from aggression. We found no effect of strain, but cages where mice were ear-notched for identification showed higher levels of wounding than cages where mice were tail-tattooed. Overall, these results emphasize the multifactorial nature of home-cage injurious aggression, and the importance of testing received wisdom when it comes to managing complex behavioral and welfare problems. In terms of practical recommendations to reduce aggressive wounding in the home cage, tail tattooing is recommended over ear notching and late weaning should be avoided.

Effect of acclimation to caging on nephrotoxic response of rats to uranium

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

Damon, E.G.; Eidson, A.F.; Hobbs, C.H.

1986-02-01

Animal studies of the toxicity and metabolism of radionuclides and chemicals often require housing of rats in metabolism cages for excreta collection. Response of rats to toxic substances may be affected by environmental factors such as the type of cage used. Dose-response studies were conducted to assess the effects of two types of cages on the nephrotoxic response of rats to uranium from implanted refined uranium ore (yellowcake). The LD50/21 days was 6 mg of uranium ore per kilogram body weight (6 mg U/kg). The 95% confidence limit (C.L.) was 3-8 mg U/kg for rats housed in metabolism cages beginningmore » on the day of implantation (naive rats). However, for rats housed in metabolism cages for 21 days before implantation (acclimated rats) the LD50/21 days was 360 mg U/kg (95% C.L. = 220-650 mg U/kg), which was the same value obtained for rats housed continuously in polycarbonate cages. This significant difference (P less than 0.01) in response of naive rats compared to response of acclimated rats appeared related to a significantly lower water consumption by the naive rats.« less

Care of captive woodcocks

USGS Publications Warehouse

Stickel, William H.; Sheldon, William G.; Stickel, Lucille F.

1965-01-01

Numbers of American woodcocks (Philohela minor) were held in cages for experimental work lasting several months. Injuries caused by birds attempting to flush were greatly reduced by clipping feathers from one wing, by making cage walls opaque, and by using high cages or false ceilings of fabric. Size of cage was found not to be important, to judge from weight changes, so long as ample food was unmistakably available. Birds were kept in both large and small cages without social conflicts. Cages on the ground proved too unsanitary for long-term use; small steel cages with removable floors were practical but did not solve the sanitation problem. Living earthworms (Lumbricus terrestris) were provided daily in amounts roughly equal to weights of birds. Birds gained on this food when worms were offered in suitable ways. The feeding tray recommended is a large roasting pan with a snap-on metal rim that retards loss of worms. Trays contained moist peat in which birds probed for worms. Two efforts to keep woodcocks on a diet of red worms (Eisenia foetide) were unsuccessful; use of this worm was considered responsible. Woodcocks were handled and transported for short periods with least injury to them when they were rolled individually in soft bags.

The effect of host relaxation and dynamics on guest molecule dynamics in H2/tetrahydrofuranhydrate.

PubMed

Peterson, Vanessa K; Shoko, Elvis; Kearley, Gordon J

2011-01-01

We use ab initio molecular dynamics simulations to obtain classically the effects of H2O cage motions on the potential-energy surface (PES) of encapsulated H2 in the H2/tetrahydrofuran-hydrate system. The significant differences between the PES for the H2 in rigid and flexible cages that we find will influence calculation of the quantum dynamics of the H2. Part of these differences arises from the relaxation of the H2O cage around the classical H2, with a second part arising from the coupling of both translational and rotational motions of H2 with the H20 cage. We find that isotopic substitution of 2H for 1H of the H2O cage affects the coupling, which has implications for experiments that require the use of 2H2O, including inelastic neutron scattering that uses 2H2O cages in order to focus on the H2 guest dynamics. Overall, this work emphasizes the importance of taking into account cage dynamics in any approach used to understand the dynamics of H2 guests in porous framework materials.

Evaluation of cage designs and feeding regimes for honey bee (Hymenoptera: Apidae) laboratory experiments.

PubMed

Huang, Shao Kang; Csaki, Tamas; Doublet, Vincent; Dussaubat, Claudia; Evans, Jay D; Gajda, Anna M; Gregorc, Alex; Hamilton, Michele C; Kamler, Martin; Lecocq, Antoine; Muz, Mustafa N; Neumann, Peter; Ozkirim, Asli; Schiesser, Aygün; Sohr, Alex R; Tanner, Gina; Tozkar, Cansu Ozge; Williams, Geoffrey R; Wu, Lyman; Zheng, Huoqing; Chen, Yan Ping

2014-02-01

The aim of this study was to improve cage systems for maintaining adult honey bee (Apis mellifera L.) workers under in vitro laboratory conditions. To achieve this goal, we experimentally evaluated the impact of different cages, developed by scientists of the international research network COLOSS (Prevention of honey bee COlony LOSSes), on the physiology and survival of honey bees. We identified three cages that promoted good survival of honey bees. The bees from cages that exhibited greater survival had relatively lower titers of deformed wing virus, suggesting that deformed wing virus is a significant marker reflecting stress level and health status of the host. We also determined that a leak- and drip-proof feeder was an integral part of a cage system and a feeder modified from a 20-ml plastic syringe displayed the best result in providing steady food supply to bees. Finally, we also demonstrated that the addition of protein to the bees' diet could significantly increase the level ofvitellogenin gene expression and improve bees' survival. This international collaborative study represents a critical step toward improvement of cage designs and feeding regimes for honey bee laboratory experiments.

Caged Protein Prenyltransferase Substrates: Tools for Understanding Protein Prenylation

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

DeGraw, Amanda J.; Hast, Michael A.; Xu, Juhua

Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterizationmore » of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.« less

Effect of acclimation to caging on nephrotoxic response of rats to uranium.

PubMed

Damon, E G; Eidson, A F; Hobbs, C H; Hahn, F F

1986-02-01

Animal studies of the toxicity and metabolism of radionuclides and chemicals often require housing of rats in metabolism cages for excreta collection. Response of rats to toxic substances may be affected by environmental factors such as the type of cage used. Dose-response studies were conducted to assess the effects of two types of cages on the nephrotoxic response of rats to uranium from implanted refined uranium ore (yellowcake). The LD50/21 days was 6 mg of uranium ore per kilogram body weight (6 mg U/kg). The 95% confidence limit (C.L.) was 3-8 mg U/kg for rats housed in metabolism cages beginning on the day of implantation (naive rats). However, for rats housed in metabolism cages for 21 days before implantation (acclimated rats) the LD50/21 days was 360 mg U/kg (95% C.L. = 220-650 mg U/kg), which was the same value obtained for rats housed continuously in polycarbonate cages. This significant difference (P less than 0.01) in response of naive rats compared to response of acclimated rats appeared related to a significantly lower water consumption by the naive rats.

COATING ALTERNATIVES GUIDE (CAGE) USER'S GUIDE

EPA Science Inventory

The guide provides instructions for using the Coating Alternatives GuidE (CAGE) software program, version 1.0. It assumes that the user is familiar with the fundamentals of operating an IBM-compatible personal computer (PC) under the Microsoft disk operating system (MS-DOS). CAGE...

A fluorescent organic cage for picric acid detection.

PubMed

Acharyya, Koushik; Mukherjee, Partha Sarathi

2014-12-25

Dynamic covalent imine chemistry has been utilized to synthesize a fluorescent [3+2] self-assembled nanoscopic organic cage. The fluorescent nature of the reduced analogue of the cage was further exploited for the highly selective detection of the explosive picric acid (PA).

Carbon-Phenolic Cages for High-Speed Bearings. Part II - Bearing Evaluation with a Multiply-Alkylated Cyclopentane (MAC) Lubricant

DTIC Science & Technology

2003-01-01

program manager for the overall effort. The carbon -phenolic cages were fabricated under the direction of Mr. Wei Shih of Allcomp Inc ., City of...friction. The early carbon -phenolic cages were hand wrapped by Allcomp . Since that time, Allcomp has added a wrapping machine that uses uniform tension...AFRL-PR-WP-TR-2003-2032 CARBON -PHENOLIC CAGES FOR HIGH-SPEED BEARINGS Part II - Bearing Evaluation with a Multiply- Alkylated Cyclopentane (MAC

Extremely Low Frequency (ELF) Vertical Electric Field Exposure of Rats: Irradiation Facility

DTIC Science & Technology

1977-05-01

altered inside an animal cage even with wet or dry litter and full food and water containers. Rats weighing approximately 300 g in adjacent cages caused...with guard circuit Field inside empty cage Field inside complete cage ( litter (wet or dry) + food + water) Field variations caused by 300 g rat...blanket 250 Iron 60 Broiler 130 Hair dryer 40 Vaporizer 40 Refrigerator 60 Color TV 30 Stereo 90 Coffee pot 30 Vacuum cleaner 16 Clock radio

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.

Exterior egg quality as affected by enrichment resources layout in furnished laying-hen cages.

PubMed

Li, Xiang; Chen, Donghua; Meng, Fanyu; Su, Yingying; Wang, Lisha; Zhang, Runxiang; Li, Jianhong; Bao, Jun

2017-10-01

This study aimed to investigate the effects of enrichment resources (a perch, dustbath, and nest) layout in furnished laying-hen cages (FC) on exterior quality of eggs. One hundred and sixty-eight (168) Hy-Line Brown laying hens at 16 weeks of age were randomly distributed to four treatments: small furnished cages (SFC), medium furnished cages type I (MFC-I), medium furnished cages type II (MFC-II), and medium furnished cages type III (MFC-III). Each treatment had 4 replicates or cages with 6 hens for SFC (24 birds for each SFC) and 12 hen/cage for MFC-I, -II, and -III (48 birds for each MFC-I, -II and -III). Following a 2-week acclimation, data collection started at 18 weeks of age and continued till 52 weeks of age. Dirtiness of egg surface or cracked shell as indicators of the exterior egg quality were recorded each week. The results showed that the proportion of cracked or dirty eggs was significantly affected by the FC type (p<0.01) in that the highest proportion of cracked or dirty eggs was found in MFC-I and the lowest proportion of dirty eggs in SFC. The results of this showed that furnished cage types affected both dirty eggs and cracked eggs (p<0.01). The results also indicated that not nest but dustbath lead to more dirty eggs. Only MFC-I had higher dirty eggs at nest than other FC (p< 0.01). The results of dirty eggs in MFC-I and MFC-II compared with SFC and MFC-III seemed suggest that a low position of dustbath led to more dirty eggs. SFC design affected exterior egg quality and the low position of dustbath in FC resulted in higher proportion of dirty eggs.

Effect of a commercial housing system on egg quality during extended storage.

PubMed

Jones, D R; Karcher, D M; Abdo, Z

2014-05-01

Egg producers in the United States are utilizing a variety of commercial egg production systems to provide consumer choice and meet legislative requirements. Consumer egg grades in the United States were developed for conventional cage production, and it is unclear what effect alternative production systems might have on egg quality during retail and consumer home storage. The current study was undertaken to determine what changes in egg quality characteristics occur during extended cold storage for commercially produced conventional cage, enriched colony cage, and cage-free aviary eggs. During 12 wk of cold storage, egg weight, albumen height, Haugh unit, static compression shell strength, vitelline membrane strength and deformation, yolk index, shell dynamic stiffness, and whole egg total solids were monitored. Overall, aviary and enriched eggs were significantly (P < 0.05) heavier than conventional cage. Albumen height and Haugh unit (P < 0.05) were significantly greater for conventional cage than enriched eggs. Static compression shell strength was greatest (P < 0.05) for enriched eggs compared with aviary. No overall housing system effects for yolk measurements, shell dynamic stiffness, or whole egg total solids were observed. Albumen height, Haugh unit, and yolk quality measurements were all greatest at 0 and lowest at 12 wk of storage (P < 0.05). The rate of quality change among the housing systems for each measured attribute at 4, 6, and 12 wk was determined. Other than differences in the change of egg weight at 4 wk, no significant differences in the rate of quality decline were found among the housing systems. The results of the current study indicate that current US egg quality standards should effectively define quality for commercially produced conventional cage, enriched colony cage, and cage-free aviary eggs.

Mosquito density, biting rate and cage size effects on repellent tests.

PubMed

Barnard, D R; Posey, K H; Smith, D; Schreck, C E

1998-01-01

Mosquito biting rates and the mean duration of protection (in hours) from bites (MDPB) of Aedes aegypti and Anopheles quadrimaculatus, using the repellent 'deet' (N,N-diethyl-3-methylbenzamide) on a 50 cm2 area of healthy human skin, were observed in small (27 l), medium (approximately 65 l) and large (125 l) cages containing low, medium or high densities of mosquitoes: respectively, 640, 128 or 49 cm3 of cage volume per female. At the initial treatment rate of approximately 0.4 microliter/cm2 (1 ml of 25% deet in ethanol on 650 cm2 of skin), the MDPB for deet against Ae. aegypti ranged from 4.5 to 6.5 h and was significantly less (5.0 +/- 0.8 h) in large cages compared with medium (6.2 +/- 0.9 h) and small (6.2 +/- 0.8 h) cages, regardless of the density. Against An. quadrimaculatus the MDPB for deet 0.4 microliter/cm2 was 1.5-8.0 h, less in small (3.7 +/- 2.3 h) and large (2.2 +/- 1.1 h) cages at medium (3.7 +/- 2.3 h) and high (2.5 +/- 1.7 h) mosquito densities, and was longest in medium cages (6.2 +/- 2.6 h) at low mosquito densities (5.8 +/- 2.8 h). With equinoxial photoperiodicity (light on 06.00-18.00 hours) the biting rate was influenced by the time of observation (08.00, 12.00, 16.00 hours) for Ae. aegypti but not for An. quadrimaculatus. For both species, the biting rate was inversely proportional to mosquito density and the MDPB. The shortest MDPBs were obtained in large cages with high densities of mosquitoes and longest protection times occurred in medium sized cages with low mosquito densities.

Self-Assembled Pyridine-Dipyrrolate Cages.

PubMed

Zhang, Huacheng; Lee, Juhoon; Lammer, Aaron D; Chi, Xiaodong; Brewster, James T; Lynch, Vincent M; Li, Hao; Zhang, Zhan; Sessler, Jonathan L

2016-04-06

An inherently nonlinear pyridine dipyrrolate ligand, namely 2,6-bis(3,4-diethyl-5-carboxy-1H-pyrrol-2yl)pyridine (compound 1), is able to distinguish between different zinc(II) cation sources, namely Zn(acac)2 and Zn(OAc)2, respectively. This differentiation is manifest both in terms of the observed fluorescent behavior in mixed organic media and the reaction chemistry. Treatment of 1 with Zn(acac)2 gives rise to a cage dimer, cage-1, wherein two molecules of compound 1 act as double bridging units to connect two individual cage subunits. As inferred from X-ray crystallographic studies, this cage system consists of discrete zinc dimers with hydroxide bridges that, with the assistance of bound DMF solvent molecules, serve to fix the geometry and orientation of the pyridine dipyrrolate building blocks. When a different zinc source, Zn(OAc)2, is used to carry out an ostensibly similar complexation reaction with compound 1, an acetate-bridged 1D abacus-like cage polymer is obtained as inferred from X-ray diffraction analysis. This extended solid state structure, cage-2, contains individual zinc dimer cage submits and appears stabilized by solvent molecules (DMF) and the counteranion (acetate). Rod-like assemblies are also observed by DLS and SEM. This construct, in contrast to cage-1, proved fluorescent in mixed organic media. The structure of the ligand itself (i.e., in the absence of Zn(II)) was confirmed by X-ray crystallographic analysis and was found to assemble into a supramolecular polymer. Conversion to a dimer form was seen upon the addition of TBAOAc. On the basis of the metric parameters, the structures seen in the solid state are stabilized via hydrogen bonding interactions involving solvent molecules.

Screening of alcohol use disorders in psychiatric outpatients: influence of gender, age, and psychiatric diagnosis.

PubMed

Sánchez Autet, Mónica; Garriga, Marina; Zamora, Francisco Javier; González, Idilio; Usall, Judith; Tolosa, Leticia; Benítez, Concepción; Puertas, Raquel; Arranz, Belén

2017-07-14

Alcohol use disorders (AUD) are 2 times higher among psychiatric patients than in the general population. The under-recognition of this dual diagnosis can entail several negative outcomes. Early assessment with a screening tool like the CAGE questionnaire could be an opportunity to improve patients' prognoses. The objective of this study is to assess AUD risk in an outpatient psychiatric sample with a modified CAGE, considering the influence of age, gender and clinical psychiatric diagnosis. An observational, multicentric, descriptive study was carried out. The 4-item CAGE scale, camouflaged in a healthy lifestyle questionnaire, was implemented, using a cut-off point of one. 559 outpatients were assessed. 54% were female and the average age was 50.07 years. 182 patients presented a CAGE score ≥1 (45.1% of men and 21.9% of women). Gender was the strongest predictor of a positive result in CAGE, as men were 3.03 times more likely to score ≥1 on the CAGE questionnaire (p < .001, 95% CI: 0.22-0.49). Patients with bipolar and personality disorders had the highest rates of CAGE scores ≥1 (45.2 and 44.9%, respectively), with a significant association between diagnosis and a positive score (p = .002). Patients above 60 years were 2.5 times less likely to score ≥1 on the CAGE (p = .017, 95% CI: 0.19-0.85). Specific screening questionnaires, like the CAGE scale, can be an easy and useful tool in the assessment of AUD risk in psychiatric outpatients. Male patients with a bipolar or personality disorder present a higher risk of AUD.

-A practical application of reduced-copper antifouling paint in marine biological research.

PubMed

Jerabek, Andrea S; Wall, Kara R; Stallings, Christopher D

2016-01-01

Biofouling of experimental cages and other field apparatuses can be problematic for scientists and has traditionally been addressed using frequent manual removal (e.g., scraping, scrubbing). Recent environmental restrictions and legislative changes have driven the development of less hazardous antifouling products, making antifouling paint a potential alternative option to manual removal. Consequently, the viability of using these newly developed products as a replacement for the manual cleaning of exclusion cages was experimentally investigated. There were six treatments tested, comprising three with settlement tiles in experimental cages coated with antifouling paint, two with settlement tiles in unpainted experimental cages, and one cage-free suspended tile. The three antifouling treatments comprised two reduced-copper paints (21% Cu2O and 40% Cu2O) and one copper-free, Econea (™)-based paint (labeled "ecofriendly"). Antifouling paints were assessed for performance of preventing fouling of the cages and whether they elicited local effects on settlement tiles contained within them. All three paints performed well to reduce fouling of the cages during the initial six weeks of the experiment, but the efficacy of "ecofriendly" paint began to decrease during an extended deployment that lasted 14 weeks. The macro-community composition, biomass, and percent cover of settled organism on tiles within cages treated with copper-based paints (21% and 40% concentrations) were indistinguishable from tiles within the manually scrubbed cages. In contrast, settlement to tiles from the "ecofriendly" treatment was different in composition of macro-community and lower in biomass, suggesting the presence of local effects and therefore rendering it unsuitable for use in settlement experiments. The results of this study suggest that reduced-copper paints have the potential to serve as an alternative to manual maintenance, which may be useful for deployments in locations that are difficult to access on a frequent schedule.

Anterior debridement and fusion followed by posterior pedicle screw fixation in pyogenic spondylodiscitis: autologous iliac bone strut versus cage.

PubMed

Pee, Yong Hun; Park, Jong Dae; Choi, Young-Geun; Lee, Sang-Ho

2008-05-01

An anterior approach for debridement and fusion with autologous bone graft has been recommended as the gold standard for surgical treatment of pyogenic spondylodiscitis. The use of anterior foreign body implants at the site of active infection is still a challenging procedure for spine surgeons. Several authors have recently introduced anterior grafting with titanium mesh cages instead of autologous bone strut in the treatment of spondylodiscitis. The authors present their experience of anterior fusion with 3 types of cages followed by posterior pedicle screw fixation. They also compare their results with the use of autologous iliac bone strut. The authors retrospectively reviewed the cases of 60 patients with pyogenic spondylodiscitis treated by anterior debridement between January 2003 and April 2005. Fusion using either cages or iliac bone struts was performed during the same course of anesthesia followed by posterior fixation. Twenty-three patients underwent fusion with autologous iliac bone strut, and 37 patients underwent fusion with 1 of the 3 types of cages. The infections resolved in all patients, as noted by normalization of their erythrocyte sedimentation rates and C-reactive protein levels. Patients in both groups were evaluated in terms of their preoperative and postoperative clinical and imaging findings. Single-stage anterior debridement and cage fusion followed by posterior pedicle screw fixation can be effective in the treatment of pyogenic spondylodiscitis. There was no difference in clinical and imaging outcomes between the strut group and cage group except for the subsidence rate. The subsidence rate was higher in the strut group than in the cage group. The duration until subsidence was also shorter in the strut group than in the cage group.

From Coordination Cages to a Stable Crystalline Porous Hydrogen-Bonded Framework

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

Ju, Zhanfeng; Liu, Guoliang; Chen, Yu-Sheng

2017-03-20

A stable framework has been constructed through multiple charge-assisted H-bonds between cationic coordination cages and chloride ions. The framework maintained its original structure upon desolvation, which has been established by single-crystal structure analysis. This is the first fully characterized stable porous framework based on coordination cages after desolvation, with a moderately high Brunauer–Emmett–Teller (BET) surface area of 1201 m2 g-1. This work will not only give a light to construct stable porous frameworks based on coordination cages and thus broaden their applications, but will also provide a new avenue to the assembly of other porous materials such as porous organicmore » cages and hydrogen-bonded organic frameworks (HOFs) through non covalent bonds.« less

More vertical etch profile using a Faraday cage in plasma etching

NASA Astrophysics Data System (ADS)

Cho, Byeong-Ok; Hwang, Sung-Wook; Ryu, Jung-Hyun; Moon, Sang Heup

1999-05-01

Scanning electron microscope images of sidewalls obtained by plasma etching of an SiO2 film with and without a Faraday cage have been compared. When the substrate film is etched in the Faraday cage, faceting is effectively suppressed and the etch profile becomes more vertical regardless of the process conditions. This is because the electric potential in the cage is nearly uniform and therefore distortion of the electric field at the convex corner of a microfeature is prevented. The most vertical etch profile is obtained when the cage is used in fluorocarbon plasmas, where faceting is further suppressed due to the decrease in the chemical sputtering yield and the increase in the radical/ion flux on the substrate.

Open-cage fullerene-like graphitic carbons as catalysts for oxidative dehydrogenation of isobutane.

PubMed

Liang, Chengdu; Xie, Hong; Schwartz, Viviane; Howe, Jane; Dai, Sheng; Overbury, Steven H

2009-06-10

We report herein a facile synthesis of fullerene-like cages, which can be opened and closed through simple thermal treatments. A glassy carbon with enclosed fullerene-like cages of 2-3 nm was synthesized through a soft-template approach that created open mesopores of 7 nm. The open mesopores provided access to the fullerene-like cages, which were opened and closed through heat treatments in air and inert gas at various temperatures. Catalytic measurements showed that the open cages displayed strikingly higher activity for the oxidative dehydrogenation of isobutane in comparison to the closed ones. We anticipate that this synthesis approach could unravel an avenue for pursuing fundamental understanding of the unique catalytic properties of graphitic carbon nanostructures.