Sample records for plasmid-based short hairpin

  1. Short hairpin-loop-structured oligodeoxynucleotides reduce HSV-1 replication

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    Heinrich Jochen


    Full Text Available Abstract The Herpes simplex virus (HSV is known as an infectious agent and widespread in the human population. The symptoms of HSV infections can range from mild to life threatening, especially in immune-compromised individuals. HSV infections are commonly treated with the guanosine analogue Aciclovir, but reports of resistance are increasing. Efforts are made to establish single-stranded antisense oligodeoxynucleotides (as and small interfering ribonucleic acids (siRNAs for antiviral treatment. Recently, another class of short interfering nucleic acids, partially double-stranded hairpin loop-structured 54 mer oligodeoxynucleotides (ODNs, was shown to allow hydrolysis of HIV RNA by binding to the viral RNA. This leads to a substrate for the viral RNase H. To assess the potential of such ODNs for inhibition of HSV-1 replication, five partially double-stranded ODNs were designed based on the sequences of known siRNAs against HSV-1 with antiviral activity. Three of them are directed against early and two against leaky late genes. Primary human lung fibroblasts, MRC-5, and African green monkey kidney cells, Vero, were transfected with ODNs and subsequently infected. The effect on HSV-1 replication was determined by analyzing the virus titer in cell culture supernatants by quantitative PCR and plaque assays. An inhibitory effect was observed with all five selected ODNs, with two cases showing statistical significance in both cell types. The observed effect was sequence-specific and dose dependent. In one case the ODN was more efficient than a previously described siRNA directed against the same target site in the mRNA of UL5, a component of the helicase/primase complex. HSV-1 virions and ODNs can be applied simultaneously without transfection reagent, but at a 50-fold higher concentration to Vero cells with similar efficiencies. The results underline the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.

  2. Short hairpin-looped oligodeoxynucleotides reduce hepatitis C virus replication

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    Broecker Felix


    Full Text Available Abstract Background Persistent infection with hepatitis C virus (HCV is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Standard therapy consists of a combination of interferon-alpha and ribavirin, but many patients respond poorly, especially those infected with HCV genotypes 1 and 4. Furthermore, standard therapy is associated with severe side-effects. Thus, alternative therapeutic approaches against HCV are needed. Findings Here, we studied the effect of a new class of antiviral agents against HCV, short, partially double-stranded oligodeoxynucleotides (ODNs, on viral replication. We targeted the 5’ nontranslated region (5’ NTR of the HCV genome that has previously been shown as effective target for small interfering RNAs (siRNAs in vitro. One of the investigated ODNs, ODN 320, significantly and efficiently reduced replication of HCV replicons in a sequence-, time- and dose-dependent manner. ODN 320 targets a genomic region highly conserved among different HCV genotypes and might thus be able to inhibit a broad range of genotypes and subtypes. Conclusions ODNs provide an additional approach for inhibition of HCV, might be superior to siRNAs in terms of stability and cellular delivery, and suitable against HCV resistant to standard therapy. This study underlines the potential of partially double-stranded ODNs as antiviral agents.

  3. Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

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    Singh Upinder


    Full Text Available Abstract Background Entamoeba histolytica is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in Entamoeba histolytica. Results An episomal vector-based system, using the E. histolytica U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in E. histolytica. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%. Conclusion Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in Entamoeba histolytica, providing a useful tool for the study of this parasite.

  4. Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

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    Sedlacek Radislav


    Full Text Available Abstract Background RNA interference (RNAi is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type. Results Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals. Conclusions We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.

  5. Enantioselective Catalysis by Using Short, Structurally Defined DNA Hairpins as Scaffold for Hybrid Catalysts. (United States)

    Marek, Jasmin J; Singh, Raghvendra P; Heuer, Andreas; Hennecke, Ulrich


    A new type of DNA metal complex hybrid catalyst, which is based on single-stranded DNA oligonucleotides, is described. It was shown that oligonucleotides as short as 14 nucleotides that fold into hairpin structures are suitable as nucleic acid components for DNA hybrid catalysts. With these catalysts, excellent enantioinduction in asymmetric Diels-Alder reactions with selectivity values as high as 96 % enantiomeric excess (ee) can be achieved. Molecular dynamics simulations indicate that a rather flexible loop combined with a rigid stem region provides DNA scaffolds with these high selectivity values. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening.

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    Kimberly Brown Dahlman

    Full Text Available There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  7. Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening. (United States)

    Dahlman, Kimberly Brown; Parker, Joel S; Shamu, Tambudzai; Hieronymus, Haley; Chapinski, Caren; Carver, Brett; Chang, Kenneth; Hannon, Gregory J; Sawyers, Charles L


    There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.

  8. Novel guanidinylated bioresponsive poly(amidoamines designed for short hairpin RNA delivery

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    Yu J


    Full Text Available Jiankun Yu,1 Jinmin Zhang,1 Haonan Xing,1 Yanping Sun,1 Zhen Yang,1 Tianzhi Yang,2 Cuifang Cai,1 Xiaoyun Zhao,3 Li Yang,1 Pingtian Ding1 1School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China; 2Department of Basic Pharmaceutical Sciences, School of Pharmacy, Husson University, Bangor, ME, USA; 3Department of Microbiology and Cell Biology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, China Abstract: Two different disulfide (SS-containing poly(amidoamine (PAA polymers were constructed using guanidino (Gua-containing monomers (ie, arginine [Arg] and agmatine [Agm] and N,N'-cystamine bisacrylamide (CBA by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the

  9. Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance. (United States)

    Chou, Shuli; Shao, Changxuan; Wang, Jiajun; Shan, Anshan; Xu, Lin; Dong, Na; Li, Zhongyu


    The β-hairpin structure has been proposed to exhibit potent antimicrobial properties with low cytotoxicity, thus, multiple β-hairpin structures have been proved to be highly stable in structures containing tightly packed hydrophobic cores. The aim of this study was to develop peptide-based synthetic strategies for generating short, but effective AMPs as inexpensive antimicrobial agents. Multiple-stranded β-hairpin peptides with the same β-hairpin unit, (WRXxRW)n where n=1, 2, 3, or 4 and Xx represent the turn sequence, were synthesized, and their potential as antimicrobial agents was evaluated. Owning to the tightly packed hydrophobic core and paired Trp of this multiple-stranded β-hairpin structure, all the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells (hRBCs), and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8μM against various tested bacteria. Not only that, but W2 also showed obvious synergy with streptomycin and chloramphenicol against Escherichia coli, and displayed synergy with ciprofloxacin against Staphylococcus aureus with the FICI values ⩽0.5. Fluorescence spectroscopy and electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Collectively, based on the multiple β-hairpin peptides, the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents. We successfully screened a peptide W2 ((WRPGRW)2) from a series of multiple-stranded β-hairpin antimicrobial peptides based on the "S-shaped" motif that induced the formation of a globular structure, and Trp zipper was used to replace the disulfide bonds to reduce the cost of production. This novel structure applied to AMPs improved cell selectivity and salt stability. The findings of this study will promote the development of peptide

  10. Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery (United States)

    Yu, Jiankun; Zhang, Jinmin; Xing, Haonan; Sun, Yanping; Yang, Zhen; Yang, Tianzhi; Cai, Cuifang; Zhao, Xiaoyun; Yang, Li; Ding, Pingtian


    Two different disulfide (SS)-containing poly(amidoamine) (PAA) polymers were constructed using guanidino (Gua)-containing monomers (ie, arginine [Arg] and agmatine [Agm]) and N,N′-cystamine bisacrylamide (CBA) by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA)-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the observed enhancement of transfection efficiency and low cytotoxicity. Overall, two newly synthesized Gua-SS-PAAs polymers demonstrated great potential to be used as shRNA carriers for gene-therapy applications. PMID:27994462

  11. Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors

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    GAN Hui-zhu; ZHENG De-ming; ZHANG Gui-zhen; ZHAO Ji-sheng; ZHANG Feng-chun; BU Li-sha; YANG Shao-juan; PIAO Song-lan; DU Zhen-wu; GAO Shen


    Background RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). Methods The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P<0.05 was considered statistically significant.Results In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P<0.05), 30.1% (P<0.01) (transient transfection) and 37.6 % (P<0.05), 28.0% (P<0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P<0.05), 54-fold (P<0.01) (transient transfection) and to 108-fold (P<0.05), 50-fold (P<0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. Conclusions shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer

  12. Transforming growth factor-β1 short hairpin RNA inhibits renal allograft fibrosis

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    YIN Zhi-kang; WU Xiao-hou; XIA Yu-guo; LUO Chun-li


    Background Transforming growth factor-β1 (TGF-β1) is known to be a key fibrogenic cytokine in a number of chronic fibrotic diseases, including chronic allograft nephropathy. We examined the effects of inhibition of TGF-β1 expression by RNA interference on renal allograft fibrosis, and explored the mechanisms responsible for these effects.Methods A Sprague-Dawley-to-Wistar rat model of accelerated kidney transplant fibrosis was used. Sixty recipient adult Wistar rats were randomly divided into four groups: group T (sham-operated group), group T (plasmid-transfected group), group H (control plasmid group), and group Y (transplant only group). Rats in group T were transfected with 200μg of TGF-β1 short hairpin RNA (shRNA). Reverse transcription-polymerase chain reaction and Western blotting were used to examine the expression of TGF-β1, Smad3/7, E-cadherin, and type I collagen. The distribution of type I collagen was measured by immunohistochemistry. The pathologic changes and extent of fibrosis were assessed by hematoxylin and eosin and Masson staining. E-cadherin and α-smooth muscle actin immunohistochemical staining were used to label tubular epithelial cells and fibroblasts, respectively.Results Plasmid transfection significantly inhibited the expression of TGF-β1, as well as that of its target gene, type I collagen (P <0.05 and P <0.01, respectively). In addition, the degree of fibrosis was mild, and its development was delayed in plasmid-transfected rats. In contrast, TGF-β1-shRNA transfection maintained the expression of E-cadherin in tubular epithelial cells while it inhibited the transformation from epithelial cells to fibroblasts. Blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P <0.05 and P<0.01, respectively).Conclusions This study suggests that transfection of a TGF-β1-shRNA plasmid could inhibit the fibrosis of renal allografts. The mechanism may be associated with the downregulation

  13. Is TNF-a-targeted short hairpin RNA (shRNA) a novel potential therapeutic tool in psoriasis treatment?

    DEFF Research Database (Denmark)

    Stenderup, Karin; Jakobsen, Maria; Rosada, Cecilia


      TNF-α is a well known target in psoriasis treatment and biological treatments targeting TNF-a are already clinically used against psoriasis and psoriasis arthritis. Attention is however given to a novel therapeutic tool: RNA interference that controls gene silencing. This study investigates...... the efficiency of targeting TNF-a with specific short hairpin RNA (shRNA) and explores its potential in treating psoriasis. ShRNAs targeting human TNF-α mRNA were generated. Their efficiency in down-regulating TNF-a protein expression was evaluated using a Renilla luciferase screening-assay and a transient co......-transfection assay. The expression cassette encoding the most efficient TNF-a shRNA was inserted into a lentiviral vector, allowing proficient gene delivery. The lentiviral vector is integrated into the host genome and establishes life-long infection and persistent shRNA expression. The lentiviral vector expressing...

  14. RNAi Codex: a portal/database for short-hairpin RNA (shRNA) gene-silencing constructs. (United States)

    Olson, A; Sheth, N; Lee, J S; Hannon, G; Sachidanandam, R


    Use of RNA interference (RNAi) in forward genetic screens is proliferating. Currently, short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) are being used to silence genes to tease out functional information. It is becoming easier to harness RNAi to silence specific genes, owing to the development of libraries of readymade shRNA and siRNA gene-silencing constructs by using a variety of sources. RNAi Codex, which consists of a database of shRNA related information and an associated website, has been developed as a portal for publicly available shRNA resources and is accessible at RNAi Codex currently holds data from the Hannon-Elledge shRNA library and allows the use of biologist-friendly gene names to access information on shRNA constructs that can silence the gene of interest. It is designed to hold user-contributed annotations and publications for each construct, as and when such data become available. We will describe features of RNAi Codex and explain the use of the tool.

  15. Inhibition of acid-induced apoptosis by targeting ASIC1a mRNA with short hairpin RNA

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    Xie-chuan WENG; Jian-quan ZHENG; Qing-e JIN; Xiao-yun MA


    Aim: To study the role of acid-sensing ion channel (ASIC) la in the cell death and apoptosis induced by extracellular acid in C6 glioma cells. Methods: The stable ASICla-silenced C6 cell line, built with RNA interference technology, were con-firmed by RT-PCR and Western blot analysis. The cell viability following acid exposure was analyzed with lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The apoptotic cells dyed with Annexin-V and propidium iodide were measured with a flow cytometer, while the changes of cell cycle were also assayed. Results: The downregulation of ASIC 1 a proteins by stable transfection of short hairpin RNA decreased the cell death percentage and increased cell viability following acid exposure with LDH and the MTT assay. The rate of apoptosis was lower in the ASIC la-silenced cell line than that in the wild-type C6 cell line. The percentage of sub-G0 cells was lower in the ASICla-silenced C6 cells than that in the wild-type cells. Conclusion: Extracellular acid induced cell death and apoptosis viaASICla mechanisms in the C6 glioma cells.

  16. Knockdown of cellular RNA helicase DDX3 by short hairpin RNAs suppresses HIV-1 viral replication without inducing apoptosis. (United States)

    Ishaq, Musarat; Hu, Jiajie; Wu, Xiaoyun; Fu, Qiong; Yang, Yalin; Liu, Qingzhen; Guo, Deyin


    The targeting of a cellular co-factor, rather than the HIV-1-specific RNAs, by small interfering RNAs holds promise as the rapid mutational ability of the HIV-1 genome may obviate the potential clinical use of RNAi against this virus. The DEAD-box RNA helicase DDX3 is an essential Rev co-factor in the CRM1-Rev-RRE complex that promotes the export of unspliced and single-spliced HIV-1 RNAs from the nucleus to cytoplasm. In this report, human DDX3 was targeted by specific short hairpin RNAs, and the down-regulation of cell's endogenous DDX3 suppressed the nuclear export of unspliced HIV-1 RNAs but did not affect the cell viability. We further showed that the knockdown of cellular DDX3 could effectively inhibit the replication of HIV-1. Therefore, the current results suggest that the RNA helicase DDX3 may become a potential target by RNAi for future genetic therapy of HIV/AIDS.

  17. Transiently Expressed Short Hairpin RNA Targeting 126 kDa Protein of Tobacco Mosaic Virus Interferes with Virus Infection

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    Ming-Min ZHAO; De-Rong AN; Jian ZHAO; Guang-Hua HUANG; Zu-Hua HE; Jiang-Ye CHEN


    RNA interference (RNAi) silences gene expression by guiding mRNA degradation in asequence-specific fashion. Small interfering RNA (siRNA), an intermediate of the RNAi pathway, has been shown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells. Here, we report that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) could inhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-associated 126 kDa protein in intact plant tissue. Our results indicate that transiently expressed shRNA efficiently interfered with TMV infection. The interference observed is sequence-specific, and time- and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumber mosaic virus (CMV), an unrelated tobamovirus. In order to interfere with TMV accumulation in tobacco leaves, it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation. Our results support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expression could be employed as a potent antiviral treatment in plants.

  18. RNA interference by expression of short hairpin RNAs suppresses bcl-xL gene expression in nasopharyngeal carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Fang LIU; Cheng-wei HE; Yue-fei ZHANG; Ke-yuan ZHOU


    Aim: To evaluate a new plasmid mediated RNA interference (RNAi) system and investigate whether knock-down of bcl-xL by short hairpin RNA (shRNA) can induce apoptosis of human nasopharyngeal carcinoma (NPC) cell line CNE-2Z in vitro. Methods: The plasmid containing mU6 promoter was subcloned to yield the pmU6 plasmid, recombinant plasmid expressing shRNA targeting bcl-xL gene was designed and constructed, and were co-transfected cells with green fluorescence protein expressing plasmid. Flow cytometry was used to evaluate transfection efficiency, and RT-PCR and Western blot were applied to analyze bcl-xL mRNA and protein levels, respectively. Results: The shRNA expressed by the recombi nant plasmid efficiently suppressed bcl-xL gene expression and induced apoptosis .of NPC cells in vitro. Conclusion: The recombinant plasmid can sufficiently mediate RNAi in CNE-2Z cells, and knock-down of the bcl-xL expression by shRNA significantly induced apoptosis in CNE-2Z cells. The results suggest this new system, mediated RNAi can be used as a tool for the study of gene function and gene therapy.

  19. P19 of Tomato Bushy Stunt Virus Suppresses RNA Silencing Induced by Short Hairpin RNA in Mammal Cells

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    Wei-xian CHEN; Juan CHEN; Zhen-zhen ZHANG; Ai-long HUANG


    To counteract the immune system in parasitic hosts, some viruses encode proteins to suppress the RNA interference (RNAi) effect. In this report, we established two RNAi systems to be easily observed with strong and obvious effect. The function of the P19 of tomato bushy stunt virus, which suppresses RNAi in mammal cells, was then studied using these two systems. Short hairpin RNAs targeting green fluorescence protein (pshRNA-GFP) and firefly luciferase (pshRNA-luc) were designed and inserted into a eukaryotic transcriptional vector pTZU6+1, respectively. The shRNA expressing vectors were co-transfected with plasmids containing the target gene with or without P19. The GFP expression level was assayed by fluorescence microscopy, Western blotting and RT-PCR. The luciferase expression level was analyzed by the dual-luciferase assay system. pshRNA designed in this study down-regulated the target gene specifically and efficiently, with a decrease of expression of both genes of about 70%, respectively. When P19 was introduced into the RNAi systems, the expression of both GFP and the luciferase were mostly recovered compared with the control groups. The RNAi systems of GFP and luciferase were constructed successfully, demonstrating that P19 of tomato bushy stunt virus has the ability to counteract the RNAi effect induced by shRNA in mammal cells.

  20. Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.

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    Kevin J Paik

    Full Text Available The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

  1. A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr. (United States)

    Kang, Shin-Young; Kim, Yeon-Gu; Lee, Hong Weon; Lee, Eun Gyo


    Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

  2. RNA interference of influenza A virus replication by microRNA-adapted lentiviral loop short hairpin RNA. (United States)

    Xu, Fang; Liu, Guanqun; Liu, Qiang; Zhou, Yan


    Limitations of the current vaccines and antivirals against influenza A virus (IAV) pandemic underscore the urgent need for developing novel anti-influenza strategies. RNA interference (RNAi) induced by small interfering RNA (siRNA) has become a powerful new means to inhibit viral infection in a gene-specific manner. However, the efficacy of the siRNA delivery platform and the relatively high cost of administration have hindered widespread application of siRNA. In this study, we developed a microRNA (miRNA)-30-based lentivirus delivery system by embedding a synthetic short hairpin RNA (shRNA) stem into the context of endogenous precursor of miRNA-30 (shRNAmir) to express a silencer of the influenza gene. We showed that the miRNA-based lentivirus vector was able to express and process a single nucleoprotein (NP)-targeting shRNAmir, which could potently inhibit IAV replication. We further showed that miRNA-based lentivirus vector carrying tandemly linked NP and polymerase PB1 shRNAmirs could express and process double shRNAmirs. Despite the relatively low levels of NP and PB1 miRNAs produced in the stably transduced cells, the combination of two miRNAs exerted a great degree of inhibition on influenza infection. Given the advantage of combinatorial RNAi in preventing emergence of mutant virus, miRNA-based lentiviral vectors are valuable tools for anitiviral activities. To the best of our knowledge, this is the first study demonstrating that a miRNA-based RNAi strategy can be applied for better control of influenza virus infection.

  3. Apoptosis induced by short hairpin RNA-mediated STAT6 gene silencing in human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ming-sheng; ZHOU Yun-feng; ZHANG Wen-jie; ZHANG Xiao-lian; PAN Qin; JI Xue-mei; LUO Zhi-guo; WU Jian-ping


    Background The relationship between signal transduction and tumors has become one of the foci in cancer research. Signal transducer and activator of the transcription 6 (STAT6) signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells (HT-29 cells) and the possible mechanism of Stat6 by RNA interference techniques.Methods Four eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the STAT6gene were designed and generated by molecular biological technology. The plasmid vectors were transfected into HT-29 cells by cation liposomes to block the Stat6 signaling pathway. The expressions of STAT6 mRNA and phosph-Stat6 protein were detected by the reverse transcriptase polymerase chain reaction (RT-PCR) method and flow cytometry respectively to screen the most effective shRNA at 72 hours after transfection. The apoptosis condition of the cells in which the expression of the STAT6 gene had been interfered was analyzed by flow cytometry and confocal microscopy. Both mRNA and protein expression of B cell lymphoma-2 (Bcl-2) and Bax were detected by RT-PCR and western blotting.Results Two effective eukaryotic expression plasmid vectors of shRNA specific for the STAT6 gene were generated successfully. One can reduce the expression of the STAT6 gene by 82.4% and the other by 56.8%(P<0.01). The apoptotic rate of colon cancer cells in which STAT6 gene expression had been interfered was significantly higher than that in controlled colon cancer cells (P<0.01). In the cells in which the Stat6 signaling pathway was blocked, the levels of mRNA and protein Bcl-2 were significantly decreased, whereas those of Bax were significantly increased (P<0.01).Conclusions The Stat6 signaling pathway can inhibit apoptosis in human colon cancer cells. The subsequent disorder of

  4. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

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    Robert J Scarborough


    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  5. Short hairpin RNA targeting insulin-like growth factor binding protein-3 restores the bioavailability of insulin-like growth factor-1 in diabetic rats

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    Zhang-Yan Zhou


    Full Text Available ABSTRACT Purpose To investigate whether intracavernosal injection of short hairpin RNA for IGFBP-3 could improve erectile function in streptozotocin-induced diabetic rats. Materials and methods After 12 weeks of IGFBP-3 short hairpin RNA injection treatment, intracavernous pressure responses to electrical stimulation of cavernous nerves were evaluated. The expression of IGFBP-3 and IGF-1 at mRNA and protein levels were detected by quantitative real-time PCR analysis and Western blot, respectively. The concentration of cavernous cyclic guanosine monophosphate was detected by enzyme-linked immunosorbent assay. Results At 12 weeks after intracavernous administration of IGFBP-3 shRNA, the cavernosal pressure was significantly increased in response to the cavernous nerves stimulation compared to the diabetic group (P<0.05. Cavernous IGFBP-3 expression at both mRNA and protein levels was significantly inhibited. At the same time, cavernous IGF-1 expression was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01. Cavernous cyclic guanosine monophosphate concentration was significantly increased in the IGFBP-3 shRNA treatment group compared to the diabetic group (P<0.01. Conclusions Gene transfer of IGFBP-3 shRNA could improve erectile function via the restoration of cavernous IGF-1 bioavailability and an increase of cavernous cGMP concentration in the pathogenesis of erectile dysfunction in streptozotocin-induced diabetic rats.

  6. Short hairpin RNA targeting NP mRNA inhibiting Newcastle disease virus production and other viral structural mRNA transcription. (United States)

    Yue, Hua; Deng, Shu; Yang, Fa-Long; Li, Ding-Fei; Fu, An-Jing; Yang, Fan; Tang, Cheng


    Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiological agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry. Better understanding of the molecular basis of viral structural genes involved with production should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here we show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding mRNA but also P, HN, F, M gene mRNA. The findings reveal that newly synthesized NP mRNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

  7. Inhibition of hepatitis C virus RNA replication by short hairpin RNA synthesized by T7 RNA polymerase in hepatitis C virus subgenomic replicons. (United States)

    Hamazaki, Hiroyuki; Ujino, Saneyuki; Miyano-Kurosaki, Naoko; Shimotohno, Kunitada; Takaku, Hiroshi


    RNA interference (RNAi) is a cellular process that induces gene silencing by which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. Here, to test the RNAi method for blocking hepatitis C virus (HCV) RNA replication, we created four short hairpin RNAs (shRNAs) targeting the HCV internal ribosome entry site/Core gene transcript using T7 RNA polymerase. shRNA suppressed the replication of HCV RNA in the HCV replicon. On the other hand, short interfering RNAs synthesized using the T7 RNA polymerase system trigger a potent induction of interferon-alpha and -beta in a variety of cells. We examined whether the shRNAs synthesized using the T7 RNA polymerase system activated double-stranded RNA-dependent protein kinase, 2'-5' oligoadenylate synthetase, or interferon-regulatory factor-3. Our results demonstrated that the T7-transcribed shRNA did not activate these proteins in Huh-7 cells and the HCV replicon. These shRNAs are a promising new strategy for anti-HCV gene therapeutics.

  8. Design and Construction of Shrimp Antiviral DNA Vaccines Expressing Long and Short Hairpins for Protection by RNA Interference. (United States)

    Chaudhari, Aparna; Pathakota, Gireesh-Babu; Annam, Pavan-Kumar


    DNA vaccines present the aquaculture industry with an effective and economically viable method of controlling viral pathogens that drastically affect productivity. Since specific immune response is rudimentary in invertebrates, the presence of RNA interference (RNAi) pathway in shrimps provides a promising new approach to vaccination. Plasmid DNA vaccines that express short or long double stranded RNA in vivo have shown protection against viral diseases. The design, construction and considerations for preparing such vaccines are discussed.

  9. Short hairpin RNA targeting 2B gene of coxsackievirus B3 exhibits potential antiviral effects both in vitro and in vivo

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    Yao Hailan


    Full Text Available Abstract Background Coxsackievirus B3 is an important infectious agent of viral myocarditis, pancreatitis and aseptic meningitis, but there are no specific antiviral therapeutic reagents in clinical use. RNA interference-based technology has been developed to prevent the viral infection. Methods To evaluate the impact of RNA interference on viral replication, cytopathogenicity and animal survival, short hairpin RNAs targeting the viral 2B region (shRNA-2B expressed by a recombinant vector (pGCL-2B or a recombinant lentivirus (Lenti-2B were tansfected in HeLa cells or transduced in mice infected with CVB3. Results ShRNA-2B exhibited a significant effect on inhibition of viral production in HeLa cells. Furthermore, shRNA-2B improved mouse survival rate, reduced the viral tissues titers and attenuated tissue damage compared with those of the shRNA-NC treated control group. Lenti-2B displayed more effective role in inhibition of viral replication than pGCL-2B in vivo. Conclusions Coxsackievirus B3 2B is an effective target of gene silencing against coxsackievirus B3 infection, suggesting that shRNA-2B is a potential agent for further development into a treatment for enterviral diseases.

  10. Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

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    Orit Wolstein


    Full Text Available Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC. CCR5-targeted shRNA (sh5 and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

  11. Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes. (United States)

    Gu, W; Putral, L; Hengst, K; Minto, K; Saunders, N A; Leggatt, G; McMillan, N A J


    In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag-/- mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

  12. Gene silencing of NR2B-containing NMDA receptor by intrathecal injection of short hairpin RNA reduces formalin-induced nociception in C57BL/6 mouse. (United States)

    Zhang, Rao-Xiang; Yan, Xue-Bin; Gu, Yong-Hong; Huang, Dong; Gan, Li; Han, Rui; Huang, Li-Hua


    Spinal NR2B-containing N-methyl-D-aspartate receptors (NR2B) play a critical role in the formation of central sensitization and persistent pain. Previous studies show that gene silencing of the spinal NR2B subunit by small interfering RNA (siRNA) could alleviate nociception in animals. The siRNA is a 19- to 23-nt RNA duplex, which can be synthesized in vitro or derived from short hairpin RNAs (shRNAs). In the present study, we investigated whether intrathecal injection of shRNAs targeting NR2B (GRIN2B shRNA) could affect nociception on formalin-induced pain in mice. Our results showed that intrathecal injection of GRIN2B shRNA could decrease NR2B mRNA and protein expression levels and hence effectively relieve formalin-induced nociception in mice, suggesting that intrathecal delivery of GRIN2B shRNA can be an efficient way to silence the target gene and provide new insights into the treatment of chronic pain.

  13. [Study on the effect of Klotho gene interferred by plasmid-mediated short hairpin RNA (shRNA) on sinoatrial node pacing channel gene]. (United States)

    Cai, Yingying; Wang, Han; Hou, Yanbin; Fang, Chenli; Tian, Peng; Wang, Guihua; Li, Lu; Deng, Juelin


    The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4 and HCN2) for studying sick sinus syndrome, with Klotho gene under the interference of Plasmid-mediated short hairpin RNA. Twenty-five C57BL/6J mice were divided into four groups, i. e, plasmid shRNA 24h group, plasmid shRNA 12h group, sodium chloride 24h group and sodium chloride 12h group. Plasmid shRNA 50microL (1microg/microL) and sodium chloride 50microl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group. After 12h or 24h respectively, all mice were executed and their sinoatrial node tissues were cut. The mRNA of Klotho, HCN4 and HCN2 gene were detected by RT-PCR. The results of RT-PCR showed that Klotho, HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval. The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

  14. Short hairpin RNA targeted to dihydrofolate reductase enhances the immunoglobulin G expression in gene-amplified stable Chinese hamster ovary cells. (United States)

    Wu, Suh-Chin; Hong, Willy W L; Liu, Jin-Hwang


    The dihydrofolate reductase (dhfr)/methotrexate (MTX) selection is a common method to conduct gene amplification in stable clones of Chinese hamster ovary (CHO) cells. We previously reported the use of a short hairpin RNA (shRNA) vector targeted to the dhfr gene resulted in improving the intracellular antigen expression in gene-amplified stable CHO cells [Hong, W.W., Wu, S.C., 2007. A novel RNA silencing vector to improve antigen expression and stability in Chinese hamster ovary cells. Vaccine 25 (20), 4103-4111]. Here we investigated the use of the dhfr-targeted shRNA vector for immunoglobulin G (IgG) expression in gene-amplified stable CHO cells. With the use of the dhfr-targeted shRNA vector, the gene-amplified CHO/dhFr(-) cells were found to increase IgG expression at 1.0 microM MTX by more than 100% and to improve the genomic stability of IgG expression in MTX-free cultures by approximately 30%. The use of the dhfr-targeted shRNA vector can enhance the IgG expression in the gene-amplified stable CHO cells and uphold the IgG expression in MTX-free cultures. Utilizing the dhfr-targeted shRNA vector may provide an alternative way to maneuver CHO cell factories for IgG production in cultures.

  15. Enhanced cardioprotective effects mediated by plasmid containing the short-hairpin RNA of angiotensin converting enzyme with a biodegradable hydrogel after myocardial infarction. (United States)

    Wan, Wei-Guo; Jiang, Xue-Jun; Li, Xiao-Yan; Zhang, Cui; Yi, Xin; Ren, Shan; Zhang, Xian-Zheng


    The expression of foreign gene was enhanced and prolonged by sustained releasing a target gene to cells from biodegradable dextran-poly(e-caprolactone)-2-hydroxylethylmethacrylate-poly(N-isopropylacrylamide) (Dex-PCL-HEMA/PNIPAAm) hydrogel in vitro. Moreover, we have demonstrated that injection of the same hydrogel improved post-infarct ventricular remodeling. Therefore, we hypothesized that intramyocardial injection of plasmid containing the short-hairpin RNA (shRNA) of angiotensin converting enzyme (ACE) with the same hydrogel enhances the cardioprotective effects superior to either alone or after rat myocardial infarction (MI). In this study, equal volume of phosphate-buffered solution (PBS), 10 μg ACE-shRNA plasmids, hydrogel containing 10 μg negative control ACE-shRNA plasmids and hydrogel containing 10 μg ACE-shRNA plasmids were shortly injected into the infarct area of rats after MI, respectively. We found that ACE-shRNA plasmid-loaded hydrogel extended the duration of gene expression in vivo. Moreover, it was shown that direct intramyocardial injection of ACE-shRNA plasmid-loaded hydrogel significantly decreased the expression of local ACE expression, inhibited cell apoptosis, reduced infarct size, and improved cardiac function compared with the injection of either alone 30 days after MI in rats. These results suggest that injection of ACE-shRNA plasmid-loaded hydrogel into impaired myocardium obtains more cardioprotective effects than either alone in rat with MI by prolonging the gene silencing of ACE. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3452-3458, 2014.

  16. Adenovirus delivered short hairpin RNA targeting a conserved site in the 5' non-translated region inhibits all four serotypes of dengue viruses.

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    Anil Babu Korrapati

    Full Text Available BACKGROUND: Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs. This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi to attenuate DENV replication may offer one approach to dengue therapy. METHODOLOGY/PRINCIPAL FINDINGS: We screened the non-translated regions (NTRs of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5' NTR that maps to the 5' upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5 vector to deliver a short-hairpin RNA (shRNA targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. CONCLUSION/SIGNIFICANCE: The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.

  17. Phosphodiesterase 5a Inhibition with Adenoviral Short Hairpin RNA Benefits Infarcted Heart Partially through Activation of Akt Signaling Pathway and Reduction of Inflammatory Cytokines.

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    Longhu Li

    Full Text Available Treatment with short hairpin RNA (shRNA interference therapy targeting phosphodiesterase 5a after myocardial infarction (MI has been shown to mitigate post-MI heart failure. We investigated the mechanisms that underpin the beneficial effects of PDE5a inhibition through shRNA on post-MI heart failure.An adenoviral vector with an shRNA sequence inserted was adopted for the inhibition of phosphodiesterase 5a (Ad-shPDE5a in vivo and in vitro. Myocardial infarction (MI was induced in male C57BL/6J mice by left coronary artery ligation, and immediately after that, the Ad-shPDE5a was injected intramyocardially around the MI region and border areas.Four weeks post-MI, the Ad-shPDE5a-treated mice showed significant mitigation of the left ventricular (LV dilatation and dysfunction compared to control mice. Infarction size and fibrosis were also significantly reduced in Ad-shPDE5a-treated mice. Additionally, Ad-shPDE5a treatment decreased the MI-induced inflammatory cytokines interleukin (IL-1β, IL-6, tumor necrosis factor-α, and transforming growth factor-β1, which was confirmed in vitro in Ad-shPDE5a transfected myofibroblasts cultured under oxygen glucose deprivation. Finally, Ad-shPDE5a treatment was found to activate the myocardial Akt signaling pathway in both in vivo and in vitro experiments.These findings indicate that PDE5a inhibition by Ad-shPDE5a via the Akt signal pathway could be of significant value in the design of future therapeutics for post-MI heart failure.

  18. Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

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    Gene-Errol E Ringpis

    Full Text Available Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA. However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR. Here, we report that human CD4(+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+ T-cells ex vivo. Furthermore, levels of gene-marked CD4(+ T-cells in peripheral blood increased despite systemic infection with either

  19. Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model. (United States)

    Andey, Terrick; Marepally, Srujan; Patel, Apurva; Jackson, Tanise; Sarkar, Shubhashish; O'Connell, Malaney; Reddy, Rakesh C; Chellappan, Srikumar; Singh, Pomila; Singh, Mandip


    The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199nm) with a slight positive charge (21.82mV); CLG-shAnx2 was of similar size (217nm) with decreased charge (12.11mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2h resulting in a 6

  20. Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes. (United States)

    Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E


    Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

  1. 靶向人原癌基因c-fos的shRNA表达质粒的构建及鉴定%Construction and Identification of Short Hairpin RNA Expression Vector Targeting to Human Proto-oncogene c-fos

    Institute of Scientific and Technical Information of China (English)

    景志杰; 刘田福; 师锐赞


    Objective To construct and identify a short hairpin RNA (shRNA) expression vector targeting to human protooncogene c-fos. Methods Recombinant shRNA expression vector Psilencer3.l-sic-fos targeting to human c-fos gene was constructed and transfected to breast cancer MCF-7 cells. The expression of c-fos mRNA in stably transfected cell was determined by RT-PCR.Results PCR, restriction analysis and sequencing proved that recombinant plasmid Psilencer3. 1-sic-fos was constructed correctly.The recombinant plasmid inhibited the expression of c-fos gene at mRNA level in MCF-7 cells significantly. Conclusion The recombinant shRNA expression vector for human c-fos gene was successfully constructed, which provided a technical tool for further study on the role of c-fos gene in genesis and progress of tumors.%目的 构建靶向人原癌基因c-los的短发夹RNA(Short hairpin RNA,shRNA)重组真核表达质粒,并进行鉴定.方法 构建靶向人c-fos基因的shRNA重组表达质粒Psilencer 3.1-sic-fos,转染乳腺癌MCF-7细胞,采用RT-PCR法检测稳定转染细胞中c-fos基因mRNA的表达.结果 重组表达质粒Psilencer 3.1-sic-fos经PCR、双酶切及测序证明构建正确;重组表达质粒在mRNA水平明显抑制了MCF-7细胞c-fos基因的表达(P<0.05).结论 已成功构建了人c-fos基因shRNA重组表达质粒,为深入研究c-fos基因在肿瘤发生、发展过程中的作用提供了技术手段.

  2. Plasmid-based generation of induced neural stem cells from adult human fibroblasts

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    Philipp Capetian


    Full Text Available Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72% and glial cells (9% astrocytes, 6% oligodendrocytes. Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts. Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside

  3. Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene. (United States)

    Zhang, Xiuchun; Sato, Shirley; Ye, Xiaohong; Dorrance, Anne E; Morris, T Jack; Clemente, Thomas E; Qu, Feng


    Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

  4. Plasmids expressing interleukin-10 short hairpin RNA mediate IL-10 knockdown and enhance tumor necrosis factor alpha and interferon gamma expressions in response to porcine reproductive and respiratory syndrome virus. (United States)

    Charerntantanakul, Wasin; Kasinrerk, Watchara


    Porcine reproductive and respiratory syndrome virus (PRRSV) has been suggested to exploit interleukin-10 (IL-10) to suppress immune defense of infected pigs. The present study constructed plasmids encoding selected short hairpin RNA specific to porcine IL-10 mRNA (pIL-10sh) to knockdown IL-10 transcription and investigated the suppressive effect of PRRSV-induced IL-10 on various immune marker expressions. Naïve blood monocytes from eight PRRSV-seronegative pigs were transfected with pIL-10sh and pNeg (plasmid vector) prior to PRRSV inoculation and subsequent lipopolysaccharide (LPS) stimulation. The mRNA expressions of IL-10, IL-1β, IL-12p40, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), transforming growth factor beta (TGFβ), CD80, and CD86 were evaluated by real-time PCR. The IL-10, TNFα, and IFNγ protein productions were determined by ELISA. Compared with non-transfected monocyte control, transfection with selected pIL-10sh (pIL-10sh1), but not other pIL-10sh nor pNeg, significantly reduced IL-10 expression and significantly enhanced TNFα and IFNγ expressions. Slight increases in IL-1β, IL-12p40, CD80, and CD86 expressions were also observed. Neither pIL-10sh1 nor pNeg transfection affected TGFβ expression. Our results indicate that PRRSV does exploit IL-10 to suppress the expressions of pro-inflammatory cytokines, mainly TNFα and IFNγ, and co-stimulatory molecules, CD80 and CD86.

  5. 干扰Fas受体表达的串联shRNA表达载体的构建%Construction of recombinant plasmid series with two Fas-targeted short hairpin RNAs

    Institute of Scientific and Technical Information of China (English)

    刘明社; 王兰; 赵中夫; 张国英; 张芸; 封江南


    目的:利用pEGFP6-1和pGenesil-1质粒构建针对Fas的2个短发夹RNA(short hairpin RNA,shRNA)串联表达的载体.方法:设计表达2个shRNA结构的互补DNA序列,经退火成双链,分别克隆至带有U6启动子的质粒载体pEGFP6-1和pGenesil-1中,构建重组质粒,转化大肠杆菌DH5α菌株,扩增,提取质粒,酶切鉴定后测序分析.分别用SacI+SalI双酶切重组质粒,胶回收酶切pEGFP6-1-siFas1大片段和酶切pGenesil-1-siFas2小片段,T4 DNA Ligase连接酶切大、小片段,得到pEGFP6-1-siFas1+siFas2重组质粒.转化感受态细胞DH5α,扩增,提取串联重组质粒进行酶切鉴定.结果:成功构建靶向Fas的2个shRNA串联重组质粒载体pEGFP6-1-siFas1+siFas2.酶切鉴定和测序分析重组质粒,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功.结论:表达2个靶向Fas的shRNA表达框成功构建在一个重组质粒载体上.

  6. Short-hairpin RNA-induced suppression of adenine nucleotide translocase-2 in breast cancer cells restores their susceptibility to TRAIL-induced apoptosis by activating JNK and modulating TRAIL receptor expression

    Directory of Open Access Journals (Sweden)

    Kim Chul-Woo


    Full Text Available Abstract Background Tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL; apo2 ligand induces apoptosis in cancer cells but has little effect on normal cells. However, many cancer cell types are resistant to TRAIL-induced apoptosis, limiting the clinical utility of TRAIL as an anti-cancer agent. We previously reported that the suppression of adenine nucleotide translocase-2 (ANT2 by short-hairpin RNA (shRNA induces apoptosis of breast cancer cells, which frequently express high levels of ANT2. In the present study, we examined the effect of RNA shRNA-induced suppression of ANT2 on the resistance of breast cancer cells to TRAIL-induced apoptosis in vitro and in vivo. Results ANT2 shRNA treatment sensitized MCF7, T47 D, and BT474 cells to TRAIL-induced apoptosis by up-regulating the expression of TRAIL death receptors 4 and 5 (DR4 and DR5 and down-regulating the TRAIL decoy receptor 2 (DcR2. In MCF7 cells, ANT2 knockdown activated the stress kinase c-Jun N-terminal kinase (JNK, subsequently stabilizing and increasing the transcriptional activity of p53 by phosphorylating it at Thr81; it also enhanced the expression and activity of DNA methyltransferase 1 (DNMT1. ANT2 shRNA-induced overexpression of DR4/DR5 and TRAIL sensitization were blocked by a p53 inhibitor, suggesting that p53 activation plays an important role in the transcriptional up-regulation of DR4/DR5. However, ANT2 knockdown also up-regulated DR4/DR5 in the p53-mutant cell lines BT474 and T47 D. In MCF7 cells, ANT2 shRNA treatment led to DcR2 promoter methylation and concomitant down-regulation of DcR2 expression, consistent with the observed activation of DNMT1. Treatment of the cells with a demethylating agent or JNK inhibitor prevented the ANT2 shRNA-induced down-regulation of DcR2 and activation of both p53 and DNMT1. In in vivo experiments using nude mice, ANT2 shRNA caused TRAIL-resistant MCF7 xenografts to undergo TRAIL-induced cell death, up-regulated DR4/DR5

  7. Construction of a short hairpin RNA interference lentiviral vector of SIRT1 gene and detection of interference effect%SIRT1基因shRNA慢病毒载体构建及干扰效应检测

    Institute of Scientific and Technical Information of China (English)

    邓昭玲; 王彦; 杜利清; 刘强; 赵辉; 陈乃耀


    BACKGROUND: SIRT1 gene plays a very important role in cell energy metabolism, apoptosis and ageing, while other studies have found that SIRT1 may play a regulatory role in the inflammatory response. OBJECTIVE: To construct short hairpin RNA interference lentiviral vector of SIRT1 gene and to evaluate their inhibitory effect in THP-1 cells. METHODS: The SIRI1 target specific oligonucleotide sequence was designed, synthesized and connected into the pGCSIL-GFP vector digested by Age I and EcoR I 293T cells were packaged to produce the lentivirus, and then transfected with THP-1 cells. The inhibitory effect on SIRT1 gene was tested by the real-time quantitative PCR and Western Blot. RESULTS AND CONCLUSION: The PCR and DNA sequencing of positive clones demonstrated that the lentivirus shRNA vector of SIRT1 gene was constructed successfully. The real-time quantitative PCR and Western Blot confirmed that the vector could suppress the expression level of SIRT1 at mRNA and protein levels.%背景:SIRT1基因在细胞能量代谢、凋亡及衰老过程中发挥极重要的作用,另有研究发现SIRT1可能在炎症反应方面起着调控作用.目的:构建SIRT1特异性的shRNA慢病毒载体,并初步检测其对THP-1细胞SIRT1基因的抑制效应.方法:设计SIRT1靶点特异性的寡核苷酸序列,连接到经Age Ⅰ和EcoR Ⅰ酶切线性化的pGCSIL-GFP载体,包装293T细胞产生慢病毒,再转染THP-1细胞,通过实时荧光定量PCR实验及Western Blot检测对SIRT1靶基因的抑制情况.结果与结论:阳性克隆PCR及测序证实SIRT1基因shRNA慢病毒载体构建成功,实时荧光定量PCR及Western Blot检测该载体在mRNA和蛋白水平能抑制THP-1细胞的SIRT1表达.

  8. General combinatorics of RNA hairpins and cloverleaves. (United States)

    Liao, Bo; Wang, Tian-ming


    For an abstract single-strand RNA, a combinatorial analysis is given for two important structures, hairpins and cloverleaves. The total number of hairpins and cloverleaves of a given length with minimal hairpin loop length m(m > 0) and with minimal stack length l(l > 0) is computed, under the assumption that all base pairs can occur.

  9. Inhibition of HIV-1 in Vitro by Combination of Vpr and Tat Specific Short Hairpin RNA via Lentiviral Vectors%慢病毒介导双shRNA靶向于HIV的抑制作用研究

    Institute of Scientific and Technical Information of China (English)

    郝彦哲; 滕智平; 杨怡姝; 孙晓娜; 马晶; 金孝华; 曾毅


    RNA干扰可以有效地抑制特定基因的表达,在HIV基因治疗中成为一种重要的方法.为了防止病毒逃逸株的出现,本研究分别构建了靶向于HIV-1调控蛋白基因vpr和tat的shRNA重组慢病毒载体以及含有这两种shRNA的双表达重组慢病毒载体,分别由U6和H1两种启动子启动,通过与靶向基因tat和vpr表达质粒在293T细胞中进行共转染,采用荧光定量PCR方法测定细胞内靶向基因的表达丰度,结果显示双表达shRNA的重组慢病毒载体对vpr和tat的表达抑制率分别可以达到89.20%和62.00%.与pNL4-3病毒包装质粒共转染,P24ELISA显示plvx-vpr-tat shRNA对病毒包装产量的抑制率可以达到99.05%,比单个shRNA的抑制率都要强,HIV-1 NL4-3体外对MT4细胞攻毒实验表明双shRNA重组慢病毒载体具有很强的抑制病毒复制的能力.%Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 pro-motor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively, with ratios of 89. 20% and 62. 00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the

  10. Single primer-mediated circular polymerase chain reaction for hairpin DNA cloning and plasmid editing. (United States)

    Huang, Jiansheng; Khan, Inamullah; Liu, Rui; Yang, Yan; Zhu, Naishuo


    We developed and validated a universal polymerase chain reaction (PCR) method, single primer circular (SPC)-PCR, using single primer to simultaneously insert and amplify a short hairpin sequence into a vector with a high success rate. In this method, the hairpin structure is divided into two parts and fused into a vector by PCR. Then, a single primer is used to cyclize the chimera into a mature short hairpin RNA (shRNA) expression vector. It is not biased by loop length or palindromic structures. Six hairpin DNAs with short 4-nucleotide loops were successfully cloned. Moreover, SPC-PCR was also applied to plasmid editing within 3 h with a success rate higher than 95%.

  11. Generation and Growth of Single Hairpin Vortices (United States)

    Haji-Haidari, Ahmad

    The behavior of selectively generated single hairpin vortices are examined within a laminar boundary layer environment over a range of Reynolds numbers, the hairpin vortices are experimentally generated by means of controlled fluid injection from a streamwise slot. Flow visualization using both dye and hydrogen bubble wire is employed in conjunction with hot film anemometry to investigate the growth characteristics and evolution of these single hairpin vortices. Qualitatively, it is established that hairpin vortices form by local destabilization at the interface between the low-speed fluid introduced through the slot and the higher speed boundary layer flow. Kinematical considerations of the hairpin vortex are established. It is observed that a hairpin vortex generally displays visualization and velocity signatures characteristic of those observed for a turbulent boundary layer. Hydrogen-bubble wire visualization results specifically indicate that hairpin vortices generate two purely turbulent-like flow patterns. The first is a low-speed streak pattern developing immediately adjacent to the surface due to surface interaction by the counter -rotating legs of the hairpin vortex; the second pattern is a turbulent pocket-like pattern farther removed from the surface. It is determined from the visualization data that hairpin vortices manifest the necessary flow characteristics which give rise to the regenerative and sustained process required for maintenance of turbulence. The regeneration and the growth process takes place through the formation of similar hairpin-like vortices by one of two means. The first is an inviscid lateral propagation of the initial disturbance which gives rise to outboard (subsidiary), vortices which cause the lateral spreading of the structure. A more complicated and eruptive process occurs by means of viscous-inviscid interactions which give rise to trailing vortices (secondary), which cause the streamwise elongation of the disturbance. A

  12. The membrane integration of a naturally occurring alpha-helical hairpin. (United States)

    Nagy, Akos; Turner, R James


    Helical hairpins, two closely spaced helical membrane spanning segments separated by a short surface turn, are thought to be common in integral membrane proteins. Here, we study the membrane integration of a naturally occurring helical hairpin from the secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1. This sequence is only slightly longer and significantly less hydrophobic than a previously identified minimal poly-leucine model hairpin structure. Using site directed mutagenesis we document the importance of the turn propensity of the amino acids in the intervening surface turn but, somewhat surprisingly, our results indicate that the formation of this natural hairpin apparently does not depend on specific helix-helix interactions. Our results suggest that helical hairpins may be formed quite readily from even minimally hydrophobic sequences separated by a short, sufficiently strong, turn signal, and that current methods for predicting integral membrane protein topology may miss many similar short helical hairpin sequences. Thus the occurrence of these structures may be much more common than presently thought.

  13. Electroanalytical determination of d(GCGAAGC) hairpin. (United States)

    Trnková, Libuse; Postbieglová, Irena; Holik, Miroslav


    Hairpins (or hairpin-like structures) may play a major role in expansion events of triplet repeat expansion diseases (X syndrome, Huntington's disease, Friedreich's ataxia). The d(GCGAAGC) fragment has been found in the replication origins of phage phiX 174 and herpes simplex virus, in a promoter region of an Escherichia coli heat-shock gene, and in rRNA genes. The paper deals with the application of electrochemical methods to the determination of the DNA heptamer-d(GCGAAGC) which forms very stable hairpin structure in aqueous solutions. On mercury electrodes, this hairpin provides voltammetric reduction signals of adenine and cytosine, and oxidation signals of guanine. Both signals have been studied by cyclic voltammetry (CV), linear sweep voltammetry (LSV), and elimination voltammetry with linear scan (EVLS) in dependence on pH, accumulation time, scan rate, and loop sequences. The EVLS in combination with the adsorptive stripping was employed to the determination of the detection limit (LD) of this mini-hairpin (2 nM). Multidimensional voltammetric data were worked up by Fourier Transform (FT) and for the first coefficient a confidence ellipse was calculated in order to drop out some outlier data. The same method was used also for detection limit determinations. The values of LD obtained by two approaches were compared.

  14. Excited singlet states of "hairpin" polyenes


    Froelich, Wolfgang; Dewey, Harry J.; Deger, Hans; Dick, Bernhard; Klingensmith, Kenneth A.; Puettmann, Wilhelm; Vogel, Emanuel; Hohlneicher, Georg; Michl, Josef


    The synthesis and UV-visible, polarized-fluorescence and MCD spectra of 6 U-shaped hairpin polyenes (e.g., I) are reported. Qual. arguments and results of p-electron calcns. permit the identification of 4 excited singlet states and their assignment to mixts. of singly and doubly excited configurations. The hairpin polyenes represent a link between the all-trans-polyenes on the 1 hand and the annulenes and acenes on the other; they have the topol. of the former and a geometry near that of the ...

  15. Ultrafast Unzipping of a Beta-Hairpin Peptide

    NARCIS (Netherlands)

    Zinth, W.; Schrader, T.E.; Schreier, W.J.; Koller, F.O.; Cordes, T.; Babitzki, G.; Denschlag, R.; Tavan, P.; Löweneck, M.; Dong, Shou-Liang; Moroder, L.; Renner, C.; Corkum, Paul; Jonas, David M.; Miller, R.J. Dwayne; Weiner, Andrew M.


    Light induced switching of a beta-hairpin structure is investigated by femtosecond IR spectroscopy. While the unzipping process comprises ultrafast kinetics and is finished within 1 ns, the folding into the hairpin structure is a much slower process.

  16. Destabilization of the TAR hairpin leads to extension of the polyA hairpin and inhibition of HIV-1 polyadenylation

    NARCIS (Netherlands)

    Vrolijk, M.M.; Harwig, A.; Berkhout, B.; Das, A.T.


    ABSTRACT: BACKGROUND: Two hairpin structures that are present at both the 5' and 3' end of the HIV-1 RNA genome have important functions in the viral life cycle. The TAR hairpin binds the viral Tat protein and is essential for Tat-mediated activation of transcription. The adjacent polyA hairpin

  17. Short (

    NARCIS (Netherlands)

    Telleman, Gerdien; den Hartog, Laurens


    Aim: This systematic review assessed the implant survival rate of short (<10 mm) dental implants installed in partially edentulous patients. A case report of a short implant in the posterior region have been added. Materials and methods: A search was conducted in the electronic databases of MEDLINE

  18. Short (

    NARCIS (Netherlands)

    Telleman, Gerdien; den Hartog, Laurens


    Aim: This systematic review assessed the implant survival rate of short (<10 mm) dental implants installed in partially edentulous patients. A case report of a short implant in the posterior region have been added. Materials and methods: A search was conducted in the electronic databases of MEDLINE

  19. Plasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    Yan-Bo Liu1; De-Qi Xu; Ling Zhang1; Ya-Xiong Guo; Li-Fang Gao; Xi-Chun Liu; Li-Juan Zhao; Bao-Feng Guo; Li-Jing Zhao; Xue-Jian Zhao


    Persistent activation of Survivin and its overexpression contribute to the formation,progression and metastasis of several different tumor types.Therefore,Survivin is an ideal target for RNA interference mediated-growth inhibition.Blockade of Survivin using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth.RNA interference does not fully ablate target gene expression,owing to the idiosyncrasies associated with shRNAs and their targets.To enhance the therapeutic efficacy of Survivin-specific shRNA,we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 (GRIM-19).Then,the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium (S.typhimurium) to treat prostate cancer in vitroand in vivo.We found that the co-expressed Survivin-specific shRNA and GRIM-19synergistically and more effectively inhibited prostate tumor proliferation and survival,when compared with treatment with either single agent alone in vitro and in vivo.This study has provided a novel cancer gene therapeutic approach for prostate cancer.

  20. Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines. (United States)

    Diebold, S S; Schulz, O; Alexopoulou, L; Leitner, W W; Flavell, R A; Reis e Sousa, C


    Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8 alpha(+) DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

  1. Evolution of outer membrane beta-barrels from an ancestral beta beta hairpin. (United States)

    Remmert, M; Biegert, A; Linke, D; Lupas, A N; Söding, J


    Outer membrane beta-barrels (OMBBs) are the major class of outer membrane proteins from Gram-negative bacteria, mitochondria, and plastids. Their transmembrane domains consist of 8-24 beta-strands forming a closed, barrel-shaped beta-sheet around a central pore. Despite their obvious structural regularity, evidence for an origin by duplication or for a common ancestry has not been found. We use three complementary approaches to show that all OMBBs from Gram-negative bacteria evolved from a single, ancestral beta beta hairpin. First, we link almost all families of known single-chain bacterial OMBBs with each other through transitive profile searches. Second, we identify a clear repeat signature in the sequences of many OMBBs in which the repeating sequence unit coincides with the structural beta beta hairpin repeat. Third, we show that the observed sequence similarity between OMBB hairpins cannot be explained by structural or membrane constraints on their sequences. The third approach addresses a longstanding problem in protein evolution: how to distinguish between a very remotely homologous relationship and the opposing scenario of "sequence convergence." The origin of a diverse group of proteins from a single hairpin module supports the hypothesis that, around the time of transition from the RNA to the protein world, proteins arose by amplification and recombination of short peptide modules that had previously evolved as cofactors of RNAs.

  2. Binding of DNA hairpins to an assembler-strand as part of a primordial translation device (United States)

    Baumann, Ulrich


    A crucial event in the process leading to the origin of life is the emergence of a simple translation device. To approach experimental realization of this device the binding ability of short DNA hairpins to complementary oligonucleotides fixed on a solid support was investigated. The binding is achieved by base pairing between the loop nucleotides of the hairpins containing different numbers of adenosine residues and oligothymidylates covalently linked to cellulose. The loop has to consist of at least five nucleotides to achieve binding. The exact number of established base pairs was determined in two ways. First, the elution temperatures of hairpins and those of oligoadenylates which had the length of the loop were compared. Secondly, the architecture of the loop was analyzed by means of the single-strand-specific nuclease from mung bean acting as structural probe. Onlyn-2 of n loop nucleotides of a hairpin are able to form base pairs. Therefore, a strong evidence for the formation of a triplet of base pairs between primeval tRNA and mRNA sufficient to stabilize the complex enzyme-free is given.

  3. Two-step cleavage of hairpin RNA with 5' overhangs by human DICER

    Directory of Open Access Journals (Sweden)

    Suzuki Harukazu


    Full Text Available Abstract Background DICER is an RNase III family endoribonuclease that processes precursor microRNAs (pre-miRNAs and long double-stranded RNAs, generating microRNA (miRNA duplexes and short interfering RNA duplexes with 20~23 nucleotides (nts in length. The typical form of pre-miRNA processed by the Drosha protein is a hairpin RNA with 2-nt 3' overhangs. On the other hand, production of mature miRNA from an endogenous hairpin RNA with 5' overhangs has also been reported, although the mechanism for this process is unknown. Results In this study, we show that human recombinant DICER protein (rDICER processes a hairpin RNA with 5' overhangs in vitro and generates an intermediate duplex with a 29 nt-5' strand and a 23 nt-3' strand, which was eventually cleaved into a canonical miRNA duplex via a two-step cleavage. The previously identified endogenous pre-miRNA with 5' overhangs, pre-mmu-mir-1982 RNA, is also determined to be a substrate of rDICER through the same two-step cleavage. Conclusions The two-step cleavage of a hairpin RNA with 5' overhangs shows that DICER releases double-stranded RNAs after the first cleavage and binds them again in the inverse direction for a second cleavage. These findings have implications for how DICER may be able to interact with or process differing precursor structures.

  4. Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication.

    Directory of Open Access Journals (Sweden)

    Shirley Lam

    Full Text Available BACKGROUND: Chikungunya virus (CHIKV is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. METHODS: Plasmid-based small hairpin RNA (shRNA was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: Taken together, these

  5. Tomographic PIV measurements of a regenerating hairpin vortex (United States)

    Sabatino, D. R.; Rossmann, T.


    The three-dimensional formation and regeneration of a hairpin vortex in a laminar boundary layer is studied in a free-surface water channel. The vortex is generated by fluid injection through a narrow slot into a laminar boundary layer (Re_{δ ^*} = 485) and recorded with tomographic particle image velocimetry. The swirling strength based on the λ _2 criterion shows that the hairpin initially forms at the upstream edge of the elongated ring vortex produced by the injection. The elongated ring vortex decays while the hairpin vortex strengthens. Because the hairpin vortex is of sufficient strength, it forms a kink in the legs as a result of inviscid induction. A bridging structure forms between the legs initially upstream of the kink. As this structure dissipates, another bridging structure forms downstream of the kink and closes the vortex loop between the legs. This pinches off the original hairpin head such that two distinct vortices result. The formation of the secondary hairpin head does not appear to be preceded by a reduction in the spanwise gap between the legs or significant change in height above the wall as has been seen when exposed to a mean turbulent profile. Instead, the formation is preceded by the stretching of the hairpin legs downstream of the kink, exposes the ejected fluid between the legs to boundary layer flow producing conditions similar to those that formed the initial hairpin vortex.

  6. Construction of short hairpin RNA eukaryotic expression vectors targeting peroxire- doxin Ⅰ and identification of their biological functions%靶向PeroxiredoxinⅠ基因的shRNA真核表达载体的构建及生物学功能鉴定

    Institute of Scientific and Technical Information of China (English)

    郭启帅; 黄曦; 张俊; 李少林


    Objective To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting peroxiredoxin Ⅰ ( Prx Ⅰ ) gene and investigate the effect of down-regulating Prx Ⅰ expression on the biological functions of breast carcinoma MCF-7 cells. Methods The eukaryotic expression vectors targeting Prx Ⅰ gene were constructed and then transfected into MCF-7 cells. Transfection efficiency was evaluated by flow cytometry. The mRNA and protein expressions of Prx Ⅰ in MCF-7 cells were detected by RT-PCR and Western blotting. The proliferation of MCF-7 cells was determined by MTT assay. The cell cycle and apoptosis were evaluated by flow cytometry. Results The eukaryotic expression vectors pGPU6-HK (negative control), pGPU6-Prxl, pGPU6-Prx2, pGPU6-Prx3 and pGPU6-Prx4 were successfully constructed and then transfected into MCF-7 cells respectively. Transfection efficiency was about 80%. The expression of Prx Ⅰ in MCF-7 cells transfected with 4 Prx vectors were inhibited significantly at both mRNA and protein levels, especially in the cells with pGPU6-Prx3, whose inhibitory rates were 82.6% and 80.5% respectively at mRNA and protein levels. When pGPU6-Prx3 group was compared with untransfected group and the group transfected with pGPU6HK, the cell proliferation was markedly delayed (P < 0. 05 ), the cell apoptosis rate was significantly increased, and the cells were arrested in G1 phase and S phase were significantly decreased (P < 0. 05 ).Conclusion Eukaryotic expression vectors of shRNA Prx Ⅰ is successfully constructed, and specifically downregulate the Prx Ⅰ expression at mRNA and protein levels. Transfection of pGPU6-Prx3 vector significantly inhibits cell proliferation, induces cell apoptosis, and regulates cell phase redistribution in MCF-7 cells.%目的 构建针对PeroxiredoxinⅠ(PrxⅠ)基因的短发夹RNA(short hairpin RNA, shRNA)真核表达载体,观察下调PrxⅠ基因表达后对乳腺癌MCF-7细胞生物学功能的影响.方法

  7. Capping β-hairpin with N-terminal d-amino acid stabilizes peptide scaffold. (United States)

    Makwana, Kamlesh M; Mahalakshmi, Radhakrishnan


    Various strategies exist to stabilize de novo designed synthetic peptide β-hairpins or β-sheets structures, especially at the non-hydrogen bonding position. However, strategies to stabilize strand termini, which are affected by fraying, are highly limited. Here, by substituting N-terminal aliphatic amino acid with its mirror image counterpart, we achieve a significant increase in scaffold stabilization, resulting from the formation of a terminal aliphatic-aromatic hydrophobic CH…pi cluster. Our extensive solution NMR studies support the incorporation of an N-terminal d-aliphatic amino acid in the design of short β-hairpins, while successfully retaining the overall structural scaffold. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 260-266, 2016.

  8. 靶向 Pokemon 基因 shRNA 重组质粒的构建及其在 CNE2细胞的筛选%Construction of short hairpin RNA recombinant plasmids targeting human Pokemon gene and screening in CNE2 cells

    Institute of Scientific and Technical Information of China (English)

    刘斐; 焦伟; 蓝娇; 肖瑞平


    Objective:To construct three short hairPin RNA( shRNA)recombinant Plasmids targeting human Pokemon gene and screen a Plasmid which can block the Pokemon exPression at maximal degree in nasoPharyngeal carcinoma( NPC)CNE2 cells. Methods:Three shRNAs were designed according to the coding sequences of the Pokemon gene. The shRNA sequences were inserted into PRNAT - U6. 1 / Neo during restriction enzyme,connection, transformation and DNA sequencing. The recombinant Plasmids and blank Plasmid were transfected into CNE2 cells using LiPofectamine 2000. The exPression of Pokemon mRNA and Protein was detected by real time Polymerase chain reaction(PCR)and Western blot,the transfected blank Plasmid CNE2 was used as a negative control,the untreated CNE2 was used as a blank control. Results:The results of DNA sequencing showed shRNA fragments were accordance with the designed sequences. ComParing with the negative control,the Pokemon mRNA and Protein exPression levels of the shRNA grouPs were down - regulated. The Pokemon results of shRNA2 grouP was lowest. Conclusion:Construc-tion and screening shRNA recombinant Plasmids targeting human Pokemon gene can Provide an effective tool for fur-ther studying the role of Pokemon gene in occurrence and develoPment of NPC.%目的:构建3个靶向 Pokemon 基因的短发夹 RNA(shRNA)真核表达载体,在鼻咽癌细胞株 CNE2中筛选出干扰效果最佳的载体。方法:依据 shRNA 设计原则,针对 Pokemon mRNA 序列设计并化学合成3对RNA 干扰序列,经酶切、连接和转化,插入 PRNAT - U6.1/ Neo 质粒,经 DNA 测序验证。脂质体介导 shRNA 重组质粒及空白质粒转染 CNE2细胞,荧光定量 PCR 和 Western blot 检测 Pokemon 在 mRNA 和蛋白水平的表达,转染空白质粒的 CNE2作为阴性对照,未加处理的 CNE2作为空白对照。结果:DNA 测序结果显示 shRNA片段与设计序列一致;转染各重组质粒后的 CNE2细胞

  9. The influence of down-regulation of TAp73 expression by short hairpin RNA on the proliferation and invasion of osteosarcoma%siRNA沉默TAp73对骨肉瘤细胞的增殖和侵袭力的作用研究

    Institute of Scientific and Technical Information of China (English)

    薛剑; 靳安民; 吕海; 闵少雄


    Objective To investigate the pro-apoptosis, anti-migration and anti-proliferation of Saos-2 cells in osteosarcoma through genetic inhibition of expression of TAp73 protein. Methods Human osteosarcoma cell line Saos-2 was cultured. A short hairpin RNA targeting TAp73 was constructed and transfected into Saos-2 cell line. The expression of TAp73 protein were examined by Western blot method. The proliferation was assayed by CCK-8. Flow cytometry was used to detect the cell cycle. Transwell test was used to detect the invasion ability of Saos-2. Results Western blot revealed notably down-regulated expression at protein levels of Tap 73. The cellular proliferation in the treatment group detected by CCK-8 was active after siRNAtreatment (P<0.05). The proportion of the cells in the S phase increased. The invasion ability increased from( 84.17±1.35) to (92.67±2.80)(P<0.01). Conclusions TAp73 is necessary for human osteosarcoma cell proliferation and invasion. It is probably a potential target for osteosareoma treatment.%目的 探讨通过siRNA沉默细胞TAp73蛋白表达对骨肉瘤Saos-2细胞的增殖、周期以及侵袭力的作用研究.方法 以骨肉瘤Saos-2细胞系为研究对象,设计合成沉默TAp73的siRNA片段,转染入Saos-2细胞,用Westem-blot法检测干扰后TAp73蛋白的表达水平,CCK-8法检测细胞增殖,利用流式细胞仪检测细胞周期,Transwell法检测细胞侵袭能力. 结果 Western-blot检测显示siRNA对TAp73蛋白表达有显著下调作用;CCK-8检测实验组细胞增殖明显(P<0.05);细胞周期检测显示细胞处于DNA合成期(S期)的细胞比例上升;穿过人工基底膜的细胞数量也明显增加,由(84.17±1.35)增加为(92.67±2.80)(P<0.05).结论 抗癌基因TAp73在骨肉瘤的增殖、凋亡和侵袭过程中发挥有重要作用,可能成为控制肿瘤增殖和转移的关键性因素.

  10. 质粒shRNA体内干扰Klotho基因对窦房结通道基因的影响%Study on the Effect of Klotho Gene Interferred by Plasmid-mediated Short Hairpin RNA (shRNA) on Sinoatrial Node Pacing Channel Gene

    Institute of Scientific and Technical Information of China (English)

    蔡盈盈; 汪汉; 侯言彬; 房晨鹂; 田鹏; 王贵华; 李璐; 邓珏琳


    通过质粒shRNA体内干扰,研究Klotho基因与窦房结起搏通道相关基因HCN4及HCN2之间的关系,为病窦综合征的研究提供新思路.取C57BL/6J小鼠20只,分为4组,每组5只,分别为:质粒shRNA 24 h组、质粒shRNA 12 h组、生理盐水24 h组、生理盐水12h组.质粒shRNA组经尾静脉注射质粒shRNA 50 μL(1 μg质粒/μL),生理盐水组经尾静脉注射生理盐水50 μL.分别于注射12h及24 h后取窦房结周围组织,行RT-PCR检测各组小鼠的Klotho、HCN2、HCN4基因的mRNA水平.RT-PCR结果显示:与生理盐水12h组比较,shRNA 12 h组的klotho、HCN4和HCN2的mRNA表达量明显降低,均有统计学差异(P<0.05).以上结果提示,小鼠Klotho基因和窦房结起搏基因可能存在一定关系.%The study was aimed to assess the effect of Klotho gene and sinoatrial node pacing channel gene (HCN4and HCN2) for studying sick sinus syndrome,with Klotho gene under the interference of Plasmid-mediated short hairpin RNA.Twenty-five C57BL/6J mice were divided into four groups,i.e,plasmid shRNA 24h group,plasmid shRNA 12h group,sodium chloride 24h group and sodium chloride 12h group.Plasmid shRNA 50μL (1μg/μL) and sodium chloride 50μl were respectively injected according to mice vena caudalis into those in plasmid shRNA group and sodium chloride group.After 12h or 24h respectively,all mice were executed and their sinoatrial node tissues were cut.The mRNA of Klotho,HCN4 and HCN2 gene were detected by RT-PCR.The results of RT-PCR showed that Klotho,HCN4 and HCN2 mRNA levels were lower compared with those in sodium chloride 12h group after 12h interference interval.The results indicated that there might be the a certain relationship between Klotho gene and sinoatrial node pacing channel gene.

  11. Inhibitory effects of conditionally replicative adenovirus armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control on human renal carcinoma OSRC cells%荷载管家基因shRNA的双调控溶瘤腺病毒抑制肾癌细胞增殖的研究

    Institute of Scientific and Technical Information of China (English)

    李连涛; 张宝福; 李慧忠; 郑骏年


    Objective To study the effects of suppressing cell proliferation and GAPDH gene expression on human renal carcinoma OSRC cells with TD - GAPDH, a conditionally replicative adenovirus ( CRAds ) armed with short hairpin RNA against GAPDH genes with hTERT promoter regulation and control . Methods Renal carcinoma OSRC cells and human proximal tubular epithelial cells ( HK - 2 ) were infected with TD - GAPDH, TD55, ZD55 - EGFP. The protein expressions of E1A were detected by Western blot. GAPDH expressing levels of OSRC cells were detected by RT -PCR and Western blot. Cell apoptosis was measured by TUNEL assay. Cell proliferation was assayed by MTT method. OSRC cells were stained with crystal violet to detect cytotoxici effect . Results Western blot assay of E1A expression indicated OSRC cells infected with TD - GAPDH, TD55, ZD55 - EGFP expressed E1A, not detected E1A protein expression in HK - 2 cells. Significant reduction of GAPDH mRNA and protein content was observed in the lysates from OSRC cells in -fected with TD - GAPDH. TD - GAPDH treatment of OSRC cells resulted in increased apoptotie cell death than TD 55 and ZD55 - EGFP treatment. Results of crystal violet stain and MTT assay demonstrated that the antitumor effect of TD-GAPDH was more potent than both TD55 and ZD55 - EGFP. Conclusion CRAds armed with shRNA against GAPDH gene could duplicate in tumor cells , which has both the lytic ability of oncolytic adenovirus and the capacity to deliver shR -NA. TD - GAPDH armed with shRNA gene can significantly inhibit tumor cell growth , which may provide a new platform for cancer gene therapy with shRNA and mayprove to be a useful novel tool for renal cancer therapy .%目的 研究荷载管家基因GAPDH shRNA的溶瘤腺病毒(TD-GAPDH)对人肾癌细胞OSRC增殖抑制的作用.方法 病毒TD-GAPDH、TD55、ZD55-EGFP感染OSRC细胞、人肾小管上皮细胞HK-2.Western blot法检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、Western blot法分别检测GAPDH

  12. Destabilization of the TAR hairpin leads to extension of the polyA hairpin and inhibition of HIV-1 polyadenylation. (United States)

    Vrolijk, Martine M; Harwig, Alex; Berkhout, Ben; Das, Atze T


    Two hairpin structures that are present at both the 5' and 3' end of the HIV-1 RNA genome have important functions in the viral life cycle. The TAR hairpin binds the viral Tat protein and is essential for Tat-mediated activation of transcription. The adjacent polyA hairpin encompasses the polyadenylation signal AAUAAA and is important for the regulation of polyadenylation. Specifically, this RNA structure represses polyadenylation at the 5' side, and enhancer elements on the 3' side overcome this suppression. We recently described that the replication of an HIV-1 variant that does not need TAR for transcription was severely impaired by destabilization of the TAR hairpin, even though a complete TAR deletion was acceptable. In this study, we show that the TAR-destabilizing mutations result in reduced 3' polyadenylation of the viral transcripts due to an extension of the adjacent polyA hairpin. Thus, although the TAR hairpin is not directly involved in polyadenylation, mutations in TAR can affect this process. The stability of the HIV-1 TAR hairpin structure is important for the proper folding of the viral RNA transcripts. This study illustrates how mutations that are designed to study the function of a specific RNA structure can change the structural presentation of other RNA domains and thus affect viral replication in an indirect way.

  13. Episomal plasmid-based generation of induced pluripotent stem cells from fetal femur-derived human mesenchymal stromal cells. (United States)

    Megges, Matthias; Oreffo, Richard O C; Adjaye, James


    Human bone mesenchymal stromal cells derived from fetal femur 55 days post-conception were reprogrammed to induced pluripotent stem cells using episomal plasmid-based expression of OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC and supplemented with the following pathway inhibitors - TGFβ receptor inhibitor (A-83-01), MEK inhibitor (PD325901), GSK3β inhibitor (CHIR99021) and ROCK inhibitor (HA-100). Successful induction of pluripotency in two iPS-cell lines was demonstrated in vitro and by the Pluritest.

  14. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    Directory of Open Access Journals (Sweden)

    Gayani N. P. Dedduwa-Mudalige


    Full Text Available Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.

  15. "Off-on" electrochemical hairpin-DNA-based genosensor for cancer diagnostics. (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt; Ferapontova, Elena E


    A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

  16. Phenotypic inheritance induced by hairpin RNA in Drosophila

    Institute of Scientific and Technical Information of China (English)

    Huaguang Li; Yi Lu


    Phenotypic inheritance induced by RNA has been docu-mented in mouse and Caenorhabditis elegans. Here we report a similar inheritance in Drosophila. Mutant phe-notypes of eye defects and antenna duplication gener-ated from the crossing of one RNA interference (RNAi)transgenic line harboring one hairpin RNA transgene with a GAL4 driver line were inherited independently of the GAL4 driver. Hairpin RNA injection exper-iments demonstrated that the hairpin RNA could induce heritable mutant-like phenotypes on the eye and antenna. The penetrance of mutant phenotypes was reduced when the mutants were crossed to agol and piwi mutants. Our data suggest that hairpin RNA can induce phenotypic inheritance in Drosophila.

  17. Transient β-hairpin formation in α-synuclein monomer revealed by coarse-grained molecular dynamics simulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hang; Ma, Wen [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Han, Wei [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Schulten, Klaus, E-mail: [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States); Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 (United States)


    Parkinson’s disease, originating from the intrinsically disordered peptide α-synuclein, is a common neurodegenerative disorder that affects more than 5% of the population above age 85. It remains unclear how α-synuclein monomers undergo conformational changes leading to aggregation and formation of fibrils characteristic for the disease. In the present study, we perform molecular dynamics simulations (over 180 μs in aggregated time) using a hybrid-resolution model, Proteins with Atomic details in Coarse-grained Environment (PACE), to characterize in atomic detail structural ensembles of wild type and mutant monomeric α-synuclein in aqueous solution. The simulations reproduce structural properties of α-synuclein characterized in experiments, such as secondary structure content, long-range contacts, chemical shifts, and {sup 3}J(H{sub N}H{sub C{sub α}})-coupling constants. Most notably, the simulations reveal that a short fragment encompassing region 38-53, adjacent to the non-amyloid-β component region, exhibits a high probability of forming a β-hairpin; this fragment, when isolated from the remainder of α-synuclein, fluctuates frequently into its β-hairpin conformation. Two disease-prone mutations, namely, A30P and A53T, significantly accelerate the formation of a β-hairpin in the stated fragment. We conclude that the formation of a β-hairpin in region 38-53 is a key event during α-synuclein aggregation. We predict further that the G47V mutation impedes the formation of a turn in the β-hairpin and slows down β-hairpin formation, thereby retarding α-synuclein aggregation.

  18. Inhibitory effect of adenovirus-mediated short hairpin RNA targeting P85 and Akt1 on growth of human gastric adenocarcinoma cell%腺病毒介导的靶向P85和Akt1短发夹RNA对人胃腺癌细胞生长抑制作用的研究

    Institute of Scientific and Technical Information of China (English)

    张靖; 付彦超; 康春生; 张庆瑜; 王涛; 张洁


    目的 构建靶向P85和蛋白激酶B1(PKB1/Akt1)的短发夹RNA(shRNA)腺病毒载体,研究其对人胃腺癌细胞SGC-7901生长的抑制效果.方法 构建腺病毒载体rAd5-P+A,体外转染SGC-7901细胞后,以实时定量PCR和Western blot分别检测P85和Akt1的mRNA和蛋白质的表达.以噻唑蓝比色分析法(MTT法)和流式细胞法评价转染后胃癌细胞的增殖活性.构建裸鼠皮下荷瘤模型进一步观察rAd5-P+A对SGC-7901细胞生长的抑制效果,并应用原位末端标记技术(TUNEL法)检测肿瘤细胞的凋亡情况.结果 成功构建的rAd5-P+A重组腺病毒载体转染SGC-7901细胞后可显著抑制p85和Akt1的mRNA表达,而P85和Aktl蛋白表达量在转染48 h、72 h后分别下调57.5%、63.7%和67.8%、75.6%,与空白对照组和通用腺病毒对照(rAd5-HK)组相比,差异具有统计学意义(P=0.005,P=0.003).与空白对照组和rAd5-HK组相比,SGC-7901细胞的增殖活性在rAd5-P+A转染后第2天明显下降(P<0.001),且rAd5-P+A转染组进入S期的细胞数减少了5.9%~7.1%,而进入G0/G1期的细胞增加了12.1%~13.7%.裸鼠皮下荷瘤模型治疗实验也显示,rAd5-P+A可抑制胃癌细胞的生长,诱导细胞的凋亡.结论 腺病毒介导的靶向P85和Akt1的shRNA可抑制人胃腺癌细胞的生长,这可能为胃腺癌靶向性联合基因治疗提供新的策略.%Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocareinoma cells. Methods P85 and Aktl shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein

  19. Structural studies on an internal loop from a hairpin ribozyme

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Z.; SantaLucia, J. Jr.; Tinoco, I. Jr. [Univ. of California, Berkeley, CA (United States)


    Ribozymes, RNA enzymes, catalyze site-specific RNA cleavage and ligation reactions. We are studying the three-dimensional structure of a hairpin ribozyme derived from the minus strand of tobacco ring spot virus satellite RNA ((-)sTRSV), which has been engineering to specifically cleave the HIV-1 RNA. The minimum structure for the catalytic reaction involves a 50-nucleotide ribozyme and a 14-nucleotide substrate. The proposed secondary structure of the ribozyme-substrate complex consists of four short helices separated by two internal loops. The relatively large size (64-nucleotide) of the ribozyme-substrate complex presents formidable problems in solving the structure using NMR. Therefore we are studying smaller structural subunits of the complex. We are determining the high resolution structure of the symmetric internal loop involving the cleavage site and the flanking helices. One strand of the internal loop was selectively {sup 13}C-labeled at C8 of each purine and C6 of each pyrimidine. By using {sup 13}C-edited two-dimensional NMR, the proton NOESY spectrum was greatly simplified. This allowed unambiguous sequential proton resonance assignments along each strand. Three-dimensional {sup 1}-{sup 13}C HMQC-NOESY was used to further facilitate resonance assignments. We are also enzymatically synthesizing the entire 50-nucleotide ribozyme and will combine it with the {sup 13}C-labeled substrate. Through comparison of the NOE connectivities of the labeled nucleotides from the internal loop alone with those from the entire complex, the differences between the two structures can be elucidated.

  20. Energy landscapes, folding mechanisms, and kinetics of RNA tetraloop hairpins. (United States)

    Chakraborty, Debayan; Collepardo-Guevara, Rosana; Wales, David J


    RNA hairpins play a pivotal role in a diverse range of cellular functions, and are integral components of ribozymes, mRNA, and riboswitches. However, the mechanistic and kinetic details of RNA hairpin folding, which are key determinants of most of its biological functions, are poorly understood. In this work, we use the discrete path sampling (DPS) approach to explore the energy landscapes of two RNA tetraloop hairpins, and provide insights into their folding mechanisms and kinetics in atomistic detail. Our results show that the potential energy landscapes have a distinct funnel-like bias toward the folded hairpin state, consistent with efficient structure-seeking properties. Mechanistic and kinetic information is analyzed in terms of kinetic transition networks. We find microsecond folding times, consistent with temperature jump experiments, for hairpin folding initiated from relatively compact unfolded states. This process is essentially driven by an initial collapse, followed by rapid zippering of the helix stem in the final phase. Much lower folding rates are predicted when the folding is initiated from extended chains, which undergo longer excursions on the energy landscape before nucleation events can occur. Our work therefore explains recent experiments and coarse-grained simulations, where the folding kinetics exhibit precisely this dependency on the initial conditions.

  1. Hybridization-based biosensor containing hairpin probes and use thereof (United States)

    Miller, Benjamin L.; Strohsahl, Christopher M.


    A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

  2. Emergence of hairpins in the conformations of a confined polymer

    CERN Document Server

    Werner, E; Muralidhar, A; Frykholm, K; Smithe, T St Clere; Fritzsche, J; Westerlund, F; Dorfman, K D; Mehlig, B


    If a semiflexible polymer confined to a narrow channel bends around by 180 degrees, the polymer is said to exhibit a hairpin. The equilibrium extension statistics in either the limit where hairpins are rare or in the limit where they are common have been characterized in detail. In this article we consider the intermediate situation, where hairpins are rare but common enough to influence the extension statistics. We study the equilibrium distribution of the extension, as well as the approach to equilibrium, by a combination of theoretical analysis, Monte Carlo simulations, and experiments on DNA coated by the protein RecA, which enhances the stiffness of DNA by approximately one order of magnitude. We find good agreement between the model and simulations. The model also provides excellent agreement with experimental data provided that we assume a persistence length of 2 $\\mu$m for the RecA-DNA filament.

  3. Electroanalysis of single-nucleotide polymorphism by hairpin DNA architectures. (United States)

    Abi, Alireza; Ferapontova, Elena E


    Genetic analysis of infectious and genetic diseases and cancer diagnostics require the development of efficient tools for fast and reliable analysis of single-nucleotide polymorphism (SNP) in targeted DNA and RNA sequences often responsible for signalling disease onset. Here, we highlight the main trends in the development of electrochemical genosensors for sensitive and selective detection of SNP that are based on hairpin DNA architectures exhibiting better SNP recognition properties compared with linear DNA probes. SNP detection by electrochemical hairpin DNA beacons is discussed, and comparative analysis of the existing SNP sensing strategies based on enzymatic and nanoparticle signal amplification schemes is presented.

  4. 脊髓源星形胶质细胞胶质原纤维酸性蛋白表达抑制最佳短发夹样RNA分子筛选及真核表达载体构建%Optimal screening of short hairpin and construction of its eukaryotic expression vector for glial fibrillary acidic protein

    Institute of Scientific and Technical Information of China (English)

    高明勇; 李振宇; 闫洪印


    BACKGROUND: The glial scar formation after spinal cord injury in mammals is the physical and chemical barriers for neural regeneration, and relieving or delaying glial scar formation can provide benefit conditions for the regeneration of injured spinal cord.OBJECTIVE: To design and screen short hairpin RNA (shRNA) interfere molecular targeting the gene coding region of glial fibrillary acidic protein (GFAP) in rat, and reconstruct the eukaryotic vector of shRNA.DESIGN: An observational animal experiment.SETTING: Department of Spine Surgery, Shenzhen Hospital affiliated to Southern Medical University.MATERIALS: Twenty-five Wistar rats of clean degree, either male or female, weighing 20-25 g, were used. DMEM/F12,lipofectamine2000, Trizol RNA separating kits); fetal bovine serum (Hyclone); BamH Ⅰ, HindⅢ, Pstl, Sail and T4 ligases;Plasmid mini preparation kit and DNA gel extraction kit.METHODS: The experiments were carried out in the Shenzhen Hospital affiliated to Southern Medical University from October 2005 to June 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of GFAP target sequence with 9 bp spacer were designed and synthesized, then they were inserted directionally into plasmid Psilencer 2.1 respectively to generate small interfering RNA (siRNA) eukaryotic expression vector. ShRNA molecules were transfected by liposome via ex vivo expression repressive model of GFAP of rat spinal astrocytes. The effects of expressive suppression were detected with real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and then the optimal shRNA eukaryotic vector of repressive expression of GFAP was screened.MAIN OUTCOME MEASURES: ① Interfering sequence specific shRNA template synthesis; ② Constructing specific recombinant plasmid eukaryotic expression vector. ③ Culturing rat spinal astrocytes in vitro; ④ Effects of expressive suppression on GFAP in primary astrocytes after RNA

  5. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes. (United States)

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping


    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer.

  6. Whirlwinds and hairpins in the atmospheric surface layer

    NARCIS (Netherlands)

    Oncley, Steven P.; Hartogensis, O.K.; Tong, Chenning


    Vortices in the atmospheric surface layer are characterized using observations at unprecedented resolution from a fixed array of 31 turbulence sensors. During the day, these vortices likely are dust devils, though no visual observations are available for confirmation. At night, hairpin vortices

  7. Light-triggered β-hairpin folding and unfolding

    NARCIS (Netherlands)

    Schrader, Tobias E.; Schreier, Wolfgang J.; Cordes, Thorben; Koller, Florian O.; Babitzki, Galina; Denschlag, Robert; Renner, Christian; Löweneck, Markus; Dong, Shou-Liang; Moroder, Luis; Tavan, Paul; Zinth, Wolfgang


    A light-switchable peptide is transformed with ultrashort pulses from a β-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular

  8. Thermodynamics of a Simple Three-Dimensional DNA Hairpin Model

    CERN Document Server

    Kremer, Kellan; Boggess, Erin; Mask, Walker; Saucedo, Tony; Hansen, JJ; Appelgate, Ian; Jurgensen, Taylor; Santos, Aaron


    We characterize the equation of state for a simple three-dimensional DNA hairpin model using a Metropolis Monte Carlo algorithm. This algorithm was run at constant temperature and fixed separation between the terminal ends of the strand. From the equation of state, we compute the compressibility, thermal expansion coefficient, and specific heat along with adiabatic path.

  9. Whirlwinds and hairpins in the atmospheric surface layer

    NARCIS (Netherlands)

    Oncley, Steven P.; Hartogensis, O.K.; Tong, Chenning


    Vortices in the atmospheric surface layer are characterized using observations at unprecedented resolution from a fixed array of 31 turbulence sensors. During the day, these vortices likely are dust devils, though no visual observations are available for confirmation. At night, hairpin vortices appe

  10. Raising the Speed Limit for β-Hairpin Formation (United States)

    Davis, Caitlin M.; Xiao, Shifeng; Raleigh, Daniel P.; Dyer, R. Brian


    Understanding the folding of the β-hairpin is a crucial step in studying how β-rich proteins fold. We have studied CLN025, an optimized ten residue synthetic peptide, which adopts a compact, well-structured β-hairpin conformation. Formation of the component β-sheet and β-turn structures of CLN025 was probed independently using a combination of equilibrium Fourier transform infrared spectroscopy and laser-induced temperature jump coupled with time-resolved infrared and fluorescence spectroscopies. We find that CLN025 is an ultrafast folder due to its small free energy barrier to folding and that it exceeds the predicted speed limit for β-hairpin formation by an order of magnitude. We also find that the folding mechanism cannot be described by a simple two-state model, but rather is a heterogeneous process involving two independent parallel processes. Formation of stabilizing cross-strand hydrophobic interactions and turn alignment occur competitively, with relaxation lifetimes of 82 ± 10 and 124 ± 10 ns, respectively, at the highest probed temperature. The ultrafast and heterogeneous folding kinetics observed for CLN025 provide evidence for folding on a nearly barrierless free energy landscape, and recalibrate the speed limit for the formation of a β-hairpin. PMID:22873643

  11. Heuristic RNA pseudoknot prediction including intramolecular kissing hairpins (United States)

    Sperschneider, Jana; Datta, Amitava; Wise, Michael J.


    Pseudoknots are an essential feature of RNA tertiary structures. Simple H-type pseudoknots have been studied extensively in terms of biological functions, computational prediction, and energy models. Intramolecular kissing hairpins are a more complex and biologically important type of pseudoknot in which two hairpin loops form base pairs. They are hard to predict using free energy minimization due to high computational requirements. Heuristic methods that allow arbitrary pseudoknots strongly depend on the quality of energy parameters, which are not yet available for complex pseudoknots. We present an extension of the heuristic pseudoknot prediction algorithm DotKnot, which covers H-type pseudoknots and intramolecular kissing hairpins. Our framework allows for easy integration of advanced H-type pseudoknot energy models. For a test set of RNA sequences containing kissing hairpins and other types of pseudoknot structures, DotKnot outperforms competing methods from the literature. DotKnot is available as a web server under PMID:21098139

  12. β-hairpin-mediated nucleation of polyglutamine amyloid formation (United States)

    Kar, Karunakar; Hoop, Cody L.; Drombosky, Kenneth W.; Baker, Matthew A.; Kodali, Ravindra; Arduini, Irene; van der Wel, Patrick C. A.; Horne, W. Seth; Wetzel, Ronald


    The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington’s disease (HD). Here we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (trpzip, disulfide, D-Pro-Gly, Coulombic attraction, L-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well-correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n* = 4 for a simple, unbroken Q23 sequence to approximate unitary n* values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of SSNMR 13C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn* values remain quite low (~ 10−10) and there is no evidence for significant embellishment of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases. PMID:23353826

  13. Plasmid-based Stat3 siRNA delivered by hydroxyapatite nanoparticles suppresses mouse prostate tumour growth in vivo

    Institute of Scientific and Technical Information of China (English)

    Zuo-Wen Liang; Ling Zhang; Bao-Xue Yang; Bao-Feng Guo; Yang Li; Xiao-Jie Li; Xin Li; Li-Ting Zhao; Ii-Fang Gao; Hao Yu; Xue-Jian Zhao


    DNA vector-based Stat3-specific RNA interference (si-Stat3) blocks Stat3 signalling and inhibits prostate tumour growth. However, the antitumour activity depends on the efficient delivery of si-Stat3. The effects on the growth of mouse prostate cancer cells of si-Stat3 delivered by hydroxyapatite were determined in this study. RM-1 tumour blocks were transplanted into C57BL/6 mice. CaCl2-modif ied hydroxyapatite carrying si-Stat3 plasmids were injected into tumours, and tumour growth and histology were determined. The expression levels of Stat3, pTyr-Stat3, Bcl-2, Bax, Caspase3, VEGF and cyclin Dl were measured by western blot analysis. Amounts of apoptosis in cancer cells were analysed with immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay. The results showed that hydroxyapatite-delivered si-Stat3 significantly suppressed tumour growth up to 74% (P<0.01). Stat3 expression was dramatically downregulated in the tumours. The immunohistochemistry and TUNEL results showed that si-Stet3-induced apoptosis (up to 42%, P<0.01). The Stat3 downstream genes Bcl-2, VEGFand cyclin Dl were also strongly downregulated in the tumour tissues that also displayed significant increases in Bax expression and Caspase3 activity. These results suggest that hydroxyapatite can be used for the in vivo delivery of plasmid-based siRNAs into tumours.

  14. Folding Dynamics of an α Helix and a β Hairpin (United States)

    Hofrichter, James


    What processes limit the rate at which proteins fold? In an effort to address this question we have begun to study the dynamics of the formation of loops, α helices and the minimal β structural element, a β hairpin, which must occur on the pathway from random coils to folded proteins. Because these processes occur on time scales of 10-5-10-9 seconds and experimental access to these time scales has been limited, the kinetics of these processes have not been extensively studied. The expectation is that a more complete understanding of the dynamics of these microprocesses will provide constraints on possible mechanisms for the overall folding of more complex structures. We have explored the kinetics of the helix-coil transition of a synthetic, 21-residue peptide: Ac-WAAAH^+(AAARA)_3A-NH2 and of the folding of a 16 residue β hairpin from protein G B1 using the nanosecond temperature jump technique. Both processes were studied by monitoring tryptophan fluorescence. In the helical peptide, the quantum yield of tryptophan decreases as a result of the interaction between tryptophan in position 1 with the protonated histidine in position 5. In the native conformation of the hairpin, it increases because it forms part of a hydrophobic cluster which stabilizes the native conformation (in a peptide in which a dansylated lysine is incorporated at the C-terminus the fluorescence is quenched). At 300 K, the relaxation time for the helix-coil transition is ~ 250 ns and that for the hairpin-coil transition is ~ 2.2 μs, about 10 times slower. The apparent activation energies are 6.8 kcal/mol for the helix and 10 kcal/mol for the hairpin. We have developed simple kinetic models for these processes which incorporate the sequence- and position-dependent properties known from equilibrium studies and the single-sequence approximation. These models provide a remarkably consistent picture of the dynamics, permitting us to extract information on both the microscopic rates for the c

  15. The Formation of Packets of Hairpins in Shear Flows (United States)

    Cohen, Jacob; Karp, Michael; Shukhman, Ilia


    In the present work we utilize a recently developed new method in an attempt to understand the generation of packets of hairpin vortices from a pair of counter rotating streamwise vortices embedded in uniform shear flow. This analytical-based solution method is capable of following (numerically) the evolution of finite-amplitude localized vortical disturbances embedded in shear flows. Due to their localization in space, the surrounding base flow is assumed to have homogeneous shear to leading order. The method can solve in a novel way the interaction between a general family of unbounded planar homogeneous shear flows and any localized disturbance. The solution is carried out using Lagrangian variables in Fourier space which is convenient and enables fast computations. The revealed mechanism for generation of packets of hairpins seems to be universal and has been observed in the past both in fully developed wall-bounded shear flows as well as in wall-bounded transitional shear flows.

  16. β-hairpin-mediated nucleation of polyglutamine amyloid formation


    Kar, Karunakar; Hoop, Cody L.; Drombosky, Kenneth W.; Baker, Matthew A.; Kodali, Ravindra; Arduini, Irene; van der Wel, Patrick C.A.; Horne, W. Seth; Wetzel, Ronald


    The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington’s disease (HD). Here we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (trpzip, disulfide, D-Pro-Gly, Coulombic attraction, L-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well...

  17. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

    Directory of Open Access Journals (Sweden)

    Tatjana P Kristensen


    Full Text Available The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural

  18. Traveling hairpin-shaped fluid vortices in plane Couette flow. (United States)

    Deguchi, K; Nagata, M


    Traveling-wave solutions are discovered in plane Couette flow. They are obtained when the so-called steady hairpin vortex state found recently by Gibson [J. Fluid Mech. 638, 243 (2009)] and Itano and Generalis [Phys. Rev. Lett. 102, 114501 (2009)] is continued to sliding Couette flow geometry between two concentric cylinders by using the radius ratio as a homotopy parameter. It turns out that in the plane Couette flow geometry two traveling waves having the phase velocities with opposite signs are associated with their appearance from the steady hairpin vortex state, where the amplitude of the phase velocities increases gradually from zero as the Reynolds number is increased. The solutions obviously inherit the streaky structure of the hairpin vortex state, but shape preserving flow patterns propagate in the streamwise direction. Other striking features of the solution are asymmetric mean flow profiles and strong quasistreamwise vortices which occupy the vicinity of only the top or bottom moving boundary, depending on the sign of the phase velocity. Furthermore, we find that the pitchfork bifurcation associated with the appearance of the solution becomes imperfect when the flow is perturbed by a Poiseuille flow component.

  19. Hairpin DNA probe with 5'-TCC/CCC-3' overhangs for the creation of silver nanoclusters and miRNA assay. (United States)

    Xia, Xiaodong; Hao, Yuanqiang; Hu, Shengqiang; Wang, Jianxiu


    A facile strategy for the assay of target miRNA using fluorescent silver nanoclusters (AgNCs) has been described. Due to the preferable interaction between cytosine residues and Ag(+), a short cytosine-rich oligonucleotide (ODN) with only six bases 5'-TCCCCC-3' served as an efficient scaffold for the creation of the AgNCs. The AgNCs displayed a bright red emission when excited at 545nm. Such ODN base-stabilized AgNCs have been exploited for miRNA sensing. Overhangs of TCC at the 5' end (5'-TCC) and CCC at the 3' end (CCC-3') (denoted as 5'-TCC/CCC-3') appended to the hairpin ODN probe which also contains recognition sequences for target miRNA were included. Interestingly, the AgNCs/hairpin ODN probe showed similar spectral properties as that templated by 5'-TCCCCC-3'. The formation of the hairpin ODN probe/miRNA duplex separated the 5'-TCC/CCC-3' overhangs, thus disturbing the optical property or structure of the AgNCs. As a result, fluorescence quenching of the AgNCs/hairpin ODN probe was obtained, which allows for facile determination of target miRNA. The proposed method is simple and cost-effective, holding great promise for clinical applications.

  20. Kinetic Monte Carlo method applied to nucleic acid hairpin folding. (United States)

    Sauerwine, Ben; Widom, Michael


    Kinetic Monte Carlo on coarse-grained systems, such as nucleic acid secondary structure, is advantageous for being able to access behavior at long time scales, even minutes or hours. Transition rates between coarse-grained states depend upon intermediate barriers, which are not directly simulated. We propose an Arrhenius rate model and an intermediate energy model that incorporates the effects of the barrier between simulated states without enlarging the state space itself. Applying our Arrhenius rate model to DNA hairpin folding, we demonstrate improved agreement with experiment compared to the usual kinetic Monte Carlo model. Further improvement results from including rigidity of single-stranded stacking.

  1. Identify Beta-Hairpin Motifs with Quadratic Discriminant Algorithm Based on the Chemical Shifts. (United States)

    YongE, Feng; GaoShan, Kou


    Successful prediction of the beta-hairpin motif will be helpful for understanding the of the fold recognition. Some algorithms have been proposed for the prediction of beta-hairpin motifs. However, the parameters used by these methods were primarily based on the amino acid sequences. Here, we proposed a novel model for predicting beta-hairpin structure based on the chemical shift. Firstly, we analyzed the statistical distribution of chemical shifts of six nuclei in not beta-hairpin and beta-hairpin motifs. Secondly, we used these chemical shifts as features combined with three algorithms to predict beta-hairpin structure. Finally, we achieved the best prediction, namely sensitivity of 92%, the specificity of 94% with 0.85 of Mathew's correlation coefficient using quadratic discriminant analysis algorithm, which is clearly superior to the same method for the prediction of beta-hairpin structure from 20 amino acid compositions in the three-fold cross-validation. Our finding showed that the chemical shift is an effective parameter for beta-hairpin prediction, suggesting the quadratic discriminant analysis is a powerful algorithm for the prediction of beta-hairpin.

  2. The discodermolide hairpin structure flows from conformationally stable modular motifs. (United States)

    Jogalekar, Ashutosh S; Kriel, Frederik H; Shi, Qi; Cornett, Ben; Cicero, Daniel; Snyder, James P


    (+)-Discodermolide (DDM), a polyketide macrolide from marine sponge, is a potent microtubule assembly promoter. Reported solid-state, solution, and protein-bound DDM conformations reveal the unusual result that a common hairpin conformational motif exists in all three microenvironments. No other flexible microtubule binding agent exhibits such constancy of conformation. In the present study, we combine force-field conformational searches with NMR deconvolution in different solvents to compare DDM conformers with those observed in other environments. While several conformational families are perceived, the hairpin form dominates. The stability of this motif is dictated primarily by steric factors arising from repeated modular segments in DDM composed of the C(Me)-CHX-C(Me) fragment. Furthermore, docking protocols were utilized to probe the DDM binding mode in beta-tubulin. A previously suggested pose is substantiated (Pose-1), while an alternative (Pose-2) has been identified. SAR analysis for DDM analogues differentiates the two poses and suggests that Pose-2 is better able to accommodate the biodata.

  3. The Role of XPG in Processing (CAGn/(CTGn DNA Hairpins

    Directory of Open Access Journals (Sweden)

    Hou Caixia


    Full Text Available Abstract Background During DNA replication or repair, disease-associated (CAGn/(CTGn expansion can result from formation of hairpin structures in the repeat tract of the newly synthesized or nicked DNA strand. Recent studies identified a nick-directed (CAGn/(CTGn hairpin repair (HPR system that removes (CAGn/(CTGn hairpins from human cells via endonucleolytic incisions. Because the process is highly similar to the mechanism by which XPG and XPF endonucleases remove bulky DNA lesions during nucleotide excision repair, we assessed the potential role of XPG in conducting (CAGn/(CTGn HPR. Results To determine if the XPG endonuclease is involved in (CAGn/(CTGn hairpin removal, two XPG-deficient cell lines (GM16024 and AG08802 were examined for their ability to process (CAGn/(CTGn hairpins in vitro. We demonstrated that the GM16024 cell line processes all hairpin substrates as efficiently as HeLa cells, and that the AG08802 cell line is partially defective in HPR. Analysis of repair intermediates revealed that nuclear extracts from both XPG-deficient lines remove CAG/CTG hairpins via incisions, but the incision products are distinct from those generated in HeLa extracts. We also show that purified recombinant XPG protein greatly stimulates HPR in XPG-deficient extracts by promoting an incision 5' to the hairpin. Conclusions Our results strongly suggest that 1 human cells possess multiple pathways to remove (CAGn/(CTGn hairpins located in newly synthesized (or nicked DNA strand; and 2 XPG, although not essential for (CAGn/(CTGn hairpin removal, stimulates HPR by facilitating a 5' incision to the hairpin. This study reveals a novel role for XPG in genome-maintenance and implicates XPG in diseases caused by trinucleotide repeat expansion.

  4. Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

    Directory of Open Access Journals (Sweden)

    Boerrigter Michaël


    Full Text Available Abstract Background Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. Methods Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP, 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls. The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. Results Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. Conclusion The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

  5. The 5'-proximal hairpin of turnip yellow mosaic virus RNA: its role in translation and encapsidation. (United States)

    Bink, Hugo H J; Schirawski, Jan; Haenni, Anne-Lise; Pleij, Cornelis W A


    The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5' untranslated region, it was observed that stabilization of the 5'-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5'-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5' cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis.

  6. Construct Hairpin RNA to Fight Against Rice Dwarf Virus

    Institute of Scientific and Technical Information of China (English)

    MAZhong-Liang; YANGHuai-Yi; WANGRong; TIENPo


    Hairpin RNA (hpRNA) can induce post-transcriptional gene silencing, and was constructed with128-754 bp of segment 8 of rice dwarf virus then placed under the control of the CaMV35S promoter andused to transform rice cultivar "Zhonghua 11" via Agrobacterium tumefaciens. A total of 12 independentlines containing hpRNA were obtained as demonstrated by Southern blotting analysis. Challenge inocula-tion with rice dwarf virus (RDV) viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plantscontaining the hpRNA transgene displayed high resistance or delayed and attenuated viral symptoms. Incontrast, transgenic lines expressing sense RNA showed severe symptoms similar to control plants trans-formed with the vector alone. These results suggest that hpRNA confers high resistance to RDV intransgenic rice.

  7. Identification and tracking of hairpin vortex auto-generation in turbulent wall-bounded flow (United States)

    Huang, Yangzi; Green, Melissa


    Hairpin vortices have been widely accepted as component structures of turbulent boundary layers. Their properties (size, vorticity, energy) and dynamic phenomena (origin, growth, breakdown) have been shown to correlate to the complex, multi-scaled turbulent motions observed in both experiments and simulations. As established in the literature, the passage of a hairpin vortex creates a wall-normal ejection of fluid, which encounters the high-speed freestream resulting in near-wall shear and increased drag. A previously generated simulation of an isolated hairpin vortex is used to study the auto-generation of a secondary vortex structure. Eulerian methods such as the Q criterion and Γ2 function, as well as Lagrangian methods are used to visualize the three-dimensional hairpin vortices and the auto-generation process. The circulation development and wall-normal location of both primary and secondary hairpin heads are studied to determine if there is a correlation between the strength and height of the primary hairpin vortex with the secondary hairpin vortex auto-generation.

  8. Stable RNA hairpins in 88 coding regions of human mRNA

    Institute of Scientific and Technical Information of China (English)

    PAN Min; WANG Chuanming; LIU Ciquan


    RNA hairpins containing UNCG, GNRA, CUUG (N=A, U, C or G, R=G or A) loops are unusually thermodynamic stable and conserved structures. The structural features of these hairpin loops are very special, and they play very important roles in vivo. They are prevalent in rRNA, catalytic RNA and non-coding mRNA. However, the 5′ C(UUCG)G 3′ hairpin is not found in the folding structure of 88 human mRNA coding regions. It is also different from rRNA in that there is no preference for certain sequences among tetraloops in these 88 mRNA folding structures.

  9. Scientific advice on the suitability of data for the assessment of DNA integration into the fish genome of a genetically modified DNA plasmid-based veterinary vaccine

    Directory of Open Access Journals (Sweden)

    European Food Safety Authority


    Full Text Available Pancreas disease caused by salmonid alphavirus in farmed Atlantic salmon (Salmo salar leads to high mortality rates post infection and histopathological lesions in several organs. As protection against pancreas disease, Novartis developed a prophylactic DNA plasmid-based vaccine to be administered to salmon as naked plasmid in a single intramuscular injection. In order to assess the legal status of the fish vaccinated with this new vaccine with regard to the legislation on genetically modified organisms, the European Commission suggested that the company carry out a scientific study on the integration/non-integration of the plasmid DNA into the fish genome. Subsequently, the European Commission requested EFSA to give scientific advice on the study design and the conclusions drawn by the company. PCR based analysis of genomic DNA from muscle samples, taken from at or around the injection site 436 days post vaccination, led the company to conclude that integration of plasmid DNA into the fish genome is extremely unlikely. After an assessment of the study, EFSA considers that the study presented by Novartis Animal Health on the integration/non-integration of DNA plasmid-based vaccine into the salmon genomic DNA provides insufficient information on the potential integration of plasmid DNA fragments into the fish genome due to a limited coverage of the plasmid DNA by the detection method provided, the limited number of samples analysed and an insufficient limit of detection and method validation. Therefore, EFSA is of the opinion that the results from the integration/non-integration study submitted by Novartis Animal Health are not sufficient to support the conclusion of non-integration of plasmid DNA into the fish genome drawn by the company.

  10. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations (United States)

    Jia, Yanwei; Sanchez, J. Aquiles; Wangh, Lawrence J.


    Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals. PMID:25082368

  11. Maintenance of the flip sequence orientation of the ears in the parvoviral left-end hairpin is a nonessential consequence of the critical asymmetry in the hairpin stem. (United States)

    Li, Lei; Cotmore, Susan F; Tattersall, Peter


    Parvoviral terminal hairpins are essential for viral DNA amplification but are also implicated in multiple additional steps in the viral life cycle. The palindromes at the two ends of the minute virus of mice (MVM) genome are dissimilar and are processed by different resolution mechanisms that selectively direct encapsidation of predominantly negative-sense progeny genomes and conserve a single Flip sequence orientation at the 3' (left) end of such progeny. The sequence and predicted structure of these 3' hairpins are highly conserved within the genus Parvovirus, exemplified by the 121-nucleotide left-end sequence of MVM, which folds into a Y-shaped hairpin containing small internal palindromes that form the "ears" of the Y. To explore the potential role(s) of this hairpin in the viral life cycle, we constructed infectious clones with the ear sequences either inverted, to give the antiparallel Flop orientation, or with multiple transversions, conserving their base composition but changing their sequence. These were compared with a "bubble" mutant, designed to activate the normally silent origin in the inboard arm of the hairpin, thus potentially rendering symmetric the otherwise asymmetric junction resolution mechanism that drives maintenance of Flip. This mutant exhibited a major defect in viral duplex and single-strand DNA replication, characterized by the accumulation of covalently closed turnaround forms of the left end, and was rapidly supplanted by revertants that restored asymmetry. In contrast, both sequence and orientation changes in the hairpin ears were tolerated, suggesting that maintaining the Flip orientation of these structures is a consequence of, but not the reason for, asymmetric left-end processing.

  12. Uridylation of RNA Hairpins by Tailor Confines the Emergence of MicroRNAs in Drosophila


    Reimão-Pinto, Madalena M.; Ignatova, Valentina; Burkard, Thomas R.; Hung, Jui-Hung; Manzenreither, Raphael A.; Sowemimo, Ivica; Herzog, Veronika A.; Reichholf, Brian; Fariña-Lopez, Sara; Ameres, Stefan L.


    Summary Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. Here, we report Tailor, an uridylyltransferase that is required for the majority of 3′ end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3′ overhang of microRNA hairpins, which...

  13. Recognition of hairpin DNA from coil DNA by electrospray mass spectrometry with annealing strategy

    Institute of Scientific and Technical Information of China (English)

    Bo Zheng; Yi Quan Liu; Gu Yuan


    This research presented an annealing strategy to identify hairpin DNA from coil DNA with the same base composition but different arrangements using electrospray mass spectrometry (ESI-MS).A series of single-stranded DNA were annealed with their complementary sequences,respectively.All the five pairs of hairpin DNA and coil DNA were unambiguously distinguished by ESIMS with annealing strategy.This research offers a potential method to probe the DNA structure by comparing with mass spectral characteristics.

  14. Simulations of beta-hairpin folding confined to spherical pores using distributed computing. (United States)

    Klimov, D K; Newfield, D; Thirumalai, D


    We report the thermodynamics and kinetics of an off-lattice Go model beta-hairpin from Ig-binding protein confined to an inert spherical pore. Confinement enhances the stability of the hairpin due to the decrease in the entropy of the unfolded state. Compared with their values in the bulk, the rates of hairpin formation increase in the spherical pore. Surprisingly, the dependence of the rates on the pore radius, R(s), is nonmonotonic. The rates reach a maximum at R(s)/R(g,N)(b) approximately equal to 1.5, where R(g,N)(b) is the radius of gyration of the folded beta-hairpin in the bulk. The denatured state ensemble of the encapsulated beta-hairpin is highly structured even at substantially elevated temperatures. Remarkably, a profound effect of confinement is evident even when the beta-hairpin occupies less than a 10th of the sphere volume. Our calculations show that the emergence of substantial structure in the denatured state of proteins in inert pores is a consequence of confinement. In contrast, the structure of the bulk denatured state ensemble depends dramatically on the extent of denaturation.

  15. Design of monomeric water-soluble β-hairpin and β-sheet peptides. (United States)

    Jiménez, M Angeles


    Since the first report in 1993 (JACS 115, 5887-5888) of a peptide able to form a monomeric β-hairpin structure in aqueous solution, the design of peptides forming either β-hairpins (two-stranded antiparallel β-sheets) or three-stranded antiparallel β-sheets has become a field of growing interest and activity. These studies have yielded great insights into the principles governing the stability and folding of β-hairpins and antiparallel β-sheets. This chapter provides an overview of the reported β-hairpin/β-sheet peptides focussed on the applied design criteria, reviews briefly the factors contributing to β-hairpin/β-sheet stability, and describes a protocol for the de novo design of β-sheet-forming peptides based on them. Guidelines to select appropriate turn and strand residues and to avoid self-association are provided. The methods employed to check the success of new designed peptides are also summarized. Since NMR is the best technique to that end, NOEs and chemical shifts characteristic of β-hairpins and three-stranded antiparallel β-sheets are given.

  16. Development and application of automated systems for plasmid-based functional proteomics to improve syntheitc biology of engineered industrial microbes for high level expression of proteases for biofertilizer production (United States)

    In addition to microarray technology, which provides a robust method to study protein function in a rapid, economical, and proteome-wide fashion, plasmid-based functional proteomics is an important technology for rapidly obtaining large quantities of protein and determining protein function across a...

  17. Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination.


    Washio, T.; Sasayama, J; Tomita, M.


    Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes. Instead of trying to detect each individual hairpin, we averaged the free energy values around the stop codons over the entire genome to predict how extensively the organism relies on hairpin formation in the process of transcription termination. The free energy values of Escherichia coli K-12 sho...

  18. Effect of DNA Hairpin Loops on the Twist of Planar DNA Origami Tiles (United States)

    Li, Zhe; Wang, Lei; Yan, Hao; Liu, Yan


    The development of scaffolded DNA origami, a technique in which a long single-stranded viral genome is folded into arbitrary shapes by hundreds of short synthetic oligonucleotides, represents an important milestone in DNA nanotechnology. Recent findings have revealed that two-dimensional (2D)DNA origami structures based on the original design parameters adopt a global twist with respect to the tile plane, which may be because the conformation of the constituent DNA (10.67 bp/turn) deviates from the natural B-type helical twist (10.4 bp/turn). Here we aim to characterize the effects of DNA hairpin loops on the overall curvature of the tile and explore their ability to control, and ultimately eliminate any unwanted curvature. A series of dumbbell-shaped DNA loops were selectively displayed on the surface of DNA origami tiles with the expectation that repulsive interactions among the neighboring dumbbell loops and between the loops and the DNA origami tile would influence the structural features of the underlying tiles. A systematic, atomic force microscopy (AFM) study of how the number and position of the DNA loops influenced the global twist of the structure was performed, and several structural models to explain the results were proposed. The observations unambiguously revealed that the first generation of rectangular shaped origami tiles adopt a conformation in which the upper right (corner 2) and bottom left (corner 4) corners bend upward out of the plane, causing linear superstructures attached by these corners to form twisted ribbons. Our experimental observations are consistent with the twist model predicted by the DNA mechanical property simulation software CanDo. Through the systematic design and organization of various numbers of dumbbell loops on both surfaces of the tile, a nearly planar rectangular origami tile was achieved. PMID:22126326

  19. Uridylation of RNA Hairpins by Tailor Confines the Emergence of MicroRNAs in Drosophila (United States)

    Reimão-Pinto, Madalena M.; Ignatova, Valentina; Burkard, Thomas R.; Hung, Jui-Hung; Manzenreither, Raphael A.; Sowemimo, Ivica; Herzog, Veronika A.; Reichholf, Brian; Fariña-Lopez, Sara; Ameres, Stefan L.


    Summary Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. Here, we report Tailor, an uridylyltransferase that is required for the majority of 3′ end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3′ overhang of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3′ guanosine. In contrast to mirtrons, conserved Drosophila precursor microRNAs are significantly depleted in 3′ guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3′ ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila. PMID:26145176

  20. Formation mechanism of hairpin vortices in the wake of a truncated square cylinder in a duct

    CERN Document Server

    Dousset, Vincent


    We investigate the laminar shedding of hairpin vortices in the wake of a truncated square cylinder placed in a duct, for Reynolds numbers around the critical threshold of the onset of vortex shedding. We single out the formation mechanism of the hairpin vortices by means of a detailed analysis of the flow patterns in the steady regime. We show that unlike in previous studies of similar structures, the dynamics of the hairpin vortices is entwined with that of the counter-rotating pair of streamwise vortices, which we found to be generated in the bottom part of the near wake (these are usually referred to as base vortices). In particular, once the hairpin structure is released, the base vortices attach to it, forming its legs, so these are streamwise, and not spanwise as previously observed in unconfined wakes or behind cylinders of lower aspect ratios. We also single out a trail of Omega-shaped vortices, generated between successive hairpin vortices through a mechanism that is analogous to that active in near-...

  1. The role of loop stacking in the dynamics of DNA hairpin formation

    CERN Document Server

    Mosayebi, Majid; Ouldridge, Thomas E; Louis, Ard A; Doye, Jonathan P K


    We study the dynamics of DNA hairpin formation using oxDNA, a nucleotide-level coarse-grained model of DNA. In particular, we explore the effects of the loop stacking interactions and non-native base pairing on the hairpin closing times. We find a non-monotonic variation of the hairpin closing time with temperature, in agreement with the experimental work of Wallace et al. [Proc. Nat. Acad. Sci. USA 2001, 98, 5584-5589]. The hairpin closing process involves the formation of an initial nucleus of one or two bonds between the stems followed by a rapid zippering of the stem. At high temperatures, typically above the hairpin melting temperature, an effective negative activation enthalpy is observed because the nucleus has a lower enthalpy than the open state. By contrast, at low temperatures, the activation enthalpy becomes positive mainly due to the increasing energetic cost of bending a loop that becomes increasingly highly stacked as the temperature decreases. We show that stacking must be very strong to induc...

  2. Phototriggered DNA hairpin formation in a stilbenediether-linked bis(oligonucleotide) conjugate

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, F.D.; Liu, X.


    Photochemical switches have found numerous biological applications, but have received only limited application to the control of DNA base pairing processes. The authors report here that a hairpin-forming bis(oligonucleotide) conjugate possessing a stilbenediether linker undergoes reversible trans {leftrightarrow} cis photoisomerization when both the cis and trans conjugates are in the random coil conformation, but undergoes one-way cis {r{underscore}arrow} trans isomerization when both are in hairpin conformations. Thus base-pairing inhibits trans {r{underscore}arrow} cis but not cis {r{underscore}arrow} trans isomerization. Moreover, photoisomerization of the random coil cis conjugate under conditions where the trans conjugate forms a stable hairpin results in phototriggered base pairing. The use of base pairing to effect one-way photoisomerization and of photoisomerization to effect base pairing are without precedent.

  3. Predicting 3D Structure, Flexibility, and Stability of RNA Hairpins in Monovalent and Divalent Ion Solutions (United States)

    Shi, Ya-Zhou; Jin, Lei; Wang, Feng-Hua; Zhu, Xiao-Long; Tan, Zhi-Jie


    A full understanding of RNA-mediated biology would require the knowledge of three-dimensional (3D) structures, structural flexibility, and stability of RNAs. To predict RNA 3D structures and stability, we have previously proposed a three-bead coarse-grained predictive model with implicit salt/solvent potentials. In this study, we further develop the model by improving the implicit-salt electrostatic potential and including a sequence-dependent coaxial stacking potential to enable the model to simulate RNA 3D structure folding in divalent/monovalent ion solutions. The model presented here can predict 3D structures of RNA hairpins with bulges/internal loops (RNA hairpins with bulge loops of different lengths at several divalent/monovalent ion conditions. In addition, the model successfully predicts the stability of RNA hairpins with various loops/stems in divalent/monovalent ion solutions. PMID:26682822

  4. DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family (United States)

    Franch, Oskar; Iacovelli, Federico; Falconi, Mattia; Juul, Sissel; Ottaviani, Alessio; Benvenuti, Claudia; Biocca, Silvia; Ho, Yi-Ping; Knudsen, Birgitta R.; Desideri, Alessandro


    In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous

  5. Linear Chromosome-generating System of Agrobacterium tumefaciens C58: Protelomerase Generates and Protects Hairpin Ends

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Wai Mun; DaGloria, Jeanne; Fox, Heather; Ruan, Qiurong; Tillou, John; Shi, Ke; Aihara, Hideki; Aron, John; Casjens, Sherwood (Utah); (UMM)


    Agrobacterium tumefaciens C58, the pathogenic bacteria that causes crown gall disease in plants, harbors one circular and one linear chromosome and two circular plasmids. The telomeres of its unusual linear chromosome are covalently closed hairpins. The circular and linear chromosomes co-segregate and are stably maintained in the organism. We have determined the sequence of the two ends of the linear chromosome thus completing the previously published genome sequence of A. tumefaciens C58. We found that the telomeres carry nearly identical 25-bp sequences at the hairpin ends that are related by dyad symmetry. We further showed that its Atu2523 gene encodes a protelomerase (resolvase) and that the purified enzyme can generate the linear chromosomal closed hairpin ends in a sequence-specific manner. Agrobacterium protelomerase, whose presence is apparently limited to biovar 1 strains, acts via a cleavage-and-religation mechanism by making a pair of transient staggered nicks invariably at 6-bp spacing as the reaction intermediate. The enzyme can be significantly shortened at both the N and C termini and still maintain its enzymatic activity. Although the full-length enzyme can uniquely bind to its product telomeres, the N-terminal truncations cannot. The target site can also be shortened from the native 50-bp inverted repeat to 26 bp; thus, the Agrobacterium hairpin-generating system represents the most compact activity of all hairpin linear chromosome- and plasmid-generating systems to date. The biochemical analyses of the protelomerase reactions further revealed that the tip of the hairpin telomere may be unusually polymorphically capable of accommodating any nucleotide.

  6. DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

    DEFF Research Database (Denmark)

    Franch, Oskar; Iacovelli, Federico; Falconi, Mattia


    and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724–9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin...... forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening–closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation...

  7. Residues in the central β-hairpin of the DNA helicase of bacteriophage T7 are important in DNA unwinding

    NARCIS (Netherlands)

    Satapathy, Ajit K.; Kochaniak, Anna B.; Mukherjee, Sourav; Crampton, Donald J.; Oijen, Antoine van; Richardson, Charles C.


    The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic β-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the β-hairpin loop to determine their role during dTTPase-driven

  8. The 5′-Proximal Hairpin of Turnip Yellow Mosaic Virus RNA: Its Role in Translation and Encapsidation (United States)

    Bink, Hugo H. J.; Schirawski, Jan; Haenni, Anne-Lise; Pleij, Cornelis W. A.


    The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5′ untranslated region, it was observed that stabilization of the 5′-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutations. A protoplast transfection assay revealed that the accumulation of plus-strand full-length RNA and subgenomic RNA, as well as protein expression levels, was affected by hairpin stability. Stabilization of this hairpin inhibited translation. A model is proposed in which a destabilized 5′-proximal hairpin allows maximal translation of the viral proteins. It is suggested that this hairpin may exist in close proximity to the 5′ cap as long as its stability is low enough to enable translation. However, at an acidic pH, the hairpin structure becomes more stable and is functionally transformed into the initiation signal for viral packaging. Slightly acidic conditions can be found in chloroplasts, where TYMV assembly is driven by a low pH generated by active photosynthesis. PMID:12805444

  9. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions. (United States)

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt


    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

  10. Immunogenicity and Protective Response Induced by Recombinant Plasmids Based on the BAB1_0267 and BAB1_0270 Open Reading Frames of Brucella abortus 2308 in BALB/c Mice (United States)

    Gómez, Leonardo A.; Alvarez, Francisco I.; Fernández, Pablo A.; Flores, Manuel R.; Molina, Raúl E.; Coloma, Roberto F.; Oñate, Angel A.


    Immunogenicity induced by recombinant plasmids based on the BAB1_0267 and BAB1_0270 open reading frames (ORFs) of Brucella abortus 2308 was evaluated. Bioinformatics analyses indicate that the BAB1_0267 and BAB1_0270 ORFs encode a protein with a SH3 domain and a Zn-dependent metalloproteinase, respectively. Both ORFs have important effects on intracellular survival and replication of B. abortus 2308, mediated via professional and non-professional phagocytic cells. Our results show that immunization with the recombinant plasmid based on the BAB1_0267 ORF significantly increases the production of IgG1, levels of IFN-γ and the lymphoproliferative response of splenocytes. However, BAB1_0267 did not provide significant levels of protection. The plasmid based on the BAB1_0270 significantly increased IgG2a production, levels of IFN-γ and TNF-α, and the lymphoproliferative response of splenocytes. These results demonstrate that immunization with the BAB1_0270 derived recombinant plasmid induce a Th1-type immune response, correlated with a heightened resistance to B. abortus 2308 infection in mice. It is concluded that the Th1-type immune response against bacterial Zn-dependent metalloproteinase induces a protective response in mice, and that pV270 recombinant plasmid is an effective candidate microbicide against brucellosis. PMID:27747197

  11. Phylogenetic study on structural elements of HIV-1 poly(A region. 1. PolyA and DSE hairpins

    Directory of Open Access Journals (Sweden)

    Zarudnaya M. I.


    Full Text Available Genome of human immunodeficiency virus type 1 (HIV-1 is highly heterogeneous. The aim of this work was a phylogenetic study on structural elements of the HIV-1 poly(A region, in particular polyA and DSE hairpins which compose a core poly(A site. Methods. The secondary structure of the HIV-1 core poly(A site has been predicted by the UNAFold program. Results. The structure of the polyA and DSE hairpins has been analysed in 1679 HIV-1 genomes of group M and 18 genomes of simian immunodeficiency virus SIVcpzPtt. We found 244 and 171 different sequences for the HIV-1 polyA and DSE hairpins, respectively. However 70 % of the HIV-1 isolates studied contain one of 7 variants of the polyA hairpin which occur with a frequency 5 % (main variants and 79 % of the isolates contain one of 7 main variants of the DSE hairpin. We also revealed subtype and country specific mutations in these hairpins. We found that the SIV polyA hairpin most closely resembles that found in HIV-1 genomes of B/C subtypes. Conclusions. The results of our large-scale phylogenetic study support some structural models of the HIV-1 5' UTR, in particular the tertiary interaction between the polyA hairpin and the matrix region in HIV-1 gRNA. Possibly, the DSE hairpin appeared in the course of viral evolution of the HIV-1 group M. An exposure of the U/GU-rich element in the apical loop of DSE hairpin could significantly increase the efficiency of pre-mRNA polyadenylation in this HIV-1 group.

  12. Beyond secondary structure: primary-sequence determinants license pri-miRNA hairpins for processing. (United States)

    Auyeung, Vincent C; Ulitsky, Igor; McGeary, Sean E; Bartel, David P


    To use microRNAs to downregulate mRNA targets, cells must first process these ~22 nt RNAs from primary transcripts (pri-miRNAs). These transcripts form RNA hairpins important for processing, but additional determinants must distinguish pri-miRNAs from the many other hairpin-containing transcripts expressed in each cell. Illustrating the complexity of this recognition, we show that most Caenorhabditis elegans pri-miRNAs lack determinants required for processing in human cells. To find these determinants, we generated many variants of four human pri-miRNAs, sequenced millions that retained function, and compared them with the starting variants. Our results confirmed the importance of pairing in the stem and revealed three primary-sequence determinants, including an SRp20-binding motif (CNNC) found downstream of most pri-miRNA hairpins in bilaterian animals, but not in nematodes. Adding this and other determinants to C. elegans pri-miRNAs imparted efficient processing in human cells, thereby confirming the importance of primary-sequence determinants for distinguishing pri-miRNAs from other hairpin-containing transcripts.

  13. Inhibition of HIV Replication by Cyclic and Hairpin PNAs Targeting the HIV-1 TAR RNA Loop

    Directory of Open Access Journals (Sweden)

    Gregory Upert


    Full Text Available Human immunodeficiency virus-1 (HIV-1 replication and gene expression entails specific interaction of the viral protein Tat with its transactivation responsive element (TAR, to form a highly stable stem-bulge-loop structure. Previously, we described triphenylphosphonium (TPP cation-based vectors that efficiently deliver nucleotide analogs (PNAs into the cytoplasm of cells. In particular, we showed that the TPP conjugate of a linear 16-mer PNA targeting the apical stem-loop region of TAR impedes Tat-mediated transactivation of the HIV-1 LTR in vitro and also in cell culture systems. In this communication, we conjugated TPP to cyclic and hairpin PNAs targeting the loop region of HIV-1 TAR and evaluated their antiviral efficacy in a cell culture system. We found that TPP-cyclic PNAs containing only 8 residues, showed higher antiviral potency compared to hairpin PNAs of 12 or 16 residues. We further noted that the TPP-conjugates of the 8-mer cyclic PNA as well as the 16-mer linear PNA displayed similar antiviral efficacy. However, cyclic PNAs were shown to be highly specific to their target sequences. This communication emphasizes on the importance of small constrained cyclic PNAs over both linear and hairpin structures for targeting biologically relevant RNA hairpins.

  14. Evolutions of hairpin vortexes over a superhydrophobic surface in turbulent boundary layer flow (United States)

    Zhang, Jingxian; Tian, Haiping; Yao, Zhaohui; Hao, Pengfei; Jiang, Nan


    Turbulent flows over a superhydrophobic surface and a smooth surface have been measured and studied by particle image velocimetry technology at Reθ = 990. The Reynolds shear stress distributions over the two surfaces are significantly different. Specifically, for the superhydrophobic surface, the Reynolds shear stress is suppressed in the near-wall region (y/δ curve. Evolutions of hairpin vortexes are analyzed to interpret differences in the Reynolds shear stress, based on some comparisons in the low-speed streaks and Q2/Q4 (ejection/sweep) events. The results show that, in the near wall region, the turbulent coherent structures (low-speed streaks and hairpin vortex) over the superhydrophobic surface are more stable and flat, due to the suppression in the strength and the lifting effect of the hairpin vortex. In the outer region, the superhydrophobic surface lifts the hairpin vortex away from the wall with a value of 0.14δ in our experiment, which makes the Q4 events occur further from the wall and contribute less to skin friction.

  15. Effect of the reflectional symmetry on the coherent hole transport across DNA hairpins (United States)

    Zarea, Mehdi; Berlin, Yuri; Ratner, Mark A.


    The coherent hole transfer in three types of DNA hairpins containing strands with adenine (A) and guanine (G) nucleobases has been studied. The investigated hairpins involve An+1GGAn, AnGAGAn, or (AG)2nA strands that connect the hole donor and hole acceptor located on opposite ends of hairpins. The positive charge transfer from the photo-excited donor to the acceptor is shown to be slower for An+1GGAn in comparison with AnGAGAn and (AG)2nA sequences. We have revealed that this is due to the reflectional symmetry of the last two sequences with respect to the axis passing through the middle base. As has been demonstrated, the symmetry of the sequence structure manifests itself in the reflectional symmetry of the energy eigenstates. In addition, it has been shown that (AG)2nA is the only symmetric sequence with a zero energy state in the middle of the LUMO tight-binding energy band. Based on our theoretical findings, we predict that the hairpin with this sequence should have the fastest coherent hole transfer rate among the class of base sequences studied.

  16. Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat. (United States)

    Sapag, Amalia; Irrazábal, Thergiory; Lobos-González, Lorena; Muñoz-Brauning, Carlos R; Quintanilla, María Elena; Tampier, Lutske


    Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism.

  17. Dodecamer d-AGATCTAGATCT and a homologous hairpin form triplex in the presence of peptide REWER.

    Directory of Open Access Journals (Sweden)

    Amrita Das

    Full Text Available We have designed a dodecamer d-AGATCTAGATCT (RY12 with alternate oligopurines and oligopyrimidines tracts and its homologous 28 bp hairpin oligomer (RY28 that forms a triple helix only in the presence of a pentapeptide REWER. An intermolecular triplex is formed by the single strand invasion of the RY28 duplex by RY12 in the presence of REWER. 5'- oligopurine end of RY12 binds to oligopurine sequence of RY28 in a parallel orientation and its oligopyrimidine stretch then changes strand and adopts an antiparallel orientation with the other strand of the duplex. Evidence for the formation of the triplex come from our studies of the UV melting curves, UV mixing curves, gel retardation assay, and chemical sequencing of 1∶1 mixture of dodecamer and hairpin oligonucleotides in the presence and absence of the peptide REWER. RY12 exists as a duplex that melts at 35°C. The hairpin (RY28 melts at 68°C. 1∶1 mixture of RY12 and RY28 in the absence of REWER gives a biphasic transition curve with thermodynamic properties corresponding to those of the melting of the duplex of RY12 and the hairpin RY28. However, the melting curve of this mixture is triphasic in the presence of the REWER; the thermodynamic parameters associated with the first phase (melting of the duplex of RY12, second phase (melting of the triplex and the third phase (melting of the hairpin show dependence on the molar ratio of peptide to oligonucleotides. Under appropriate conditions, gel retardation assay showed a shifted band that corresponds to a possible triplex. Chemical sequencing of KMnO4 and DEPC treated mixture of RY12, RY28 and REWER revealed the footprint of triplex.

  18. Simulation of infrared spectra for beta-hairpin peptides stabilized by an Aib-Gly turn sequence: correlation between conformational fluctuation and vibrational coupling. (United States)

    Kim, Joohyun; Huang, Rong; Kubelka, Jan; Bou Rcaron, Petr; Keiderling, Timothy A


    Vibrational spectra of a 12-residue beta-hairpin peptide, RYVEVBGKKILQ (HBG), stabilized by an Aib-Gly turn sequence (B = Aib) were investigated theoretically using a combination of molecular dynamics (MD) and density functional theory (DFT) calculations. Selected conformations of HBG were extracted from a classical MD trajectory and used for spectral simulations. DFT calculations, based on the Cartesian coordinate spectral property transfer protocol, were carried out for peptide structures in which all residues are replaced with Ala, except for the Aib and Gly residues, but the backbone (phi, psi, omega) structure of the original configuration is retained. The simulations provide a basis for interpretation of the HBG amide I infrared spectra in terms of structural variables such as detailed secondary structure and thermal conformational fluctuation as well as vibrational coupling as indicated by spectra of 13C isotope-labeled variants. The characteristic amide I band shape of such small beta-hairpin peptides appears to arise from the structure of the short antiparallel beta-sheet strands. The role of structural parameter fluctuation in vibrational coupling is evaluated by comparison of DFT-derived amide coupling constants for selected configurations and from transition dipole coupling calculations of coupling parameters between (13)C isotopically labeled residues for a MD-derived ensemble of configurations. Calculated results were compared with the experimentally obtained spectra for several (13)C isotope-labeled peptides of this sequence.

  19. Stable Polyglutamine Dimers Can Contain β-Hairpins with Interdigitated Side Chains—But Not α-Helices, β-Nanotubes, β-Pseudohelices, or Steric Zippers (United States)

    Miettinen, Markus S.; Monticelli, Luca; Nedumpully-Govindan, Praveen; Knecht, Volker; Ignatova, Zoya


    A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, β-nanotube, and β-pseudohelix conformers are also too short-lived to initiate aggregation. The β-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which β-hairpin-containing conformers act as templates for fibril formation. PMID:24739171

  20. Stable polyglutamine dimers can contain β-hairpins with interdigitated side chains-but not α-helices, β-nanotubes, β-pseudohelices, or steric zippers. (United States)

    Miettinen, Markus S; Monticelli, Luca; Nedumpully-Govindan, Praveen; Knecht, Volker; Ignatova, Zoya


    A common thread connecting nine fatal neurodegenerative protein aggregation diseases is an abnormally expanded polyglutamine tract found in the respective proteins. Although the structure of this tract in the large mature aggregates is increasingly well described, its structure in the small early aggregates remains largely unknown. As experimental evidence suggests that the most toxic species along the aggregation pathway are the small early ones, developing strategies to alleviate disease pathology calls for understanding the structure of polyglutamine peptides in the early stages of aggregation. Here, we present a criterion, grounded in available experimental data, that allows for using kinetic stability of dimers to assess whether a given polyglutamine conformer can be on the aggregation path. We then demonstrate that this criterion can be assessed using present-day molecular dynamics simulations. We find that although the α-helical conformer of polyglutamine is very stable, dimers of α-helices lack the kinetic stability necessary to support further oligomerization. Dimers of steric zipper, β-nanotube, and β-pseudohelix conformers are also too short-lived to initiate aggregation. The β-hairpin-containing conformers, instead, invariably form very stable dimers when their side chains are interdigitated. Combining these findings with the implications of recent solid-state NMR data on mature fibrils, we propose a possible pathway for the initial stages of polyglutamine aggregation, in which β-hairpin-containing conformers act as templates for fibril formation.

  1. The 5′-Proximal Hairpin of Turnip Yellow Mosaic Virus RNA: Its Role in Translation and Encapsidation


    Bink, Hugo H. J.; Schirawski, Jan; Haenni, Anne-Lise; Pleij, Cornelis W. A.


    The RNA genome of turnip yellow mosaic virus (TYMV) consists of more than 6,000 nucleotides. During a study of the roles of the two hairpins located in its 90-nucleotide 5′ untranslated region, it was observed that stabilization of the 5′-proximal hairpin leads to a delay in the development of symptoms on plants. This delay in symptom development for both locally and systemically infected leaves was found to be dependent on a change in the free energy of the hairpin caused by introduced mutat...

  2. Proton-coupled electron transfer in tyrosine and a β-hairpin maquette: reaction dynamics on the picosecond time scale. (United States)

    Pagba, Cynthia V; Chi, San-Hui; Perry, Joseph; Barry, Bridgette A


    In proteins, proton-coupled electron transfer (PCET) can involve the transient oxidation and reduction of the aromatic amino acid tyrosine. Due to the short life time of tyrosyl radical intermediates, transient absorption spectroscopy provides an important tool in deciphering electron-transfer mechanisms. In this report, the photoionization of solution tyrosine and tyrosinate was investigated using transient, picosecond absorption spectroscopy. The results were compared to data acquired from a tyrosine-containing β-hairpin peptide. This maquette, peptide A, is an 18-mer that exhibits π-π interaction between tyrosine (Y5) and histidine (H14). Y5 and H14 carry out an orthogonal PCET reaction when Y5 is oxidized in the mid-pH range. Photolysis of all samples (280 nm, instrument response: 360 fs) generated a solvated electron signal within 3 ps. A signal from the S1 state and a 410 nm signal from the neutral tyrosyl radical were also formed in 3 ps. Fits to S1 and tyrosyl radical decay profiles revealed biphasic kinetics with time constants of 10-50 and 400-1300 ps. The PCET reaction at pH 9 was associated with a significant decrease in the rate of tyrosyl radical and S1 decay compared to electron transfer (ET) alone (pH 11). This pH dependence was observed both in solution and peptide samples. The pH 9 reaction may occur with a sequential electron-transfer, proton-transfer (ETPT) mechanism. Alternatively, the pH 9 reaction may occur by coupled proton and electron transfer (CPET). CPET would be associated with a reorganization energy larger than that of the pH 11 reaction. Significantly, the decay kinetics of S1 and the tyrosyl radical were accelerated in peptide A compared to solution samples at both pH values. These data suggest either an increase in electronic coupling or a specific, sequence-dependent interaction, which facilitates ET and PCET in the β hairpin.

  3. Cationic modified nucleic acids for use in DNA hairpins and parallel triplexes

    DEFF Research Database (Denmark)

    Bomholt, Niels; Filichev, Vyacheslav V; Pedersen, Erik Bjerregaard


    Non-nucleosidic DNA monomers comprising partially protonated amines at low pH have been designed and synthesized. The modifications were incorporated into DNA oligonucleotides via standard DNA phosphoramidite synthesis. The ability of cationic modifications to stabilize palindromic DNA hairpins...... and parallel triplexes were evaluated using gel electrophoresis, circular dichroism and thermal denaturation measurements. The non-nucleosidic modifications were found to increase the thermal stability of palindromic hairpins at pH 8.0 as compared with a nucleosidic tetraloop (TCTC). Incorporation...... of modifications at the 5'-end of a triplex forming oligonucleotide resulted in a significant increase in thermal stability at low pH when the modifications were placed as the 5'-dangling end....

  4. A new tool in peptide engineering: a photoswitchable stilbene-type beta-hairpin mimetic. (United States)

    Erdélyi, Máté; Karlén, Anders; Gogoll, Adolf


    Peptide secondary structure mimetics are important tools in medicinal chemistry, as they provide analogues of endogenous peptides with new physicochemical and pharmacological properties. The development, synthesis, photochemical investigation, and conformational analysis of a stilbene-type beta-hairpin mimetic capable of light-triggered conformational changes have been achieved. In addition to standard spectroscopic techniques (nuclear Overhauser effects, amide temperature coefficients, circular dichroism spectroscopy), the applicability of self-diffusion measurements (longitudinal eddy current delay pulsed-field gradient spin echo (LED-PGSE) NMR technique) in conformational studies of oligopeptides is demonstrated. The title compound shows photoisomerization of the stilbene chromophore, resulting in a change in solution conformation between an unfolded structure and a folded beta-hairpin.

  5. Holographic particle image velocimetry measurements of hairpin vortices in a subcritical air channel flow (United States)

    Svizher, Alexander; Cohen, Jacob


    A holographic particle image velocimetry (HPIV) system is employed to study the evolution of coherent structures artificially generated in a plane Poiseuille air flow. As a first step the hot-wire technique and two-dimensional flow visualization are used to determine the generation conditions and dimensions of the coherent structures, their shedding frequency, trajectory, and convection velocity. Then, the HPIV method is utilized to obtain the instantaneous topology of the hairpin vortex and its associated three-dimensional distribution of the two (streamwise and spanwise) velocity components as well as the corresponding wall-normal vorticity. Finally, the experimental data are compared with results of related experimental and numerical studies. The present experimental results support the view that the generation of hairpins under various base flow conditions is governed by a basic mechanism, the important common elements of which are the shear of the base flow and an initial disturbance having a sufficiently large amplitude.

  6. Mechanical unfolding of RNA: From hairpins to structures with internal multiloops

    CERN Document Server

    Hyeon, Changbong


    Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer (LOT) experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force ($f$), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer (SOP) model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying complexity. Our work shows (i) the response of RNA to force is largely determined by the native structure; (ii) only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored.

  7. Effect of hydrophobic interactions on the folding mechanism of β-hairpins. (United States)

    Popp, Alexander; Wu, Ling; Keiderling, Timothy A; Hauser, Karin


    Hydrophobic interactions are essential in stabilizing protein structures. How they affect the folding pathway and kinetics, however, is less clear. We used time-resolved infrared spectroscopy to study the dynamics of hydrophobic interactions of β-hairpin variants of the sequence Trpzip2 (SWTWENGKWTWK-NH2) that is stabilized by two cross-strand Trp-Trp pairs. The hydrophobicity strength was varied by substituting the tryptophans pairwise by either tyrosines or valines. Relaxation dynamics were induced by a laser-excited temperature jump, which separately probed for the loss of the cross-strand β-hairpin interaction and the rise of the disordered structure. All substitutions tested result in reduced thermal stability, lower transition temperatures, and faster dynamics compared to Trpzip2. However, the changes in folding dynamics depend on the amino acid substituted for Trp. The aromatic substitution of Tyr for Trp results in the same kinetics for the unfolding of sheet and growth of disorder, with similar activation energies, independent of the substitution position. Substitution of Trp with a solely hydrophobic Val results in even faster kinetics than substitution with Tyr but is additionally site-dependent. If the hairpin has a Val pair close to its termini, the rate constants for loss of sheet and gain of disorder are the same, but if the pair is close to the turn, the sheet and disorder components show different relaxation kinetics. The Trp → Val substitutions reveal that hydrophobic interactions alone weakly stabilize the hairpin structure, but adding edge-to-face aromatic interaction strengthens it, and both modify the complex folding process.

  8. Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination

    National Research Council Canada - National Science Library

    Washio, T; Sasayama, J; Tomita, M


    Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes...

  9. Ultrasensitive detection of nucleic acids by template enhanced hybridization followed by rolling circle amplification and catalytic hairpin assembly. (United States)

    Song, Weiling; Zhang, Qiao; Sun, Wenbo


    An ultrasensitive protocol for fluorescent detection of DNA is designed by combining the template enhanced hybridization process (TEHP) with Rolling Circle Amplification (RCA) and Catalytic Hairpin Assembly (CHA), showing a remarkable amplification efficiency.

  10. Prediction of the beta-hairpins in proteins using support vector machine. (United States)

    Hu, Xiu Zhen; Li, Qian Zhong


    By using of the composite vector with increment of diversity and scoring function to express the information of sequence, a support vector machine (SVM) algorithm for predicting beta-hairpin motifs is proposed. The prediction is done on a dataset of 3,088 non homologous proteins containing 6,027 beta-hairpins. The overall accuracy of prediction and Matthew's correlation coefficient are 79.9% and 0.59 for the independent testing dataset. In addition, a higher accuracy of 83.3% and Matthew's correlation coefficient of 0.67 in the independent testing dataset are obtained on a dataset previously used by Kumar et al. (Nuclic Acid Res 33:154-159). The performance of the method is also evaluated by predicting the beta-hairpins of in the CASP6 proteins, and the better results are obtained. Moreover, this method is used to predict four kinds of supersecondary structures. The overall accuracy of prediction is 64.5% for the independent testing dataset.

  11. Effectiveness of hairpin probe in increasing the limit of detection for gold nanowire based-biosensor (United States)

    Tien Cao, Huu; Pham, Xuan Thanh Tung; Linh Ha, Van; Hieu Le, Van


    Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless and readily integrated into microelectronics and microfluidics, appear a promising alternative to optical methods for the detection of specific nucleic acid sequences. In keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain, signal-off behavior or instability. To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The observed signal gain is sufficient to achieve a demonstrated detection limit of 25 pM.

  12. Opening of the TAR hairpin in the HIV-1 genome causes aberrant RNA dimerization and packaging

    Directory of Open Access Journals (Sweden)

    Das Atze T


    Full Text Available Abstract Background The TAR hairpin is present at both the 5′ and 3′ end of the HIV-1 RNA genome. The 5′ element binds the viral Tat protein and is essential for Tat-mediated activation of transcription. We recently observed that complete TAR deletion is allowed in the context of an HIV-1 variant that does not depend on this Tat-TAR axis for transcription. Mutations that open the 5′ stem-loop structure did however affect the leader RNA conformation and resulted in a severe replication defect. In this study, we set out to analyze which step of the HIV-1 replication cycle is affected by this conformational change of the leader RNA. Results We demonstrate that opening the 5′ TAR structure through a deletion in either side of the stem region caused aberrant dimerization and reduced packaging of the unspliced viral RNA genome. In contrast, truncation of the TAR hairpin through deletions in both sides of the stem did not affect RNA dimer formation and packaging. Conclusions These results demonstrate that, although the TAR hairpin is not essential for RNA dimerization and packaging, mutations in TAR can significantly affect these processes through misfolding of the relevant RNA signals.

  13. Effect of Polypurine Reverse Hoogsteen Hairpins on Relevant Cancer Target Genes in Different Human Cell Lines. (United States)

    Villalobos, Xenia; Rodríguez, Laura; Solé, Anna; Lliberós, Carolina; Mencia, Núria; Ciudad, Carlos J; Noé, Véronique


    We studied the ability of polypurine reverse Hoogsteen hairpins (PPRHs) to silence a variety of relevant cancer-related genes in several human cell lines. PPRHs are hairpins formed by two antiparallel polypurine strands bound by intramolecular Hoogsteen bonds linked by a pentathymidine loop. These hairpins are able to bind to their target DNA sequence through Watson-Crick bonds producing specific silencing of gene expression. We designed PPRHs against the following genes: BCL2, TOP1, mTOR, MDM2, and MYC and tested them for mRNA levels, cytotoxicity, and apoptosis in prostate, pancreas, colon, and breast cancer cell lines. Even though all PPRHs were effective, the most remarkable results were obtained with those against BCL2 and mammalian target of rapamycin (mTOR) in decreasing cell survival and mRNA levels and increasing apoptosis in prostate, colon, and pancreatic cancer cells. In the case of TOP1, MDM2, and MYC, their corresponding PPRHs produced a strong effect in decreasing cell viability and mRNA levels and increasing apoptosis in breast cancer cells. Thus, we confirm that the PPRH technology is broadly useful to silence the expression of cancer-related genes as demonstrated using target genes involved in metabolism (DHFR), proliferation (mTOR), DNA topology (TOP1), lifespan and senescence (telomerase), apoptosis (survivin, BCL2), transcription factors (MYC), and proto-oncogenes (MDM2).

  14. Cloning of Novel Repeat-associated Small RNAs Derived from Hairpin Precursors in Oryza sativa

    Institute of Scientific and Technical Information of China (English)

    Chengguo YAO; Botao ZHAO; Wei LI; Yang LI; Wenming QIN; Bing HUANG; Youxin JIN


    Plant small non-coding RNAs including microRNAs (miRNAs), small interfering RNAs (siRNAs) and trans-acting siRNAs, play important roles in modulating gene expression in cells. Here we isolated 21 novel endogenous small RNA molecules, ranging from 18 to 24 nucleotides, in Oryza sativa that can be mapped to 111 hairpin precursors. Further analysis indicated that most of these hairpin sequences originated from putative miniature inverted-repeat transposable elements, a major type of DNA transposon.Considering that miRNA is characteristic of hairpin-like precursor and plant endogenous siRNAs are often located at transposon regions, we hypothesized that our cloned small RNAs might represent the intermediate product in the evolutionary process between siRNAs and miRNAs. Northern blot analysis indicated that five of them were much more abundantly expressed in flower compared to other tissues, implying their potential function in inflorescence. In conclusion, our results enrich rice small RNA data and provide a meaningful perspective for small RNA annotation in plants.

  15. Characterization of a [open quotes]kissing[close quotes] hairpin complex derived from the human immunodeficiency virus genome

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kung-Yao; Tinoco, Ignacio Jr. (Lawrence Berkeley Lab., CA (United States))


    Base-pair formation between hairpin loops-a [open quotes]kissing[close quotes] complex-is an RNA-folding motif that links two elements of RNA secondary structure. It is also a unique protein recognition site involved in regulation of ColE1 plasmid DNA replication. The trans-activation response element (TAR), a hairpin and bulge at the 5[prime] end of the untranslated leader region of the human immunodeficiency virus 1 mRNA, enhances the transcription of the virus and is necessary for viral replication. Gel electrophoresis and absorbance melting curves indicate that a synthesized RNA hairpin (Tar*-16) with a loop sequence complementary to the TAR loop sequence (CUGGGA) associates specifically with a 16-nucleotide TAR hairpin (Tar-16) to form a stable complex. RNase T1 probing indicates that the three guanines in the Tar-16 loop become inaccessible in the complex. NMR imino proton spectra reveal that 5 base pairs are formed between the two hairpin loops (Tar-16 and Tar*-16); only the adenine at the 3[prime] terminus of the TAR loop does not form a base pair with the 5[prime]-terminal uracil of the complementary loop. A 14-nucleotide hairpin [CCUA-(UCCCAG)UAGG] with a loop sequence complementary to the TAR loop is conserved within the gag gene of human immunodeficiency virus 1. A synthesized RNA hairpin corresponding to this conserved sequence also binds to the Tar-16 hairpin with high affinity. It is possible that the same RNA loop-loop interaction occurs during the viral life cycle. 40 refs., 7 figs.

  16. Analysis of complete genomes suggests that many prokaryotes do not rely on hairpin formation in transcription termination. (United States)

    Washio, T; Sasayama, J; Tomita, M


    Free energy values of mRNA tertiary structures around stop codons were systematically calculated to surmise the hairpin-forming potential for all genes in each of the 16 complete prokaryote genomes. Instead of trying to detect each individual hairpin, we averaged the free energy values around the stop codons over the entire genome to predict how extensively the organism relies on hairpin formation in the process of transcription termination. The free energy values of Escherichia coli K-12 shows a sharp drop, as expected, at 30 bp downstream of the stop codon, presumably due to hairpin-forming sequences. Similar drops are observed for Haemophilus influenzae Rd, Bacillus subtilis and Chlamydia trachomatis, suggesting that these organisms also form hairpins at their transcription termination sites. On the other hand, 12 other prokaryotes- Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis PCC6803, Helicobacter pylori, Borrelia burgdorferi, Methanococcus jannaschii, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, Aquifex aeolicus, Pyrococcus horikoshii, Mycobacterium tuberculosis and Treponema pallidum -show no apparent decrease in free energy value at the corresponding regions. This result suggests that these prokaryotes, or at least some of them, may never form hairpins at their transcription termination sites.

  17. Fluorescence-based characterization of genetically encoded peptides that fold in live cells: progress toward a generic hairpin scaffold (United States)

    Cheng, Zihao; Campbell, Robert E.


    Binding proteins suitable for expression and high affinity molecular recognition in the cytoplasm or nucleus of live cells have numerous applications in the biological sciences. In an effort to add a new minimal motif to the growing repertoire of validated non-immunoglobulin binding proteins, we have undertaken the development of a generic protein scaffold based on a single β-hairpin that can fold efficiently in the cytoplasm. We have developed a method, based on the measurement of fluorescence resonance energy transfer (FRET) between a genetically fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), that allows the structural stability of recombinant β-hairpin peptides to be rapidly assessed both in vitro and in vivo. We have previously reported the validation of this method when applied to a 16mer tryptophan zipper β-hairpin. We now describe the use of this method to evaluate the potential of a designed 20mer β-hairpin peptide with a 3rd Trp/Trp cross-strand pair to function as a generic protein scaffold. Quantitative analysis of the FRET efficiency, resistance to proteolysis (assayed by loss of FRET), and circular dichroism spectra revealed that the 20mer peptide is significantly more tolerant of destabilizing mutations than the 16mer peptide. Furthermore, we experimentally demonstrate that the in vitro determined β-hairpin stabilities are well correlated with in vivo β-hairpin stabilities as determined by FRET measurements of colonies of live bacteria expressing the recombinant peptides flanked by CFP and YFP. Finally, we report on our progress to develop highly folded 24mer and 28mer β-hairpin peptides through the use of fluorescence-based library screening.

  18. Characterizing the formation and regeneration of hairpin vortices in a laminar boundary layer (United States)

    Sabatino, Daniel R.; Maharjan, Rijan


    A free surface water channel is used to study hairpin vortex formation created by fluid injection through a narrow slot into a laminar boundary layer. Particle image velocimetry flow-field measurements of injections into quiescent cross-flow conditions confirm that elongated ring vortices are produced with a nondimensionalized circulation strength that is approximately linear with formation time. Unlike circular ring vortices, a limiting strength is not observed at a nondimensional formation time of 4 due to the proximity of the counter-rotating vortex pair. Identical injections are made into a laminar boundary layer at different free-stream velocities and streamwise slot positions (485 ≤ Reδ∗ ≤ 584) with average injection velocity ratios between 0.08 and 0.16. Visualizations indicate that the shear layer between the low x-momentum injected fluid and the boundary layer creates a Kelvin-Helmholtz instability that forms the hairpin vortex head which then monotonically decreases in circulation strength with downstream distance. A similar process can form, or regenerate, a secondary hairpin vortex upstream of the primary vortex with a circulation strength of the head that is comparable to the strength of the primary head at the time of regeneration. However, the legs of the primary vortex continue to strengthen up to regeneration. The peak circulation in the legs is not directly correlated to the strength of the original elongated ring vortex. However, when the circulation is scaled with the injection momentum ratio it is linearly related to scaled injection time. It is proposed that the injection momentum ratio and nondimensionalized injection time based on the wall normal penetration time can be used to identify threshold conditions which produce a secondary vortex. It is suggested that this criterion may be used to identify the minimum strength of flow structures that would be capable of regeneration and thus transition initiation.

  19. Stability of an amphipathic helix-hairpin surfactant peptide in liposomes. (United States)

    Waring, Alan J; Gupta, Monik; Gordon, Larry M; Fujii, Gary; Walther, Frans J


    Surfactant protein B (SP-B; 79 residues) is a member of the saposin superfamily and plays a pivotal role in lung function. The N- and C-terminal regions of SP-B, cross-linked by two disulfides, were theoretically predicted to fold as charged amphipathic helices, suggesting participation in surfactant activities. Previous studies with oxidized Super Mini-B (SMB), a construct based on the N- and C-regions of SP-B (i.e., residues 1-25 and 63-78) joined with a designer turn (-PKGG-) and two disulfides, indicated that freshly prepared SMB in lipids folded as a surface active, α-helix-hairpin. Because other peptides modeled on α-helical SP domains lost helicity and surfactant activity on storage, experiments were here performed on oxidized SMB in surfactant liposomes stored at ~2-8°C for ≤5.5years. Captive bubble surfactometry confirmed low minimum surface tensions for fresh and stored SMB preparations. FTIR spectroscopy of fresh and stored SMB formulations showed secondary structures compatible with the peptide folding as α-helix-hairpin. A homology (I-TASSER) model of oxidized SMB demonstrated a globular protein, exhibiting a core of hydrophobic residues and a surface of polar residues. Since mass spectroscopy indicated that the disulfides were maintained on storage, the stability of SMB may be partly due to the disulfides bringing the N- and C-α-helices closer. Mass spectroscopy of stored SMB preparations showed some methionine oxidation, and also partial deacylation of surfactant phospholipids to form lyso-derivatives. However, the stable conformation and activity of stored SMB surfactant suggest that the active helix-hairpin resists these chemical changes which otherwise may lead to surfactant inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Role of the DIS hairpin in replication of human immunodeficiency virus type 1. (United States)

    Berkhout, B; van Wamel, J L


    The virion-associated genome of human immunodeficiency virus type 1 consists of a noncovalently linked dimer of two identical, unspliced RNA molecules. A hairpin structure within the untranslated leader transcript is postulated to play a role in RNA dimerization through base pairing of the autocomplementary loop sequences. This hairpin motif with the palindromic loop sequence is referred to as the dimer initiation site (DIS), and the type of interaction is termed loop-loop kissing. Detailed phylogenetic analysis of the DIS motifs in different human and simian immunodeficiency viruses revealed conservation of the hairpin structure with a 6-mer palindrome in the loop, despite considerable sequence divergence. This finding supports the loop-loop kissing mechanism. To test this possibility, proviral genomes with mutations in the DIS palindrome were constructed. The appearance of infectious virus upon transfection into SupT1 T cells was delayed for the DIS mutants compared with that obtained by transfection of the wild-type provirus (pLAI), confirming that this RNA motif plays an important role in virus replication. Surprisingly, the RNA genome extracted from mutant virions was found to be fully dimeric and to have a normal thermal stability. These results indicate that the DIS motif is not essential for human immunodeficiency virus type 1 RNA dimerization and suggest that DIS base pairing does not contribute to the stability of the mature RNA dimer. Instead, we measured a reduction in the amount of viral RNA encapsidated in the mutant virions, suggesting a role of the DIS motif in RNA packaging. This result correlates with the idea that the processes of RNA dimerization and packaging are intrinsically linked, and we propose that DIS pairing is a prerequisite for RNA packaging.

  1. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer. (United States)

    Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa


    A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

  2. Numerical simulation of quasi-streamwise hairpin-like vortex generation in turbulent boundary layer

    Institute of Scientific and Technical Information of China (English)

    ZHANG Nan; LU Li-peng; DUAN Zhen-zhen; YUAN Xiang-jiang


    A mechanism for generation of near wall quasi-streamwise hairpin-like vortex (QHV) and secondary quasi-streamwise vortices (SQV) is presented. The conceptual model of resonant triad in the theory of hydrodynamic instability and direct numerical simulation of a turbulent boundary layer were applied to reveal the formation of QHV and SQV. The generation procedures and the characteristics of the vortex structures are obtained, which share some similarities with previous numerical simulations. The research using resonant triad conceptual model and numerical simulation provides a possibility for investigating and controling the vortex structures, which play a dominant role in the evolution of coherent structures in the near-wall region.

  3. Formation and evolution of a hairpin vortex induced by subharmonic sinuous low-speed streaks

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jian; Dong, Gang; Lu, Ziheng, E-mail: [State Key Laboratory of Transient Physics, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu Province, People’s Republic of China (China)


    In this paper, a process of the formation and evolution of hairpin vortices, which originated from the interaction between the spanwise-aligned low-speed streaks with a subharmonic sinuous (SS) oscillation mode, is studied using a direct numerical simulation method in a small periodic local region of an incompressible plane channel flow. The initial artificial perturbations are used to excite the SS-mode oscillation of two spanwise-aligned low-speed streaks in such a flow. A new mechanism of formation and decay of the hairpin vortices is proposed in which the shear layer induced by the spanwise collision and merging between the low-speed streaks is emphasized. Our results show that the streamwise vortices can be induced by the SS-mode streaks and then developed into an X-like pattern at the initial stage due to the mutual induction effect. The X-like vortices further enhance the spanwise oscillation and lift-up of the two streaks that thus lead to the spanwise collision and merging of the low-speed streaks and produce a low-speed region in high-speed fluid. The strong shear layer between the high- and low-speed fluids gives rise to the spanwise vorticity that connects the X-like streamwise vortices and forms the Λ-like vortex. Once the low-speed region entirely enters the high-speed fluid, the shear layer shows the ring shape and results in the transition from a Λ-like vortex to Ω-like one. After that, the viscous diffusion of the low-speed region in the high-speed fluid leads to the decay of the Ω-like vortex; the collision and merging of the low-speed streaks simultaneously reoccur upstream and give birth to a secondary Λ-like vortex, which exhibits behavior that is nearly similar with that of the primary one. Although the hairpin vortex packet is not observed in the present plane channel flow, the regeneration of the hairpin vortex suggests that this type of vortical structure plays an important role in the wall-bounded flow. (paper)

  4. A β-hairpin-binding protein for three different disease-related amyloidogenic proteins. (United States)

    Shaykhalishahi, Hamed; Mirecka, Ewa A; Gauhar, Aziz; Grüning, Clara S R; Willbold, Dieter; Härd, Torleif; Stoldt, Matthias; Hoyer, Wolfgang


    Amyloidogenic proteins share a propensity to convert to the β-structure-rich amyloid state that is associated with the progression of several protein-misfolding disorders. Here we show that a single engineered β-hairpin-binding protein, the β-wrapin AS10, binds monomers of three different amyloidogenic proteins, that is, amyloid-β peptide, α-synuclein, and islet amyloid polypeptide, with sub-micromolar affinity. AS10 binding inhibits the aggregation and toxicity of all three proteins. The results demonstrate common conformational preferences and related binding sites in a subset of the amyloidogenic proteins. These commonalities enable the generation of multispecific monomer-binding agents.

  5. Knockdown of homeobox A5 by small hairpin RNA inhibits proliferation and enhances cytarabine chemosensitivity of acute myeloid leukemia cells. (United States)

    Li, Na; Jia, Xiuhong; Wang, Jianyong; Li, Youjie; Xie, Shuyang


    Homeobox genes encode transcription factors that are essential for embryonic morphogenesis and differentiation. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. Previous studies have suggested that the increased expression levels of homeobox (HOX)A genes was correlated with the cytogenetic findings associated with poor prognosis in acute myeloid leukemia and mixed lineage leukemia. The aim of the present study was to investigate the role of HOXA5 in leukemia. The U937 human leukemia cell line was transfected with a HOXA5‑targeted short hairpin RNA (shRNA) to determine the effects of downregulation of the HOXA5 on proliferation, apoptosis, cell cycle distribution and chemoresistance in leukemia cells. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses demonstrated that the mRNA and protein expression levels of HOXA5 were markedly suppressed following transfection with an shRNA‑containing vector. Knockdown of HOXA5 significantly inhibited cell proliferation, as determined by Cell Counting kit‑8 assay. Flow cytometry revealed that reduced HOXA5 expression levels resulted in cell cycle arrest at the G1 phase, and induced apoptosis. In addition, western blot analysis demonstrated that HOXA5 knockdown increased the expression levels of caspase‑3, and reduced the expression levels of survivin in the U937 cells. Furthermore, knockdown of HOXA5 in the U937 cells enhanced their chemosensitivity to cytarabine. The results of the present study suggested that downregulation of HOXA5 by shRNA may trigger apoptosis and overcome drug resistance in leukemia cells. Therefore, HOXA5 may serve as a potential target for developing novel therapeutic strategies for leukemia.

  6. Retrospective monitoring of minimal residual disease using hairpin-shaped clone specific primers in B-cell lymphoma affected dogs. (United States)

    Gentilini, Fabio; Turba, Maria E; Forni, Monica


    Lymphoma is one of the most common forms of cancer in dogs as it is in humans but, unlike humans, the cure rates in canines are still very low. Despite the fact that high grade B-cell lymphomas are considered to be chemotherapy responsive, almost all treated dogs ultimately relapse and die due to the residual malignant lymphocytes, namely minimal residual disease (MRD). It would be extremely valuable for clinicians to detect, monitor and quantify MRD for risk group stratification, effective treatment intervention and outcome prediction. The PCRs targeting the Ig gene rearrangements constitute one of the most reliable tools to this end. We have recently validated a method which exploits hairpin-shaped primers for quantifying MRD. In the present study, that method is conveniently used for retrospectively monitoring MRD in the peripheral blood of 8 dogs diagnosed with B-cell lymphoma who underwent chemotherapy. All dogs attained complete remission. The median disease-free interval was 254.5 days (range 63-774) while the median survival time was 313.5 days (range 143-817 days). At admission, all dogs, except one which had already been treated with prednisone, had circulating neoplastic cells. All dogs attained complete remission (CR) which was almost always matched with a complete MRD response. The persistence of MRD despite apparent CR indicated a worse prognosis and a short duration of CR. Finally, the relapse is consistently anticipated by the reappearance of MRD in the peripheral blood. The study confirmed the suitability of an MRD monitoring assay as a clinical decision-making tool.

  7. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection. (United States)

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao


    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay.

  8. A combinatorial approach to the repertoire of RNA kissing motifs; towards multiplex detection by switching hairpin aptamers. (United States)

    Durand, Guillaume; Dausse, Eric; Goux, Emma; Fiore, Emmanuelle; Peyrin, Eric; Ravelet, Corinne; Toulmé, Jean-Jacques


    Loop-loop (also known as kissing) interactions between RNA hairpins are involved in several mechanisms in both prokaryotes and eukaryotes such as the regulation of the plasmid copy number or the dimerization of retroviral genomes. The stability of kissing complexes relies on loop parameters (base composition, sequence and size) and base combination at the loop-loop helix - stem junctions. In order to identify kissing partners that could be used as regulatory elements or building blocks of RNA scaffolds, we analysed a pool of 5.2 × 10(6) RNA hairpins with randomized loops. We identified more than 50 pairs of kissing RNA hairpins. Two kissing motifs, 5'CCNY and 5'RYRY, generate highly stable complexes with KDs in the low nanomolar range. Such motifs were introduced in the apical loop of hairpin aptamers that switch between unfolded and folded state upon binding to their cognate target molecule, hence their name aptaswitch. The aptaswitch-ligand complex is specifically recognized by a second RNA hairpin named aptakiss through loop-loop interaction. Taking advantage of our kissing motif repertoire we engineered aptaswitch-aptakiss modules for purine derivatives, namely adenosine, GTP and theophylline and demonstrated that these molecules can be specifically and simultaneously detected by surface plasmon resonance or by fluorescence anisotropy. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Parvoviral left-end hairpin ears are essential during infection for establishing a functional intranuclear transcription template and for efficient progeny genome encapsidation. (United States)

    Li, Lei; Cotmore, Susan F; Tattersall, Peter


    The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex.

  10. Identification of short hairpin RNA targeting foot-and-mouth disease virus with transgenic bovine fetal epithelium cells.

    Directory of Open Access Journals (Sweden)

    Hongmei Wang

    Full Text Available BACKGROUND: Although it is known that RNA interference (RNAi targeting viral genes protects experimental animals, such as mice, from the challenge of Foot-and-mouth disease virus (FMDV, it has not been previously investigated whether shRNAs targeting FMDV in transgenic dairy cattle or primary transgenic bovine epithelium cells will confer resistance against FMDV challenge. PRINCIPAL FINDING: Here we constructed three recombinant lentiviral vectors containing shRNA against VP2 (RNAi-VP2, VP3 (RNAi-VP3, or VP4 (RNAi-VP4 of FMDV, and found that all of them strongly suppressed the transient expression of a FLAG-tagged viral gene fusion protein in 293T cells. In BHK-21 cells, RNAi-VP4 was found to be more potent in inhibition of viral replication than the others with over 98% inhibition of viral replication. Therefore, recombinant lentiviral vector RNAi-VP4 was transfected into bovine fetal fibroblast cells to generate transgenic nuclear donor cells. With subsequent somatic cell cloning, we generated forty transgenic blastocysts, and then transferred them to 20 synchronized recipient cows. Three transgenic bovine fetuses were obtained after pregnant period of 4 months, and integration into chromosome in cloned fetuses was confirmed by Southern hybridization. The primary tongue epithelium cells of transgenic fetuses were isolated and inoculated with 100 TCID(50 of FMDV, and it was observed that shRNA significantly suppressed viral RNA synthesis and inhibited over 91% of viral replication after inoculation of FMDV for 48 h. CONCLUSION: RNAi-VP4 targeting viral VP4 gene appears to prevent primary epithelium cells of transgenic bovine fetus from FMDV infection, and it could be a candidate shRNA used for cultivation of transgenic cattle against FMDV.

  11. Is TNF-a-targeted short hairpin RNA (shRNA) a novel potential therapeutic tool in psoriasis treatment?

    DEFF Research Database (Denmark)

    Stenderup, Karin; Jakobsen, Maria; Rosada, Cecilia


    TNF-a shRNA was used to transduce HEK293 cells and verify vector-derived TNF-a knockdown in vitro. In vivo, psoriasis skin was exposed to lentiviral TNF-a shRNAs by a single intra-dermal injection. Psoriasis skin for the in vivo study was obtained from psoriatic plaque skin biopsies that were...... transplanted onto SCID mice employing the psoriasis xenograft model. Three weeks after exposure, biopsies were taken and the epidermal thickness as an endpoint for evaluating psoriasis and the levels of TNF-a mRNA were measured. The lentiviral TNF-a shRNAs down-regulated the TNF-a expression in vitro...

  12. Adenoviral short hairpin RNA targeting phosphodiesterase 5 attenuates cardiac remodeling and cardiac dysfunction following myocardial infarction in mice

    Institute of Scientific and Technical Information of China (English)



    Objective To observe the impact of PDE5shRNA on cardiac remodeling and heart function following myocardial infarction in mice.Methods Myocardial infarction(MI)was induced in mice by left coronary artery ligation.Mice were randomly assigned to sham operation group(n=6),PDE5shRNA group(n=12),common adenovirus group(n=15)and DMEM group(n=8).Four weeks post-MI,the survival rate was evaluated.

  13. Knockdown of NAPA using short-hairpin RNA sensitizes cancer cells to cisplatin: implications to overcome chemoresistance. (United States)

    Wu, Zchong-Zcho; Chao, Chuck C-K


    Cisplatin is a widely used anti-cancer drug which targets DNA in replicating cells. In the present study, we found that NAPA--a protein found in the endoplasmic reticulum (ER) and implicated in protein trafficking--protects cells against cisplatin. Accordingly, knockdown of NAPA using lentivirus-encoded shRNA (shNAPA) induced ER stress similar to cisplatin treatment in HEK293 cells. A low dose of cisplatin also elicited a mild ER stress response associated with the accumulation of the protective proteins BiP and NAPA. Remarkably, knockdown of NAPA induced apoptosis and enhanced cisplatin-induced cytotoxicity/apoptosis, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of NAPA increased resistance to cisplatin by reducing cisplatin-induced ER stress and apoptosis. The modulatory effects of shNAPA required the tumor suppressor p53 since the effects of NAPA knockdown were reduced by the p53 inhibitor PFT-alpha and in H1299 cells which are p53-null. A partial reversal of cisplatin resistance was also observed in cisplatin-resistant HeLa cells following knockdown of NAPA. Our results also indicated that calpain is required for ER-mediated apoptosis. Importantly, combined cisplatin/shNAPA treatment suppressed tumor growth in vivo in xenograph experiments performed in nude mice. Taken together, these observations suggest that NAPA represents a target of cisplatin, and that knockdown of NAPA may improve cisplatin-based cancer therapy.

  14. [Preparation and identification of recombinant adenoviruses carrying short hairpin RNA targeting parathyroid hormone related protein of goat]. (United States)

    Xing, Ruifang; Zheng, Huiling; Liu, Xuemei; Yan, Linhui; An, Junhui; Yang, Zhenyu; Zhu, Zhenzhen


    Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL-DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0 x 1(9) PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.

  15. Short hairpin RNA screen indicates that Klotho beta/FGF19 protein overcomes stasis in human colonic epithelial cells. (United States)

    Kim, Jinyong; Eskiocak, Ugur; Stadler, Guido; Lou, Zhenjun; Kuro-o, Makoto; Shay, Jerry W; Wright, Woodring E


    Normal human colonic epithelial cells (HCECs) are not immortalized by telomerase alone but also require CDK4. Some human cell types growth-arrest due to stress- or aberrant signaling-induced senescence (stasis). Stasis represents the consequences of growth conditions culture that are inadequate to maintain long-term proliferation. Overexpressed CDK4 titers out p16 and allows cells to ignore the growth arrest signals produced by stasis. To identify factors contributing to the inadequate culture environment, we used a 62,000-member shRNA library to knock down factors cooperating with human telomerase reverse transcriptase (hTERT) in the immortalization of HCECs. Knockdown of Klotho gamma (KLG; also known as KLPH and LCTL) allowed hTERT to immortalize HCECs. KLG is one isoform of the Klotho family of factors that coordinate interaction between different FGF ligands and the FGF receptor. We also found that knockdown of KLG induced another member of the Klotho family, Klotho beta (KLB). Induction of KLB was maintained and could activate ERK1/2 in immortalized cells. Supplementation of the culture medium with the KLB ligand FGF19 had a similar effect on hTERT-expressing HCECs as knockdown of KLG regarding both immortalization and down-regulation of the tumor suppressor Klotho alpha. Together, these data suggest that KLB is an important regulator in the immortalization of HCECs by facilitating FGF19 growth factor signaling.

  16. A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus. (United States)

    Chen, Huixin; Takei, Fumie; Koay, Evelyn Siew-Chuan; Nakatani, Kazuhiko; Chu, Justin Jang Hann


    Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples.

  17. Ultrasensitive detection of microRNAs based on hairpin fluorescence probe assisted isothermal amplification. (United States)

    Ma, Cuiping; Liu, Sen; Shi, Chao


    A hairpin fluorescence probe assisted isothermal amplification strategy was used for microRNAs (miRNAs) detection. The fluorescence hairpin probe was rationally designed by software NUPACK to reduce background signal. This isothermal amplification method consisted of two circuits. The amplification strategy not only could detect miRNA, but also amplified and reversely transcribed miRNA into DNA to enhance the stability of the target. The approach was ultrasensitive and as low as 8.5×10(-15)mol/L miR-Let-7a, corresponding to 8.5×10(-20)mol miR-Let-7a in 10µL, was able to be detected within 20min at 37°C. Moreover, successful detection of miR-Let-7a in a total RNA sample was also achieved. Thus, the rapid, simple, isothermal, and highly sensitive approach should be a promising tool for on-the-spot detection.

  18. Grooved nanowires from self-assembling hairpin molecules for solar cells. (United States)

    Tevis, Ian D; Tsai, Wei-Wen; Palmer, Liam C; Aytun, Taner; Stupp, Samuel I


    One of the challenges facing bulk heterojunction organic solar cells is obtaining organized films during the phase separation of intimately mixed donor and acceptor components. We report here on the use of hairpin-shaped sexithiophene molecules to generate by self-assembly grooved nanowires as the donor component in bulk heterojunction solar cells. Photovoltaic devices were fabricated via spin-casting to produce by solvent evaporation a percolating network of self-assembled nanowires and fullerene acceptors. Thermal annealing was found to increase power conversion efficiencies by promoting domain growth while still maintaining this percolating network of nanostructures. The benefits of self-assembly and grooved nanowires were examined by building devices from a soluble sexithiophene derivative that does not form one-dimensional structures. In these systems, excessive phase separation caused by thermal annealing leads to the formation of defects and lower device efficiencies. We propose that the unique hairpin shape of the self-assembling molecules allows the nanowires as they form to interact well with the fullerenes in receptor-ligand type configurations at the heterojunction of the two domains, thus enhancing device efficiencies by 23%.

  19. Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids. (United States)

    Lv, Yifan; Cui, Liang; Peng, Ruizi; Zhao, Zilong; Qiu, Liping; Chen, Huapei; Jin, Cheng; Zhang, Xiao-Bing; Tan, Weihong


    Here, we propose an efficient strategy for enzyme- and hairpin-free nucleic acid detection called an entropy beacon (abbreviated as Ebeacon). Different from previously reported DNA hybridization/displacement-based strategies, Ebeacon is driven forward by increases in the entropy of the system, instead of free energy released from new base-pair formation. Ebeacon shows high sensitivity, with a detection limit of 5 pM target DNA in buffer and 50 pM in cellular homogenate. Ebeacon also benefits from the hairpin-free amplification strategy and zero-background, excellent thermostability from 20 °C to 50 °C, as well as good resistance to complex environments. In particular, based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs, Ebeacon also shows high selectivity toward base mutations, such as substitution, insertion, and deletion and, therefore, is an efficient nucleic acid detection method, comparable to most reported enzyme-free strategies.

  20. Predicting 3D structure, flexibility and stability of RNA hairpins in monovalent and divalent ion solutions

    CERN Document Server

    Shi, Ya-Zhou; Wang, Feng-Hua; Zhu, Xiao-Long; Tan, Zhi-Jie


    A full understanding of RNA-mediated biology would require the knowledge of three-dimensional (3D) structures, structural flexibility and stability of RNAs. To predict RNA 3D structures and stability, we have previously proposed a three-bead coarse-grained predictive model with implicit salt/solvent potentials. In this study, we will further develop the model by improving the implicit-salt electrostatic potential and involving a sequence-dependent coaxial stacking potential to enable the model to simulate RNA 3D structure folding in divalent/monovalent ion solutions. As compared with the experimental data, the present model can predict 3D structures of RNA hairpins with bulge/internal loops (<77nt) from their sequences at the corresponding experimental ion conditions with an overall improved accuracy, and the model also makes reliable predictions for the flexibility of RNA hairpins with bulge loops of different length at extensive divalent/monovalent ion conditions. In addition, the model successfully pred...

  1. Construction and identification of plasmid-based replicon vector system derived from Alphavirus%质粒型甲病毒复制子载体系统的构建及其功能鉴定

    Institute of Scientific and Technical Information of China (English)

    张凤娟; 唐丽; 尉研; 王焕琴; 杨蕴芝; 吴萌; 张鹏; 梁国栋; 朱武洋


    目的 构建质粒型甲病毒复制子载体系统,并对其功能进行鉴定.方法 在XJ-160病毒感染性cDNA克隆的基础上,将病毒非结构基因序列分为三个片段扩增,分步克隆至真核表达载体pVAX1 CMV启动子下游,并用多克隆位点序列替代病毒的结构基因构建XJ-160病毒质粒型复制子载体.将病毒核蛋白基因及包膜糖蛋白基因分别克隆至pVAX1 CMV启动子下游构建载体的两个辅助质粒.通过绿色荧光蛋白报告基因及海肾荧光素酶报告基因的表达检验该质粒型载体系统的功能特性.结果 成功构建了包括复制子载体和辅助质粒的质粒型甲病毒复制子载体系统,且该复制子载体系统可成功表达绿色荧光蛋白报告基因及海肾荧光素酶报告基因.结论 本研究构建了能够表达外源基因的质粒型甲病毒复制子载体系统,为目标基因表达、重组病毒颗粒制备等奠定了基础.%Objective To construct a plasmid-based replicon vector system derived from Alphavirus,and its functions were identified.Methods based on XJ-160 viral infectious cDNA clone,the virus non-structural gene sequence were devided into three fragments to amplify and then cloned into the eukaryotic expression vector pVAX1 CMV promoter downstream by step clone.A multiple cloning site sequence replacing the virus structural gene,plasmid-based replicon vector derived from XJ-160 virus was constructed.Virus nucleoprotein gene and envelope glycoprotein gene were respectively cloned into pVAX1 CMV promoter downstream,constructing two helper plasmid vectors.Then by the expression of green fluorescent protein reporter gene and renilla luciferase reporter gene we test the function of the plasmid-based replicon vector system.Results The plasmid-based replicon vector system derived from XJ-160 virus was successfully constructed,and green fluorescent protein reporter gene and renilla luciferase reporter gene were successfully expressed

  2. Phylogenetic study on structural elements of HIV-1 poly(A region. 2. USE domain and TAR hairpin

    Directory of Open Access Journals (Sweden)

    Zarudnaya M. I.


    Full Text Available Aim Phylogenetic study on structural elements in the poly(A region of human immunodeficiency virus type 1 (HIV-1, in particular the major upstream sequence element (USE, which stimulates polyadenylation of HIV-1 transcript, and the TAR (trans-activation response hairpin, which juxtaposes spatially the AAUAAA and USE signals. Methods. The secondary structure of these elements has been predicted by UNA Fold program. Results. The structure of USE domain and TAR hairpin has been analysed in 1679 HIV-1 genomes and 17 genomes of simian immunodeficiency virus SIVcpzPtt. We found 376 and 588 different sequences for these elements, respectively, and revealed the most frequent base changes and subtypeand country-specific mutations. Only 43 % of HIV-1 isolates contain variants of the USE domain which occur with a frequency 5 % (the main variants and 35 % of isolates contain main variants of the TAR hairpin. We found that the SIV USE domain and TAR hairpin most closely resemble those found in HIV-1 genomes of A/G-containing subtypes. Conclusions. The results of our large-scale phylogenetic study support a hypothesis on the interaction between tRNA3Lys and the 3' end of HIV-1 genomic RNA and a controversial supposition of HIV-1 genome dimerization by the TAR-TAR kissing mechanism. Since the TAR hairpin is a target for developing antiviral drugs based on the inhibition of signal elements, the data on specific structural features of this hairpin may be useful for new antivirals design.

  3. Chemical syntheses of inhibitory substrates of the RNA-RNA ligation reaction catalyzed by the hairpin ribozyme. (United States)

    Massey, Archna P; Sigurdsson, Snorri Th


    The chemical syntheses of RNA oligomers containing modifications on the 5'-carbon of the 5'-terminal nucleoside for crystallographic and mechanistic studies of the hairpin ribozyme are reported. Phosphoramidites 4 and 8 were prepared and used in solid phase syntheses of RNA oligomers containing the sequence 5'-N'UCCUCUCC, where N' indicates either 5'-chloro-5'-deoxyguanosine or 5'-amino-5'-deoxyguanosine, respectively. A ribozyme ligation assay with the 5'-chloro- and 5'-amino-modified RNA oligomers demonstrated their inhibition of the hairpin-catalyzed RNA-RNA ligation reaction.

  4. Microsecond resolved electron density measurements with a hairpin resonator probe in a pulsed ICP discharge

    CERN Document Server

    Peterson, David; Larson, Lynda; Shannon, Steven


    Time resolved electron density measurements in pulsed RF discharges are shown using a hairpin resonance probe using low cost electronics, on par with normal Langmuir probe boxcar mode operation. Time resolution of less than one microsecond has been demonstrated. A signal generator produces the applied microwave frequency; the reflected waveform is passed through a directional coupler and filtered to remove the RF component. The signal is heterodyned with a frequency mixer and read by an oscilloscope. At certain points during the pulse, the plasma density is such that the applied frequency is the same as the resonance frequency of the probe/plasma system, creating a dip in the reflected signal. The applied microwave frequency is shifted in small increments in a frequency boxcar routine to determine the density as a function of time. The system uses a grounded probe to produce low cost, high fidelity, and highly reproducible electron density measurements that can work in harsh chemical environments. Measurement...

  5. Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures

    Energy Technology Data Exchange (ETDEWEB)

    Nandi, Chayan K.; Parui, Partha P.; Brutschy, Bernhard [University of Frankfurt, Institute for Physical and Theoretical Chemistry, Frankfurt (Germany); Schmidt, Thorsten L.; Heckel, Alexander [University of Frankfurt, Cluster of Excellence Macromolecular Complexes, c/o Institute for Organic Chemistry and Chemical Biology, Frankfurt (Germany)


    We have recently constructed a ''DNA strut'' consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson-Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions. (orig.)

  6. Nanoplasmonic Tweezers Visualize Protein p53 Suppressing Unzipping of Single DNA-Hairpins

    CERN Document Server

    Kotnala, Abhay


    Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein-DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be $2\\times 10^{-20}$ J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein-DNA interactions to revolutionize the fight against genetic diseases.

  7. Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide. (United States)

    Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin


    A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Double nanohole optical tweezers visualize protein p53 suppressing unzipping of single DNA-hairpins. (United States)

    Kotnala, Abhay; Gordon, Reuven


    Here we report on the use of double-nanohole (DNH) optical tweezers as a label-free and free-solution single-molecule probe for protein-DNA interactions. Using this approach, we demonstrate the unzipping of individual 10 base pair DNA-hairpins, and quantify how tumor suppressor p53 protein delays the unzipping. From the Arrhenius behavior, we find the energy barrier to unzipping introduced by p53 to be 2 × 10(-20) J, whereas cys135ser mutant p53 does not show suppression of unzipping, which gives clues to its functional inability to suppress tumor growth. This transformative approach to single molecule analysis allows for ultra-sensitive detection and quantification of protein-DNA interactions to revolutionize the fight against genetic diseases.

  9. Structure of the antimicrobial beta-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Kaznessis, Yiannis N


    All atom molecular dynamics simulations of the 18-residue beta-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH(2)) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure of t...

  10. Long inverted repeat transiently stalls DNA replication by forming hairpin structures on both leading and lagging strands. (United States)

    Lai, Pey Jiun; Lim, Chew Theng; Le, Hang Phuong; Katayama, Tsutomu; Leach, David R F; Furukohri, Asako; Maki, Hisaji


    Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading- and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCD-sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.

  11. Structural classification of the amide I sites of a beta-hairpin with isotope label 2DIR spectroscopy.

    NARCIS (Netherlands)

    Roy, S.; Jansen, T.L.Th.A.; Knoester, J.


    We present a theoretical study of the possibility to use isotope label two-dimensional infrared (2DIR) spectroscopy to obtain site specific structural information in trpzip2. This small beta-hairpin peptide was designed as a model system for studying protein folding in beta-sheet structures. In

  12. Dual hairpin-like molecular beacon based on coralyne-adenosine interaction for sensing melamine in dairy products. (United States)

    Wang, Guangfeng; Zhu, Yanhong; Chen, Ling; Zhang, Xiaojun


    This study presents a novel dual hairpin-like molecular beacon (MB) for the selective and sensitive detection of melamine (MA) based on the conjugation of MA and thymine. In this protocol, the coordination between coralyne and adenosine (A) leaded a dual hairpin-like MB and the fluorophore-quencher pair is close proximity resulting in the fluorescence quenching. With the addition of MA, it conjugated with thymine in the loop part of dual hairpin-like MB by triple H-bonds, triggering the dissociation of the dual hairpin-like MB. The resulting spatial separation of the fluorophore from quencher induced the enhancement in fluorescence emission. Under the optimized conditions, the sensor exhibited a wide linear range of 8×10(-9)-1.6×10(-5) M (R(2)=0.9969) towards MA, with a low detection limit of 5 nM, approximately 4000 times lower than the Drug Administration and the US Food estimated MA safety limit. The real milk samples were also investigated with a satisfying result.

  13. A Comparison of Lu You’s Phoenix Hairpin and Elizabeth Barrett Browning’s Sonnets from the Portuguese (43)

    Institute of Scientific and Technical Information of China (English)



    Both Lu You and Elizabeth Barrett Browning enjoy great popularity as a poet.In this paper I compare and contract Lu You’s well-known love ci-poem Phoenix Hairpin with Elizabeth Barrett Browning’s famous Sonnets from the Portuguese (43) in respect of theme,style,and way of expression.

  14. Combinatorial pattern discovery approach for the folding trajectory analysis of a beta-hairpin.

    Directory of Open Access Journals (Sweden)

    Laxmi Parida


    Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity c in RO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the

  15. Combinatorial Pattern Discovery Approach for the Folding Trajectory Analysis of a beta-Hairpin.

    Directory of Open Access Journals (Sweden)


    Full Text Available The study of protein folding mechanisms continues to be one of the most challenging problems in computational biology. Currently, the protein folding mechanism is often characterized by calculating the free energy landscape versus various reaction coordinates, such as the fraction of native contacts, the radius of gyration, RMSD from the native structure, and so on. In this paper, we present a combinatorial pattern discovery approach toward understanding the global state changes during the folding process. This is a first step toward an unsupervised (and perhaps eventually automated approach toward identification of global states. The approach is based on computing biclusters (or patterned clusters-each cluster is a combination of various reaction coordinates, and its signature pattern facilitates the computation of the Z-score for the cluster. For this discovery process, we present an algorithm of time complexity cinRO((N + nm log n, where N is the size of the output patterns and (n x m is the size of the input with n time frames and m reaction coordinates. To date, this is the best time complexity for this problem. We next apply this to a beta-hairpin folding trajectory and demonstrate that this approach extracts crucial information about protein folding intermediate states and mechanism. We make three observations about the approach: (1 The method recovers states previously obtained by visually analyzing free energy surfaces. (2 It also succeeds in extracting meaningful patterns and structures that had been overlooked in previous works, which provides a better understanding of the folding mechanism of the beta-hairpin. These new patterns also interconnect various states in existing free energy surfaces versus different reaction coordinates. (3 The approach does not require calculating the free energy values, yet it offers an analysis comparable to, and sometimes better than, the methods that use free energy landscapes, thus validating the

  16. Water isotope effect on the thermostability of a polio viral RNA hairpin: A metadynamics study (United States)

    Pathak, Arup K.; Bandyopadhyay, Tusar


    Oral polio vaccine is considered to be the most thermolabile of all the common childhood vaccines. Despite heavy water (D2O) having been known for a long time to stabilise attenuated viral RNA against thermodegradation, the molecular underpinnings of its mechanism of action are still lacking. Whereas, understanding the basis of D2O action is an important step that might reform the way other thermolabile drugs are stored and could possibly minimize the cold chain problem. Here using a combination of parallel tempering and well-tempered metadynamics simulation in light water (H2O) and in D2O, we have fully described the free energy surface associated with the folding/unfolding of a RNA hairpin containing a non-canonical basepair motif, which is conserved within the 3'-untranslated region of poliovirus-like enteroviruses. Simulations reveal that in heavy water (D2O) there is a considerable increase of the stability of the folded basin as monitored through an intramolecular hydrogen bond (HB), size, shape, and flexibility of RNA structures. This translates into a higher melting temperature in D2O by 41 K when compared with light water (H2O). We have explored the hydration dynamics of the RNA, hydration shell around the RNA surface, and spatial dependence of RNA-solvent collective HB dynamics in the two water systems. Simulation in heavy water clearly showed that D2O strengthens the HB network in the solvent, lengthens inter-residue water-bridge lifetime, and weakens dynamical coupling of the hairpin to its solvation environment, which enhances the rigidity of solvent exposed sites of the native configurations. The results might suggest that like other added osmoprotectants, D2O can act as a thermostabilizer when used as a solvent.

  17. A regenerative electrochemical biosensor for mercury(II) by using the insertion approach and dual-hairpin-based amplification

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Jing; Ling, Yu; Gao, Zhong Feng [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Lei, Jing Lei [College of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044 (China); Luo, Hong Qun, E-mail: [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Li, Nian Bing, E-mail: [Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)


    Highlights: • The dual-hairpin structure as a signal amplifier is label-free and handy. • The strategy uses the insertion approach to improve the hybridization efficiency. • This biosensor has a low detection limit (28 pM) for detection of Hg{sup 2+}. • This biosensor can be easily regenerated by using L-cysteine. - Abstract: A simple and effective biosensor for Hg{sup 2+} determination was investigated. The novel biosensor was prepared by the insertion approach that the moiety-labeled DNA inserted into a loosely packed cyclic-dithiothreitol (DTT) monolayer, improving the hybridization efficiency. Electrochemical impedance spectroscopy studies of two biosensors (single-hairpin and dual-hairpin structure DNA modified electrodes) used for Hg{sup 2+} detection indicated that the dual-hairpin modified electrode had a larger electron transfer resistance change (ΔR{sub ct}). Consequently, the dual-hairpin structure was used as a signal amplifier for the preparation of a selective Hg{sup 2+} biosensor. This biosensor exhibited an excellent selectivity toward Hg{sup 2+} over Cd{sup 2+}, Pd{sup 2+}, Co{sup 2+} etc. Also, a linear relation was observed between the ΔR{sub ct} and Hg{sup 2+} concentrations in a range from 0.1 nM to 5 μM with a detection limit of 28 pM under optimum conditions. Moreover, the biosensor can be reused by using L-cysteine and successfully applied for detecting Hg{sup 2+} in real samples.

  18. Interstrand side chain--side chain interactions in a designed beta-hairpin: significance of both lateral and diagonal pairings. (United States)

    Syud, F A; Stanger, H E; Gellman, S H


    The contributions of interstrand side chain-side chain contacts to beta-sheet stability have been examined with an autonomously folding beta-hairpin model system. RYVEV(D)PGOKILQ-NH2 ((D)P = D-proline, O = ornithine) has previously been shown to adopt a beta-hairpin conformation in aqueous solution, with a two-residue loop at D-Pro-Gly. In the present study, side chains that display interstrand NOEs (Tyr-2, Lys-9, and Leu-11) are mutated to alanine or serine, and the conformational impact of the mutations is assessed. In the beta-hairpin conformation Tyr-2 and Leu-11 are directly across from one another (non-hydrogen bonded pair). This "lateral" juxtaposition of two hydrophobic side chains appears to contribute to beta-hairpin conformational stability, which is consistent with results from other beta-sheet model studies and with statistical analyses of interstrand residue contacts in protein crystal structures. Interaction between the side chains of Tyr-2 and Lys-9 also stabilizes the beta-hairpin conformation. Tyr-2/Lys-9 is a "diagonal" interstrand juxtaposition because these residues are not directly across from one another in terms of the hydrogen bonding registry between the strands. This diagonal interaction arises from the right-handed twist that is commonly observed among beta-sheets. Evidence of diagonal side chain-side chain contacts has been observed in other autonomously folding beta-sheet model systems, but we are not aware of other efforts to determine whether a diagonal interaction contributes to beta-sheet stability.

  19. A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)


    The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

  20. Hydrogels constructed via self-assembly of beta-hairpin molecules (United States)

    Ozbas, Bulent

    There is a recent and growing interest in hydrogel materials that are formed via peptide self-assembly for tissue engineering applications. Peptide based materials are excellent candidates for diverse applications in biomedical field due to their responsive behavior and complex self-assembled structures. However, there is very limited information on the self-assembly and resultant network and mechanical properties of these types of hydrogels. The main goal of this dissertation is to investigate the self-assembly mechanism and viscoelastic properties of hydrogels that can be altered by changing solution conditions as well as the primary structure of the peptide. These hydrogels are formed via intramolecular folding and consequent self-assembly of 20 amino acid long beta-hairpin peptide molecules (Max1). The peptide molecules are locally amphiphilic with two linear strands of alternating hydrophobic valine and hydrophilic lysine amino acids connected with a Dproline-LProline turn sequence. Circular dichroism and FTIR spectroscopy show that at physiological conditions peptides are unfolded in the absence of salt. By raising the ionic strength of the solution electrostatic interactions between charged lysines are screened and the peptide arms are forced into a beta-sheet secondary structure stabilized by the turn sequence. These folded molecules intermolecularly assemble via hydrophobic collapse and hydrogen bonding into a three dimensional network. Folding and self-assembly of these molecules can also be triggered by increasing temperature and/or pH of the peptide solution. In addition, the random-coil to beta-sheet transition of the beta-hairpin peptides is pH and, with proper changes in the peptide sequence, thermally reversible. Rheological measurements demonstrate that the resultant supramolecular structure forms an elastic material, whose structure, and thus modulus, can be tuned by magnitude of the stimulus. Hydrogels recover their initial viscoelastic

  1. Molecular dynamics of β-hairpin models of epigenetic recognition motifs. (United States)

    Zheng, Xiange; Wu, Chuanjie; Ponder, Jay W; Marshall, Garland R


    The conformations and stabilities of the β-hairpin model peptides of Waters (Riemen, A. J.; Waters, M. L. Biochemistry 2009, 48, 1525; Hughes, R. M.; Benshoff, M. L.; Waters, M. L. Chemistry 2007, 13, 5753) have been experimentally characterized as a function of lysine ε-methylation. These models were developed to explore molecular recognition of known epigenetic recognition motifs. This system offered an opportunity to computationally examine the role of cation-π interactions, desolvation of the ε-methylated ammonium groups, and aromatic/aromatic interactions on the observed differences in NMR spectra. AMOEBA, a second-generation force field (Ponder, J. W.; Wu, C.; Ren, P.; Pande, V. S.; Chodera, J. D.; Schnieders, M. J.; Haque, I.; Mobley, D. L.; Lambrecht, D. S.; DiStasio, R. A., Jr.; Head-Gordon, M.; Clark, G. N.; Johnson, M. E.; Head-Gordon, T. J. Phys. Chem. B 2010, 114, 2549), was chosen as it includes both multipole electrostatics and polarizability thought to be essential to accurately characterize such interactions. Independent parametrization of ε-methylated amines was required from which aqueous solvation free energies were estimated and shown to agree with literature values. Molecular dynamics simulations (100 ns) using the derived parameters with model peptides, such as Ac-R-W-V-W-V-N-G-Orn-K(Me)(n)-I-L-Q-NH(2), where n = 0, 1, 2, or 3, were conducted in explicit solvent. Distances between the centers of the indole rings of the two-tryptophan residues, 2 and 4, and the ε-methylated ammonium group on Lys-9 as well as the distance between the N- and C-termini were monitored to estimate the strength and orientation of the cation-π and aromatic/aromatic interactions. In agreement with the experimental data, the stability of the β-hairpin increased significantly with lysine ε-methylation. The ability of MD simulations to reproduce the observed NOEs for the four peptides was further estimated for the monopole-based force fields, AMBER, CHARMM, and

  2. Chitosan-plasmid DNA nanoparticles encoding small hairpin RNA targeting MMP-3 and -13 to inhibit the expression of dedifferentiation related genes in expanded chondrocytes. (United States)

    Zhao, Jingxin; Fan, Xiangli; Zhang, Qiang; Sun, Fangfei; Li, Xiaojian; Xiong, Chuan; Zhang, Chunli; Fan, Hongbin


    Overexpression of matrix metalloproteinase (MMP)-3 and -13 can lead to the dedifferentiation of expanded chondrocytes. After implanting dedifferentiated cells for cartilage defect repair, graft failure may occur. Short hairpin RNA (shRNA) is a powerful genetic tool to reduce the expression of target genes. This study investigated the effects of chitosan-plasmid DNA (pDNA) nanoparticles encoding shRNA targeting MMP-3 and -13 on the dedifferentiation of expanded chondrocytes. The objective was to optimize the parameters of chitosan-pDNA formulation for achieving higher efficiency of pDNA delivery and gene silencing. The chitosan-pDNA nanoparticles were prepared using a complex coacervation process. Then the characteristics including size, shape, stability, and transfection efficiency were compared in different groups. The results indicated that chitosan of 800 kDa at N/P ratio of 4 and pH 7.0 was optimal to prepare chitosan-pDNA nanoparticles. These nanoparticles showed high DNA loading efficiency (95.8 ± 1.5%) and high gene transfection efficiency (24.5 ± 1.6%). After the expanded chondrocytes were transfected by chitosan-pDNA nanoparticles, MMP-3-610 and MMP-13-2024 groups showed greater suppression in mRNA and protein levels. The results indicated that chitosan-pDNA nanoparticles encoding shRNA targeting MMP-3 and -13 had great potential in silencing the dedifferentiation-related genes for regenerating prolonged and endurable cartilage.

  3. Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing (United States)

    Butardo, Vito M.; Fitzgerald, Melissa A.; Bird, Anthony R.; Gidley, Michael J.; Flanagan, Bernadine M.; Larroque, Oscar; Resurreccion, Adoracion P.; Laidlaw, Hunter K. C.; Jobling, Stephen A.; Morell, Matthew K.; Rahman, Sadequr


    The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed. PMID:21791436

  4. Determination of low-energy structures of a small RNA hairpin using Monte Carlo–based techniques

    Indian Academy of Sciences (India)

    Sudhanshu Shanker; Pradipta Bandyopadhyay


    The energy landscape of RNA is known to be extremely rugged, and hence finding low-energy structures starting from a random structure is a challenging task for any optimization algorithm. In the current work, we have investigated the ability of one Monte Carlo–based optimization algorithm, Temperature Basin Paving, to explore the energy landscape of a small RNA T-loop hairpin. In this method, the history of the simulation is used to increase the probability of states less visited in the simulation. It has been found that using both energy and end-to-end distance as the biasing parameters in the simulation, the partially folded structure of the hairpin starting from random structures could be obtained.

  5. β-Sheet 13C Structuring Shifts Appear only at the H-bonded Sites of Hairpins (United States)

    Shu, Irene; Stewart, James M.; Scian, Michele; Kier, Brandon L.


    The 13C chemical shifts measured for designed β hairpins indicate that the structuring shifts (upfield for Cα and C′, downfield for Cβ) previously reported as diagnostic for β structuring in protein appear only at the H-bonded strand residues. The resulting periodicity of structuring shift magnitudes is not, however, a consequence of H-bonding status; rather, it reflects a previously unrecognized alternation in the backbone torsion angles of β strands. This feature of hairpins is also likely to be present in proteins. The study provides reference values for the expectation shifts for 13C sites in β structures that should prove useful in the characterization of the folding equilibria of β sheet models. PMID:21214243

  6. Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots. (United States)

    Liu, Siming; Yoder, John I


    Understanding the functions encoded by plant genes can be facilitated by reducing transcript levels by hairpin RNA (hpRNA) mediated silencing. A bottleneck to this technology occurs when a gene encodes a phenotype that is necessary for cell viability and silencing the gene inhibits transformation. Here we compared the use of two chemically inducible plant promoter systems to drive hpRNA mediated gene silencing in transgenic, hairy roots. We cloned the gene encoding the Yellow Fluorescence Protein (YFP) into the dexamethasone inducible vector pOpOff2 and into the estradiol induced vector pER8. We then cloned a hpRNA targeting YFP under the regulation of the inducible promoters, transformed Medicago truncatula roots, and quantified YFP fluorescence and mRNA levels. YFP fluorescence was normal in pOpOff2 transformed roots without dexamethasone but was reduced with dexamethasone treatment. Interestingly, dexamethasone removal did not reverse YFP inhibition. YFP expression in roots transformed with pER8 was low even in the absence of inducer. We used the dexamethasone system to silence acetyl-CoA carboxylase gene and observed prolific root growth when this construct was transformed into Medicago until dexamethasone was applied. Our study shows that dexamethasone inducibility can be useful to silence vital genes in transgenic roots.

  7. Efficient production of superior dumbbell-shaped DNA minimal vectors for small hairpin RNA expression. (United States)

    Yu, Han; Jiang, Xiaoou; Tan, Kar Tong; Hang, Liting; Patzel, Volker


    Genetic therapy holds great promise for the treatment of inherited or acquired genetic diseases; however, its breakthrough is hampered by the lack of suitable gene delivery systems. Dumbbell-shaped DNA minimal vectors represent an attractive, safe alternative to the commonly used viral vectors which are fraught with risk, but dumbbell generation appears to be costly and time-consuming. We developed a new PCR-based method for dumbbell production which comprises only two steps. First, PCR amplification of the therapeutic expression cassette using chemically modified primers to form a ready-to-ligate DNA structure; and second, a highly efficient intramolecular ligation reaction. Compared with conventional strategies, the new method produces dumbbell vectors more rapidly, with higher yields and purity, and at lower costs. In addition, such produced small hairpin RNA expressing dumbbells triggered superior target gene knockdown compared with conventionally produced dumbbells or plasmids. Our novel method is suitable for large-scale dumbbell production and can facilitate clinical applications of this vector system.

  8. Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides. (United States)

    Oparin, Peter B; Mineev, Konstantin S; Dunaevsky, Yakov E; Arseniev, Alexander S; Belozersky, Mikhail A; Grishin, Eugene V; Egorov, Tsezi A; Vassilevski, Alexander A


    A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

  9. Differential effects of alpha-helical and beta-hairpin antimicrobial peptides against Acanthamoeba castellanii. (United States)

    Sacramento, R S; Martins, R M; Miranda, A; Dobroff, A S S; Daffre, S; Foronda, A S; De Freitas, D; Schenkman, S


    In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and P6, corresponding to the N-terminus of the pore-forming protein from Triatoma infestans, a blood-sucking insect, and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells. P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.

  10. Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus

    Directory of Open Access Journals (Sweden)

    Fangquan Wang


    Full Text Available Rice black-streaked dwarf virus (RBSDV belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21–24 nt small interfering RNA (siRNA. By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

  11. The inverse autotransporter intimin exports its passenger domain via a hairpin intermediate. (United States)

    Oberhettinger, Philipp; Leo, Jack C; Linke, Dirk; Autenrieth, Ingo B; Schütz, Monika S


    Autotransporter proteins comprise a large family of virulence factors that consist of a β-barrel translocation unit and an extracellular effector or passenger domain. The β-barrel anchors the protein to the outer membrane of Gram-negative bacteria and facilitates the transport of the passenger domain onto the cell surface. By inserting an epitope tag into the N terminus of the passenger domain of the inverse autotransporter intimin, we generated a mutant defective in autotransport. Using this stalled mutant, we could show that (i) at the time point of stalling, the β-barrel appears folded; (ii) the stalled autotransporter is associated with BamA and SurA; (iii) the stalled intimin is decorated with large amounts of SurA; (iv) the stalled autotransporter is not degraded by periplasmic proteases; and (v) inverse autotransporter passenger domains are translocated by a hairpin mechanism. Our results suggest a function for the BAM complex not only in insertion and folding of the β-barrel but also for passenger translocation.

  12. Comparison of Cytotoxic Activity in Leukemic Lineages Reveals Important Features of β-Hairpin Antimicrobial Peptides. (United States)

    Buri, Marcus V; Torquato, Heron F Vieira; Barros, Carlos Castilho; Ide, Jaime S; Miranda, Antonio; Paredes-Gamero, Edgar J


    Several reports described different modes of cell death triggered by antimicrobial peptides (AMPs) due to direct effects on membrane disruption, and more recently by apoptosis and necrosis-like patterns. Cytotoxic curves of four β-hairpin AMPs (gomesin, protegrin, tachyplesin, and polyphemusin) were obtained from several human leukemic lineages and normal monocytes and Two cell lines were then selected based on their cytotoxic sensitivity. One was sensitive to AMPs (K562) and the other resistant (KG-1) and their effect compared between these lineages. Thus, these lineages were chosen to further investigate biological features related with their cytotoxicities to AMPs. Stimulation with AMPs produced cell death, with activation of caspase-3, in K562 lineage. Increase on the fluidity of plasmatic membrane by reducing cholesterol potentiated cytotoxicity of AMPs in both lineages. Quantification of internal and external gomesin binding to the cellular membrane of both K562 and KG-1 cells showed that more peptide is accumulated inside of K562 cells. Additionally, evaluation of multi-drug resistant pumps activity showed that KG-1 has more activity than K562 lineage. A comparison of intrinsic gene patterns showed great differences between K562 and KG-1, but stimulation with gomesin promoted few changes in gene expression patterns. Differences in internalization process through the plasma membrane, multidrug resistance pumps activity, and gene expression pattern are important features to AMPs regulated cell death. J. Cell. Biochem. 118: 1764-1773, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  13. Stability and immunogenicity properties of the gene-silencing polypurine reverse Hoogsteen hairpins. (United States)

    Villalobos, Xenia; Rodríguez, Laura; Prévot, Jeanne; Oleaga, Carlota; Ciudad, Carlos J; Noé, Véronique


    Gene silencing by either small-interference RNAs (siRNA) or antisense oligodeoxynucleotides (aODN) is widely used in biomedical research. However, their use as therapeutic agents is hindered by two important limitations: their low stability and the activation of the innate immune response. Recently, we developed a new type of molecule to decrease gene expression named polypurine reverse Hoogsteen hairpins (PPRHs) that bind to polypyrimidine targets in the DNA. Herein, stability experiments performed in mouse, human, and fetal calf serum and in PC3 cells revealed that the half-life of PPRHs is much longer than that of siRNAs in all cases. Usage of PPRHs with a nicked-circular structure increased the binding affinity to their target sequence and their half-life in FCS when bound to the target. Regarding the innate immune response, we determined that the levels of the transcription factors IRF3 and its phosphorylated form, as well as NF-κB were increased by siRNAs and not by PPRHs; that the expression levels of several proinflammatory cytokines including IL-6, TNF-α, IFN-α, IFN-ß, IL-1ß, and IL-18 were not significantly increased by PPRHs; and that the cleavage and activation of the proteolytic enzyme caspase-1 was not triggered by PPRHs. These determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response.

  14. Stereopositional Outcome in the Packing of Dissimilar Aromatics in Designed β-Hairpins. (United States)

    Makwana, Kamlesh Madhusudan; Mahalakshmi, Radhakrishnan


    A popular strategy in the de novo design of stable β-sheet structures for various biomedical applications is the incorporation of aromatic pairs at the non-hydrogen-bonding (NHB) position. However, it is important to explicitly understand how aryl pair packing at the NHB region is coordinated with backbone structural rearrangements, and to delineate the benefits and drawbacks associated with stereopositional choice of dissimilar aromatic pairs. Here, we probe the consequences of flipped Trp/Tyr pairs by using engineered permutants at the NHB position of dodecapeptide β-hairpins, proximal and distal to the turn. Extensive conformational analysis of these peptides using NMR and CD spectroscopy reveal that a classic Edge-to-Face and Face-to-Edge geometry at the proximal and distal aromatic pairs, respectively, in YW-WY, is the most stabilizing. Such a preferred packing geometry in YW-WY results in a highly twisted β-sheet backbone, with Trp always providing a 'Face' orientation to its dissimilar aromatic partner Tyr. Flipping the proximal and/or distal aromatic pair distorts the ideal T-shaped geometry, and results in alternate aryl arrangements that can adversely affect strand twist and β-sheet stability. Our study reveals the existence of a strong stereopositional influence on the packing of dissimilar aromatic pairs. Our findings highlight the importance of modeling physical interaction forces while designing protein and peptide structures for functional applications.

  15. High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants.

    Directory of Open Access Journals (Sweden)

    Pu Yan

    Full Text Available With the wide use of double-stranded RNA interference (RNAi for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.

  16. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core. (United States)

    Hoop, Cody L; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M; Boatz, Jennifer C; Mandal, Abhishek; Lewandowski, Józef R; Wetzel, Ronald; van der Wel, Patrick C A


    Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

  17. The effect of active fluctuations on the dynamics of particles, motors and hairpins

    CERN Document Server

    Vandebroek, Hans


    Inspired by recent experiments on the dynamics of particles and polymers in artificial cytoskeletons and in cells, we introduce a modified Langevin equation for a particle in an environment that is a viscoelastic medium and that is brought out of equilibrium by the action of active fluctuations caused by molecular motors. We show that within such a model, the motion of a free particle crosses over from superdiffusive to subdiffusive as observed for tracer particles in an {\\it in vitro} cytoskeleton or in a cell. We investigate the dynamics of a particle confined by a harmonic potential as a simple model for the motion of the tethered head of kinesin-1. We find that the probability that the head is close to its binding site on the microtubule can be enhanced by a factor of two due to active forces. Finally, we study the dynamics of a particle in a double well potential as a model for the dynamics of DNA-hairpins. We show that the active forces effectively lower the potential barrier between the two minima and ...

  18. Mutational analysis of an archaeal minichromosome maintenance protein exterior hairpin reveals critical residues for helicase activity and DNA binding

    Directory of Open Access Journals (Sweden)

    Brewster Aaron S


    Full Text Available Abstract Background The mini-chromosome maintenance protein (MCM complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7, the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM, six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp. We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

  19. Coexpression of Escherichia coli RNase Ⅲ in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs

    Institute of Scientific and Technical Information of China (English)

    Jae Man Lee; Yoshito Kojin; Tsuneyuki Tatsuke; Hiroaki Mon; Yoshitaka Miyagawa; Takahiro Kusakabe


    Long hairpin dsRNA transcribed from chromosomal DNA can induce RNA interference in Bombyx mori cells,although its gene silencing efficiency is lower than that of exogenously introduced double-stranded RNAs(dsRNAs).To solve this problem,we monitored the nuclear cytoplasmic translocation of the transcribed hairpin dsRNA and analyzed the processing efficiency into mature small interfering RNA(siRNA).Northern blot analysis revealed that the transcribed hairpin dsRNAs were spliced and transported into the cytoplasm,but were not effectively diced into siRNAs.Interestingly,RNAi with hairpin dsRNAs from genome-integrated IR transgene was stimulated by the coexpression of Escherichia coli RNase Ⅲ,although this exogenous enzyme seemed to bring about nonspecific cleavage of cellular mRNA.

  20. Oligodeoxynucleotide binding to (CTG) · (CAG) microsatellite repeats inhibits replication fork stalling, hairpin formation, and genome instability. (United States)

    Liu, Guoqi; Chen, Xiaomi; Leffak, Michael


    (CTG)(n) · (CAG)(n) trinucleotide repeat (TNR) expansion in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)(n) · (CAG)(n) structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)(45) · (CAG)(45) causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)(45) · (CAG)(45) lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)(45) · (CAG)(45) expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.

  1. Probing the Gaseous Structure of a β-Hairpin Peptide with H/D Exchange and Electron Capture Dissociation (United States)

    Straus, Rita N.; Jockusch, Rebecca A.


    An improved understanding of the extent to which native protein structure is retained upon transfer to the gas phase promises to enhance biological mass spectrometry, potentially streamlining workflows and providing fundamental insights into hydration effects. Here, we investigate the gaseous conformation of a model β-hairpin peptide using gas-phase hydrogen-deuterium (H/D) exchange with subsequent electron capture dissociation (ECD). Global gas-phase H/D exchange levels, and residue-specific exchange levels derived from ECD data, are compared among the wild type 16-residue peptide GB1p and several variants. High protection from H/D exchange observed for GB1p, but not for a truncated version, is consistent with the retention of secondary structure of GB1p in the gas phase or its refolding into some other compact structure. Four alanine mutants that destabilize the hairpin in solution show levels of protection similar to that of GB1p, suggesting collapse or (re)folding of these peptides upon transfer to the gas phase. These results offer a starting point from which to understand how a key secondary structural element, the β-hairpin, is affected by transfer to the gas phase. This work also demonstrates the utility of a much-needed addition to the tool set that is currently available for the investigation of the gaseous conformation of biomolecules, which can be employed in the future to better characterize gaseous proteins and protein complexes.

  2. Streak instability and generation of hairpin-vortices by a slotted jet in channel crossflow: Experiments and linear stability analysis (United States)

    Philip, Jimmy; Karp, Michael; Cohen, Jacob


    Streaks and hairpin-vortices are experimentally generated in a laminar plane Poiseuille crossflow by injecting a continuous jet through a streamwise slot normal to the crossflow, with air as the working media. Small disturbances form stable streaks, however, higher disturbances cause the formation of streaks which undergo instability leading to the generation of hairpin vortices. Particular emphasis is placed on the flow conditions close to the generation of hairpin-vortices. Measurements are carried out in the cases of natural and phase-locked disturbance employing smoke visualisation, particle image velocimetry, and hot-wire anemometry, which include, the dominant frequency, wavelength, and the disturbance shape (or eigenfunctions) associated with the coherent part of the velocity field. A linear stability analysis for both one- and two-dimensional base-flows is carried out to understand the mechanism of instability and good agreement of wavelength and eigenfunctions are obtained when compared to the experimental data, and a slight under-prediction of the growth-rates by the linear stability analysis consistent with the final nonlinear stages in transitional flows. Furthermore, an energy analysis for both the temporal and spatial stability analysis revels the dominance of the symmetric varicose mode, again, in agreement with the experiments, which is found to be governed by the balance of the wallnormal shear and dissipative effects rather than the spanwise shear. In all cases the anti-symmetric sinuous modes governed by the spanwise shear are found to be damped both in analysis and in our experiments.

  3. Probing the Gaseous Structure of a β-Hairpin Peptide with H/D Exchange and Electron Capture Dissociation (United States)

    Straus, Rita N.; Jockusch, Rebecca A.


    An improved understanding of the extent to which native protein structure is retained upon transfer to the gas phase promises to enhance biological mass spectrometry, potentially streamlining workflows and providing fundamental insights into hydration effects. Here, we investigate the gaseous conformation of a model β-hairpin peptide using gas-phase hydrogen-deuterium (H/D) exchange with subsequent electron capture dissociation (ECD). Global gas-phase H/D exchange levels, and residue-specific exchange levels derived from ECD data, are compared among the wild type 16-residue peptide GB1p and several variants. High protection from H/D exchange observed for GB1p, but not for a truncated version, is consistent with the retention of secondary structure of GB1p in the gas phase or its refolding into some other compact structure. Four alanine mutants that destabilize the hairpin in solution show levels of protection similar to that of GB1p, suggesting collapse or (re)folding of these peptides upon transfer to the gas phase. These results offer a starting point from which to understand how a key secondary structural element, the β-hairpin, is affected by transfer to the gas phase. This work also demonstrates the utility of a much-needed addition to the tool set that is currently available for the investigation of the gaseous conformation of biomolecules, which can be employed in the future to better characterize gaseous proteins and protein complexes.

  4. Short tunnels.

    NARCIS (Netherlands)

    Schreuder, D.A.


    Before dealing with the question of lighting short tunnels, it is necessary define what is meant by a tunnel and when it should be called 'short'. Confined to motorized road traffic the following is the most apt definition of a tunnel: every form of roofing-over a road section, irrespective of it le

  5. SKOV-3 cell imaging by paramagnetic particles labeled with hairpin cell-penetrating peptides

    Institute of Scientific and Technical Information of China (English)

    ZHAI Xiao-hui; LIU Min; GUO Xiao-juan; WANG Si-cen; ZHANG Hong-xia; GUO You-min


    Background The hairpin cell-penetrating peptides (hCPPs) demonstrate an interesting characteristic of conditioned activation by molecules. We hypothesized that hCPPs have the potential to selectively deliver a paramagnetic gadolinium probe into the matrix metalloproteinase 2 (MMP-2) positive human ovary adenocarcinoma cell lines,SKOV-3.Methods hCPPs were synthesized and labeled with 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid gadolinium (Ⅲ) (Gd-DOTA) and fluorescein isothiocyanate (FITC) by f-moc strategy using a standard solid phase peptide synthesis protocol. MMP-2 expression and activity were demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) and zymography. Internalization and location of hCPPs in SKOV-3 cells were observed by fluorescein imaging and flow cytometery. Selective delivery of Gd-DOTA in SKOV-3 cells was observed by magnetic resonance imaging (MRI) and transmission electron microscopy (TEM).Results The uptake of hCPPs by SKOV-3 cells depended on the activity of MMP-2. T1WI signals of SKOV-3 cells treated with Gd-DOTA-hCPPs suggested the uptake of Gd-DOTA-hCPPs increased in a time- (r=0.990, P <0.01) and concentration-dependent manner (r=0.964, P <0.001), but was inhibited by a MMP-2 inhibitor. Electron-dense particles observed in the cytoplasm and nucleus by transmission electron microscopy proved the intracellular penetration of gadolinium.Conclusions hCPPs can be used as an effective vector for an MRI molecular probe to assess the activity of MMP-2.

  6. Function and anatomy of plant siRNA pools derived from hairpin transgenes

    Directory of Open Access Journals (Sweden)

    Lee Kevin AW


    Full Text Available Abstract Background RNA interference results in specific gene silencing by small-interfering RNAs (siRNAs. Synthetic siRNAs provide a powerful tool for manipulating gene expression but high cost suggests that novel siRNA production methods are desirable. Strong evolutionary conservation of siRNA structure suggested that siRNAs will retain cross-species function and that transgenic plants expressing heterologous siRNAs might serve as useful siRNA bioreactors. Here we report a detailed evaluation of the above proposition and present evidence regarding structural features of siRNAs extracted from plants. Results Testing the gene silencing capacity of plant-derived siRNAs in mammalian cells proved to be very challenging and required partial siRNA purification and design of a highly sensitive assay. Using the above assay we found that plant-derived siRNAs are ineffective for gene silencing in mammalian cells. Plant-derived siRNAs are almost exclusively double-stranded and most likely comprise a mixture of bona fide siRNAs and aberrant partially complementary duplexes. We also provide indirect evidence that plant-derived siRNAs may contain a hitherto undetected physiological modification, distinct from 3' terminal 2-O-methylation. Conclusion siRNAs produced from plant hairpin transgenes and extracted from plants are ineffective for gene silencing in mammalian cells. Thus our findings establish that a previous claim that transgenic plants offer a cost-effective, scalable and sustainable source of siRNAs is unwarranted. Our results also indicate that the presence of aberrant siRNA duplexes and possibly a plant-specific siRNA modification, compromises the gene silencing capacity of plant-derived siRNAs in mammalian cells.

  7. Short stature (United States)

    ... condition. Bone or skeletal disorders, such as: Rickets Achondroplasia Chronic diseases, such as: Asthma Celiac disease Congenital ... growth seems slow or your child seems small. TREATMENT Your child's short stature may affect her self- ...

  8. Short communication

    African Journals Online (AJOL)

    preferred customer

    ABSTRACT: Nutrients, metabolizable energy (ME), anti-nutritional factors, digestible organic ... for animals are in short supply and expensive, ... peels from hotels (Hawassa); sisal waste from ..... chain omega-3 fatty acids in livestock meat and.

  9. Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe


    A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

  10. Dendritic structure DNA for specific metal ion biosensor based on catalytic hairpin assembly and a sensitive synergistic amplification strategy. (United States)

    Zhao, Jianmin; Jing, Pei; Xue, Shuyan; Xu, Wenju


    In this work, a sensitive electrochemical biosensing to Pb(2+) was proposed based on the high specificity of DNAzymes to Pb(2+). The response signal was efficiently amplified by the catalytic hairpin assembly induced by strand replacement reaction and the formation of dendritic structure DNA (DSDNA) by layer-by-layer assembly. Firstly, in the presence of Pb(2+), the substrate strand (S1) of the Pb(2+)-specific DNAzymes was specifically cleaved by Pb(2+). Secondly, one of the two fragments (rS1) introduced into the electrode surface was hybridized with a hairpin DNA (H1) and further replaced by another hairpin DNA (H2) by the hybridization reaction of H1 with H2. The released rS1 then induced the next hybridization with H1. After repeated cycles, the catalytic recycling assembly of H2 with H1 was completed. Thirdly, two bioconjugates of Pt@Pd nanocages (Pt@PdNCs) labeled with DNA S3/S4 and electroactive toluidine blue (Tb) (Tb-S3-Pt@PdNCs and Tb-S4-Pt@PdNCs) were captured onto the resultant electrode surface through the hybridization of S3 and H2, S3 and S4, resulting in the formation of DSDNA triggered by layer-by-layer assembly. This formed DSDNA greatly facilitated the immobilization of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrin (MnTMPyP) as mimicking enzyme. Under the synergistic catalysis of Pt@PdNCs and MnTMPyP to H2O2 reduction, the effective signal amplification of the developed Pb(2+) biosensor was achieved. As a result, the sensitive detection of the proposed electrochemical strategy for Pb(2+) was greatly improved in the range of 0.1pM-200nM with a detection limit of 0.033pM.

  11. The mitochondrial genomes of sponges provide evidence for multiple invasions by Repetitive Hairpin-forming Elements (RHE

    Directory of Open Access Journals (Sweden)

    Lavrov Dennis V


    Full Text Available Abstract Background The mitochondrial (mt genomes of sponges possess a variety of features, which appear to be intermediate between those of Eumetazoa and non-metazoan opisthokonts. Among these features is the presence of long intergenic regions, which are common in other eukaryotes, but generally absent in Eumetazoa. Here we analyse poriferan mitochondrial intergenic regions, paying particular attention to repetitive sequences within them. In this context we introduce the mitochondrial genome of Ircinia strobilina (Lamarck, 1816; Demospongiae: Dictyoceratida and compare it with mtDNA of other sponges. Results Mt genomes of dictyoceratid sponges are identical in gene order and content but display major differences in size and organization of intergenic regions. An even higher degree of diversity in the structure of intergenic regions was found among different orders of demosponges. One interesting observation made from such comparisons was of what appears to be recurrent invasions of sponge mitochondrial genomes by repetitive hairpin-forming elements, which cause large genome size differences even among closely related taxa. These repetitive hairpin-forming elements are structurally and compositionally divergent and display a scattered distribution throughout various groups of demosponges. Conclusion Large intergenic regions of poriferan mt genomes are targets for insertions of repetitive hairpin- forming elements, similar to the ones found in non-metazoan opisthokonts. Such elements were likely present in some lineages early in animal mitochondrial genome evolution but were subsequently lost during the reduction of intergenic regions, which occurred in the Eumetazoa lineage after the split of Porifera. Porifera acquired their elements in several independent events. Patterns of their intra-genomic dispersal can be seen in the mt genome of Vaceletia sp.

  12. Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin

    Directory of Open Access Journals (Sweden)

    Jakobsen Maria


    Full Text Available Abstract Background Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα, interleukin-12 (IL-12, and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs. Methods Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels. Results Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimick the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by

  13. An O(n(5)) algorithm for MFE prediction of kissing hairpins and 4-chains in nucleic acids. (United States)

    Chen, Ho-Lin; Condon, Anne; Jabbari, Hosna


    Efficient methods for prediction of minimum free energy (MFE) nucleic secondary structures are widely used, both to better understand structure and function of biological RNAs and to design novel nano-structures. Here, we present a new algorithm for MFE secondary structure prediction, which significantly expands the class of structures that can be handled in O(n(5)) time. Our algorithm can handle H-type pseudoknotted structures, kissing hairpins, and chains of four overlapping stems, as well as nested substructures of these types.

  14. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin. (United States)

    Springer, Tzvia I; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L


    Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD's β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD's β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (Rh) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the β-hairpin's role in stabilizing interactions between CyaA-ACD and N-CaM.

  15. Structure of 23S rRNA hairpin 35 and its interaction with the tylosin-resistance methyltransferase RlmAII (United States)

    Lebars, Isabelle; Yoshizawa, Satoko; Stenholm, Anne R.; Guittet, Eric; Douthwaite, Stephen; Fourmy, Dominique


    The bacterial rRNA methyltransferase RlmAII (formerly TlrB) contributes to resistance against tylosin-like 16-membered ring macrolide antibiotics. RlmAII was originally discovered in the tylosin-producer Streptomyces fradiae, and members of this subclass of methyltransferases have subsequently been found in other Gram-positive bacteria, including Streptococcus pneumoniae. In all cases, RlmAII methylates 23S rRNA at nucleotide G748, which is situated in a stem–loop (hairpin 35) at the macrolide binding site of the ribosome. The conformation of hairpin 35 recognized by RlmAII is shown here by NMR spectroscopy to resemble the anticodon loop of tRNA. The loop folds independently of the rest of the 23S rRNA, and is stabilized by a non-canonical G–A pair and a U-turn motif, rendering G748 accessible. Binding of S.pneumoniae RlmAII induces changes in NMR signals at specific nucleotides that are involved in the methyltransferase–RNA interaction. The conformation of hairpin 35 that interacts with RlmAII is radically different from the structure this hairpin adopts within the 50S subunit. This indicates that the hairpin undergoes major structural rearrangement upon interaction with ribosomal proteins during 50S assembly. PMID:12514124

  16. Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition,Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

    Institute of Scientific and Technical Information of China (English)

    Ting Zhang; Ge Cui; Yun-Liang Yao; Yue Guo; Qi-Chun Wang; Xi-Ning Li; Wen-Ming Feng


    Background:Protein arginine methyltransferases 1 (PRMT1) is over-expressed in a variety of cancers,including lung cancer,and is correlated with a poor prognosis of tumor development.This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer (NSCLC) migration in vitro.Methods:In this study,PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs.Cell migration was measured using both scratch wound healing and transwell cell migration assays.The mRNA expression levels of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor ofmetalloproteinase 1,2 (TIMP l,2) were measured using quantitative real-time reverse transcription-polymerase chain reaction.The expression levels of protein markers for epithelial-mesenchymal transition (EMT) (E-cadherin,N-cadherin),focal adhesion kinase (FAK),Src,AKT,and their corresponding phosphorylated states were detected by Western blot.Results:Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group.The mRNA expression of MMP-2 decreased while TIMP 1 and TIMP2 increased significantly.E-cadherin mRNA expression also increased while N-cadherin decreased.Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged.Conclusions:PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT,extracellular matrix degradation,and Src phosphorylation in vitro.

  17. Virus-derived transgenes expressing hairpin RNA give immunity to Tobacco mosaic virus and Cucumber mosaic virus

    Directory of Open Access Journals (Sweden)

    Liu Yong


    Full Text Available Abstract Background An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses. Results Inverted repeats of the partial Tobacco mosaic virus (TMV movement protein (MP gene and the partial Cucumber mosaic virus (CMV replication protein (Rep gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines (MP16-17-3, MP16-17-29 and MP16-17-58 immune to TMV infection and three transgenic tobacco lines (Rep15-1-1, Rep15-1-7 and Rep15-1-32 immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃ did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA. Conclusions We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection.

  18. Kinetic network models of tryptophan mutations in β-hairpins reveal the importance of non-native interactions. (United States)

    Razavi, Asghar M; Voelz, Vincent A


    We present an analysis of the most extensive explicit-solvent simulations of β-hairpins to date (9.4 ms in aggregate), with the aim of probing the effects of tryptophan mutations on folding. From molecular simulations of GB1 hairpin, trpzip4, trpzip5, and trpzip6 performed on Folding@home, Markov State Models (MSMs) were constructed using a unified set of metastable states, enabling objective comparison of folding mechanisms. MSM models display quantitative agreement with experimental structural observables and folding kinetics, and predict multimodal kinetics due to specific non-native kinetic traps, which be identified as on- or off-pathway from the network topology. We quantify kinetic frustration by several means, including the s-ensemble method to evaluate glasslike behavior. Free-energy profiles and transition state movement clearly show stabilization of non-native states as Trp mutations are introduced. Remarkably, we find that "β-capped" sequences (trpzip4 and trpzip5) are able to overcome this frustration and remain cooperative two-state folders with a large time-scale gap. These results suggest that, while β-capping motifs are robust, fold stabilization by tryptophan generally may require overcoming significant non-native kinetic traps, perhaps explaining their under-representation in natural proteins.

  19. Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants. (United States)

    Han, Yong-Woon; Matsumoto, Tomoko; Yokota, Hiroaki; Kashiwazaki, Gengo; Morinaga, Hironobu; Hashiya, Kaori; Bando, Toshikazu; Harada, Yoshie; Sugiyama, Hiroshi


    N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py-Im polyamides 1-5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k(a)) of the Py-Im polyamides with their target DNA decreased with the number of Im in the Py-Im polyamides. The structures of four-ring Py-Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py-Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py-Im polyamide induced by the incorporation of Im reduces the association rate of Py-Im polyamides. This fundamental knowledge of the binding of Py-Im polyamides to DNA will facilitate the design of hairpin Py-Im polyamides as synthetic DNA-binding modules.

  20. A novel DNA tetrahedron-hairpin probe for in situ"off-on" fluorescence imaging of intracellular telomerase activity. (United States)

    Feng, Qiu-Mei; Zhu, Meng-Jiao; Zhang, Ting-Ting; Xu, Jing-Juan; Chen, Hong-Yuan


    A novel three-dimensionally structured DNA probe is reported to realize in situ"off-on" imaging of intracellular telomerase activity. The probe consists of a DNA tetrahedron and a hairpin DNA on one of the vertices of the DNA tetrahedron. It is composed of four modified DNA segments: S1-Au nanoparticle (NP) inserting a telomerase strand primer (TSP) and S2-S4, three Cy5 dye modified DNA segments. Fluorescence of Cy5 at three vertices of the DNA tetrahedron is quenched by the Au NP at the other vertex due to the effective fluorescence resonance energy transfer (FRET) ("off" state). When the probe meets telomerase, the hairpin structure changes to rod-like through complementary hybridization with the telomerase-triggered stem elongation product, resulting in a large distance between the Au NP and Cy5 and the recovery of Cy5 fluorescence ("on" state). The molar ratio of 3 : 1 between the reporter (Cy5) and the target related TSP makes the probe show high sensitivity and recovery efficiency of Cy5 in the presence of telomerase extracted from HeLa cells. Given the functional and compact nanostructure, the mechanically stable and noncytotoxic nature of the DNA tetrahedron, this FRET-based probe provides more opportunities for biosensing, molecular imaging and drug delivery.

  1. The geometry and efficacy of cation-pi interactions in a diagonal position of a designed beta-hairpin. (United States)

    Tatko, Chad D; Waters, Marcey L


    Cation-pi interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation-pi interactions in a diagonal position of a beta-hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from -0.20 to -0.48 kcal/mole. Our experimental values for the diagonal cation-pi interactions are similar to those found in alpha-helical studies. Upfield shifting of the Lys and Arg side chains indicates that the geometries of cation-pi interactions adopted in the 12-residue beta-hairpin are comparable to those found in protein structures. The Lys was found to interact through the polarized Cepsilon, and the Arg is stacked against the aromatic ring of Phe or Trp. Folding of these peptides was found to be enthalpically favorable (DeltaH degrees equals approximately -3 kcal/mole) and entropically unfavorable (DeltaS degrees equals approximately -8 cal mole(-1) K(-1)).

  2. Short Review

    DEFF Research Database (Denmark)

    Lynnerup, Niels; Rühli, Frank


    modality in ancient mummy research. The aim of this short review is to address the advantages and pitfalls of this particular technique for such unique samples. We recommend that when results of X-ray examination of mummies are presented, the specific recording data should be listed, and any given finds......, for example, of Paleopathology, should be cross-checked against other sources, for example, CT-scanning, direct inspection (also by endoscopy), and so forth....

  3. Short esophagus. (United States)

    Kunio, Nicholas R; Dolan, James P; Hunter, John G


    In the presence of long-standing and severe gastroesophageal reflux disease, patients can develop various complications, including a shortened esophagus. Standard preoperative testing in these patients should include endoscopy, esophagography, and manometry, whereas the objective diagnosis of a short esophagus must be made intraoperatively following adequate mediastinal mobilization. If left untreated, it is a contributing factor to the high recurrence rate following fundoplications or repair of large hiatal hernias. A laparoscopic Collis gastroplasty combined with an antireflux procedure offers safe and effective therapy.

  4. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)


    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  5. A cell-permeable hairpin peptide inhibits hepatitis C viral nonstructural protein 5A-mediated translation and virus production. (United States)

    Khachatoorian, Ronik; Arumugaswami, Vaithilingaraja; Ruchala, Piotr; Raychaudhuri, Santanu; Maloney, Eden M; Miao, Edna; Dasgupta, Asim; French, Samuel W


    NS5A is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly, and translation. We and others have shown that NS5A augments HCV internal ribosomal entry site (IRES)-mediated translation. Furthermore, Quercetin treatment and heat shock protein (HSP) 70 knockdown inhibit the NS5A-driven augmentation of IRES-mediated translation and infectious virus production. We have also coimmunoprecipitated HSP70 with NS5A and demonstrated cellular colocalization, leading to the hypothesis that the NS5A/HSP70 complex formation is important for IRES-mediated translation. Here, we have identified the NS5A region responsible for complex formation through in vitro deletion analyses. Deletion of NS5A domains II and III failed to reduce HSP70 binding, whereas domain I deletion eliminated complex formation. NS5A domain I alone also bound HSP70. Deletion mapping of domain I identified the C-terminal 34 amino acids (C34) as the interaction site. Furthermore, addition of C34 to domains II and III restored complex formation. C34 expression significantly reduced intracellular viral protein levels, in contrast to same-size control peptides from other NS5A domains. C34 also competitively inhibited NS5A-augmented IRES-mediated translation, whereas controls did not. Triple-alanine scan mutagenesis determined that an exposed beta-sheet hairpin in C34 was primarily responsible for NS5A-augmented IRES-mediated translation. Moreover, treatment with a 10-amino acid peptide derivative of C34 suppressed NS5A-augmented IRES-mediated translation and significantly inhibited intracellular viral protein synthesis, with no associated cytotoxicity. These results support the hypothesis that the NS5A/HSP70 complex augments viral IRES-mediated translation, identify a sequence-specific hairpin element in NS5A responsible for complex formation, and demonstrate the functional significance of C34 hairpin-mediated NS5A/HSP70 interaction. Identification of this element may allow

  6. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase. (United States)

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu


    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  7. Copper(II)-Mediated Self-Assembly of Hairpin Peptides and Templated Synthesis of CuS Nanowires. (United States)

    Wang, Chengdong; Sun, Yawei; Wang, Jiqian; Xu, Hai; Lu, Jian R


    The self-assembly of peptides and proteins under well-controlled conditions underlies important nanostructuring processes that could be harnessed in practical applications. Herein, the synthesis of a new hairpin peptide containing four histidine residues is reported and the self-assembly process mediated by metal ions is explored. The work involves the combined use of circular dichroism, NMR spectroscopy, UV/Vis spectroscopy, AFM, and TEM to follow the structural and morphological details of the metal-coordination-mediated folding and self-assembly of the peptide. The results indicate that by forming a tetragonal coordination geometry with four histidine residues, copper(II) ions selectively trigger the peptide to fold and then self-assemble into nanofibrils. Furthermore, the copper(II)-bound nanofibrils template the synthesis of CuS nanowires, which display a near-infrared laser-induced thermal effect.

  8. Cross-strand coupling of a beta-hairpin peptide stabilized with an Aib-Gly turn studied using isotope-edited IR spectroscopy. (United States)

    Huang, Rong; Setnicka, Vladimir; Etienne, Marcus A; Kim, Joohyun; Kubelka, Jan; Hammer, Robert P; Keiderling, Timothy A


    Isotope-edited IR spectroscopy was used to study a series of singly and doubly 13C=O-labeled beta-hairpin peptides stabilized by an Aib-Gly turn sequence. The double-labeled peptides have amide I' IR spectra that show different degrees of vibrational coupling between the 13C-labeled amides due to variations in the local geometry of the peptide structure. The single-labeled peptides provide controls to determine frequencies characteristic of the diagonal force field (FF) contributions at each position for the uncoupled 13C=O modes. Separation of diagonal FF and coupling effects on the spectra are used to explain the cross-strand labeled spectral patterns. DFT calculations based on an idealized model beta-hairpin peptide correctly predict the vibrational coupling patterns. Extending these model results by consideration of frayed ends and the hairpin conformational flexibility yields an alternate interpretation of details of the spectra. Temperature-dependent isotopically labeled IR spectra reveal differences in the thermal stabilities of the individual isotopically labeled sites. This is the first example of using an IR-based isotopic labeling technique to differentiate structural transitions at specific sites along the peptide backbone in model beta-hairpin peptides.

  9. Investigating the role of chain and linker length on the catalytic activity of an H 2 production catalyst containing a β-hairpin peptide

    Energy Technology Data Exchange (ETDEWEB)

    Reback, Matthew L.; Ginovska, Bojana; Buchko, Garry W.; Dutta, Arnab; Priyadarshani, Nilusha; Kier, Brandon L.; Helm, Monte L.; Raugei, Simone; Shaw, Wendy J.


    Building on our recent report of an active H2 production catalyst [Ni(PPh2NProp-peptide)2]2+ (Prop=para-phenylpropionic acid, peptide (R10)=WIpPRWTGPR-NH2, p=D-proline, and P2N=1-aza-3,6-diphosphacycloheptane) that contains structured -hairpin peptides, here we investigate how H2 production is effected by: (1) the length of the hairpin (eight or ten residues) and (2) limiting the flexibility between the peptide and the core complex by altering the length of the linker: para-phenylpropionic acid (three carbons) or para-benzoic acid (one carbon). Reduction of the peptide chain length from ten to eight residues increases or maintains the catalytic current for H2 production for all complexes, suggesting a non-productive steric interaction at longer peptide lengths. While the structure of the hairpin appears largely intact for the complexes, NMR data are consistent with differences in dynamic behavior which may contribute to the observed differences in catalytic activity. Molecular dynamics simulations demonstrate that complexes with a one-carbon linker have the desired effect of restricting the motion of the hairpin relative to the complex; however, the catalytic currents are significantly reduced compared to complexes containing a three-carbon linker as a result of the electron withdrawing nature of the -COOH group. These results demonstrate the complexity and interrelated nature of the outer coordination sphere on catalysis.

  10. Charge and geometry of residues in the loop 2 β hairpin differentially affect agonist and ethanol sensitivity in glycine receptors. (United States)

    Perkins, Daya I; Trudell, James R; Asatryan, Liana; Davies, Daryl L; Alkana, Ronald L


    Recent studies highlighted the importance of loop 2 of α1 glycine receptors (GlyRs) in the propagation of ligand-binding energy to the channel gate. Mutations that changed polarity at position 52 in the β hairpin of loop 2 significantly affected sensitivity to ethanol. The present study extends the investigation to charged residues. We found that substituting alanine with the negative glutamate at position 52 (A52E) significantly left-shifted the glycine concentration response curve and increased sensitivity to ethanol, whereas the negative aspartate substitution (A52D) significantly right-shifted the glycine EC₅₀ but did not affect ethanol sensitivity. It is noteworthy that the uncharged glutamine at position 52 (A52Q) caused only a small right shift of the glycine EC₅₀ while increasing ethanol sensitivity as much as A52E. In contrast, the shorter uncharged asparagine (A52N) caused the greatest right shift of glycine EC₅₀ and reduced ethanol sensitivity to half of wild type. Collectively, these findings suggest that charge interactions determined by the specific geometry of the amino acid at position 52 (e.g., the 1-Å chain length difference between aspartate and glutamate) play differential roles in receptor sensitivity to agonist and ethanol. We interpret these results in terms of a new homology model of GlyR based on a prokaryotic ion channel and propose that these mutations form salt bridges to residues across the β hairpin (A52E-R59 and A52N-D57). We hypothesize that these electrostatic interactions distort loop 2, thereby changing agonist activation and ethanol modulation. This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs.

  11. Differential RNAi responses of Nicotiana benthamiana individuals transformed with a hairpin-inducing construct during Plum pox virus challenge. (United States)

    Montes, Christian; Castro, Álvaro; Barba, Paola; Rubio, Julia; Sánchez, Evelyn; Carvajal, Denisse; Aguirre, Carlos; Tapia, Eduardo; DelÍ Orto, Paola; Decroocq, Veronique; Prieto, Humberto


    Gene silencing and large-scale small RNA analysis can be used to develop RNA interference (RNAi)-based resistance strategies for Plum pox virus (PPV), a high impact disease of Prunus spp. In this study, a pPPViRNA hairpin-inducing vector harboring two silencing motif-rich regions of the PPV coat protein (CP) gene was evaluated in transgenic Nicotiana benthamiana (NB) plants. Wild-type NB plants infected with a chimeric PPV virus (PPV::GFP) exhibited affected leaves with mosaic chlorosis congruent to GFP fluorescence at 21 day post-inoculation; transgenic lines depicted a range of phenotypes from fully resistant to susceptible. ELISA values and GFP fluorescence intensities were used to select transgenic-resistant (TG-R) and transgenic-susceptible (TG-S) lines for further characterization of small interfering RNAs (siRNAs) by large-scale small RNA sequencing. In infected TG-S and untransformed (WT) plants, the observed siRNAs were nearly exclusively 21- and 22-nt siRNAs that targeted the whole PPV::GFP genome; 24-nt siRNAs were absent in these individuals. Challenged TG-R plants accumulated a full set of 21- to 24-nt siRNAs that were primarily associated with the selected motif-rich regions, indicating that a trans-acting siRNAs process prevented viral multiplication. BLAST analysis identified 13 common siRNA clusters targeting the CP gene. 21-nt siRNA sequences were associated with the 22-nt siRNAs and the scarce 23- and 24-nt molecules in TG-S plants and with most of the observed 22-, 23-, and 24-nt siRNAs in TG-R individuals. These results validate the use of a multi-hot spot silencing vector against PPV and elucidate the molecules by which hairpin-inducing vectors initiate RNAi in vivo.

  12. Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon. (United States)

    Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing


    Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Synthesis and characterization of a beta-hairpin peptide that represents a 'core module' of bovine pancreatic trypsin inhibitor (BPTI). (United States)

    Carulla, N; Woodward, C; Barany, G


    A new strategy for the design and construction of peptide fragments that can achieve defined, nativelike secondary structure is presented. The strategy is based upon the hypothesis that 'core elements' of a protein, synthesized in a single polypeptide molecule, will favor nativelike structure, and that by incorporating a cross-link, nativelike core structure will dominate the ensemble as the more extended conformations are excluded. 'Core elements' are the elements of packed secondary structure that contain the slowest exchanging backbone amide protons in the native protein. The 'core elements' in bovine pancreatic trypsin inhibitor (BPTI) are the two long strands of antiparallel beta-sheet (residues 18-24 and 29-35) and the small beta-bridge (residues 43-44). To test the design strategy, we synthesized an 'oxidized core module', which contains the antiparallel strands connected by a modified reverse turn (A27 replaced by D), a natural disulfide cross-link at the open end of the hairpin, and N- and C-termini blocking groups. A peptide with identical sequence but lacking the disulfide cross-link at the open end was used as the 'reduced core module' control. The conformational behavior of both peptides was examined using (1)H NMR spectroscopy. Chemical shift dispersion, chemical shift deviation from random coil values, sequential and long-range NOEs, and H/D amide exchange rates were compared for the two peptides. We conclude that the ensemble of oxidized and reduced core module conformations samples both nativelike 4:4 and non-native 3:5 beta-hairpin structure, and that the oxidized module samples nativelike structure for a greater fraction of the time than the reduced module.

  14. Short ROSE-like RNA thermometers control IbpA synthesis in Pseudomonas species. (United States)

    Krajewski, Stefanie S; Nagel, Miriam; Narberhaus, Franz


    The bacterial small heat shock protein IbpA protects client proteins from aggregation. Due to redundancy in the cellular chaperone network, deletion of the ibpA gene often leads to only a mild or no phenotypic defect. In this study, we show that a Pseudomonas putida ibpA deletion mutant has a severe growth defect under heat stress conditions and reduced survival during recovery revealing a critical role of IbpA in heat tolerance. Transcription of the ibpA gene depends on the alternative heat shock sigma factor σ(32). Production of IbpA protein only at heat shock temperatures suggested additional translational control. We conducted a comprehensive structural and functional analysis of the 5' untranslated regions of the ibpA genes from P. putida and Pseudomonas aeruginosa. Both contain a ROSE-type RNA thermometer that is substantially shorter and simpler than previously reported ROSE elements. Comprised of two hairpin structures only, they inhibit translation at low temperature and permit translation initiation after a temperature upshift. Both elements regulate reporter gene expression in Escherichia coli and ribosome binding in vitro in a temperature-dependent manner. Structure probing revealed local melting of the second hairpin whereas the first hairpin remained unaffected. High sequence and structure conservation of pseudomonal ibpA untranslated regions and their ability to confer thermoregulation in vivo suggest that short ROSE-like thermometers are commonly used to control IbpA synthesis in Pseudomonas species.

  15. Short ROSE-like RNA thermometers control IbpA synthesis in Pseudomonas species.

    Directory of Open Access Journals (Sweden)

    Stefanie S Krajewski

    Full Text Available The bacterial small heat shock protein IbpA protects client proteins from aggregation. Due to redundancy in the cellular chaperone network, deletion of the ibpA gene often leads to only a mild or no phenotypic defect. In this study, we show that a Pseudomonas putida ibpA deletion mutant has a severe growth defect under heat stress conditions and reduced survival during recovery revealing a critical role of IbpA in heat tolerance. Transcription of the ibpA gene depends on the alternative heat shock sigma factor σ(32. Production of IbpA protein only at heat shock temperatures suggested additional translational control. We conducted a comprehensive structural and functional analysis of the 5' untranslated regions of the ibpA genes from P. putida and Pseudomonas aeruginosa. Both contain a ROSE-type RNA thermometer that is substantially shorter and simpler than previously reported ROSE elements. Comprised of two hairpin structures only, they inhibit translation at low temperature and permit translation initiation after a temperature upshift. Both elements regulate reporter gene expression in Escherichia coli and ribosome binding in vitro in a temperature-dependent manner. Structure probing revealed local melting of the second hairpin whereas the first hairpin remained unaffected. High sequence and structure conservation of pseudomonal ibpA untranslated regions and their ability to confer thermoregulation in vivo suggest that short ROSE-like thermometers are commonly used to control IbpA synthesis in Pseudomonas species.

  16. Amplified fluorescence detection of adenosine via catalyzed hairpin assembly and host-guest interactions between β-cyclodextrin polymer and pyrene. (United States)

    Huang, Haihua; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Guo, Qiuping; Huang, Jin; Liu, Jianbo; Song, Chunxia


    Nowadays, enzyme-free nucleic acid-based signal amplification strategies are frequently utilized in the design of biosensors due to their excellent sensitivity. Developing more extended analytical methods is fundamental for basic studies in the biological and biomedical fields. Herein, we introduce an enzyme-free amplified detection strategy for the small molecule adenosine. The approach is based on adenosine-aptamer binding triggered catalyzed hairpin assembly and host-guest interactions between β-cyclodextrin polymer (β-CDP) and pyrene. Two hairpin probes (probe H1 and probe H2) and an aptamer-trigger/inhibitor duplex probe were employed in the system and the pyrene-labeled probe H1 was chosen as the signal unit. In the absence of adenosine, the aptamer-trigger was inhibited by the inhibitor strand. The hairpin probes were in the closed hairpin formation without activation of the trigger strand. Pyrene labeled at the 5'-termini of the single-stranded stem of probe H1 could be easily trapped in the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to a significant fluorescence enhancement. Once the hairpin assembly was catalyzed by the adenosine-aptamer binding event, a hybridized DNA duplex H1/H2 was created continuously. Pyrene labeled at the 5'-termini of the DNA duplex H1/H2 finds it difficult to enter the cavity of β-CDP due to steric hindrance, leading to a weaker fluorescence signal. Thus, the target could be detected by this simple mix-and-detect amplification method without a need for expensive and perishable protein enzymes. As low as 42 nM of adenosine was detected by this assay, which is comparable to that of some reported colorimetric methods. Meanwhile, the proposed method was further successfully applied to detect adenosine in human serum samples, showing great potential for adenosine detection from complex fluids.

  17. 构建干扰RhoA的短发夹RNA真核表达载体%Construction of recombinant plasmid with RhoA -targeted short hairpin RNA

    Institute of Scientific and Technical Information of China (English)

    卢永清; 苏继荣; 董红霖; 李文龙; 武伟; 张治军; 杨涛; 李久池


    目的 利用pGenesil-1质粒构建针对RboA的短发夹RNA(shRNA)表达载体.方法 设计2个shRNA结构的互补DNA序列,经退火成双链,胶回收酶切pGenesil-1大片段,T4 DNA Ligase连接酶切大片段和DNA序列,得到质粒pGenesil-1-RhoA1和pGenesil-1-RhoA2.转化感受态细胞DH5a,扩增,提取重组质粒进行酶切鉴定.结果 成功构建靶向RhoA的shRNA重组质粒载体.酶切鉴定和测序分析重组质粒,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功.结论 表达靶向RhoA的shRNA表达框成功构建在重组质粒载体上.

  18. Short-hairpin RNA-mediated Heat shock protein 90 gene silencing inhibits human breast cancer cell growth in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Zuo, Keqiang [Department of General Surgery, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Li, Dan [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Pulli, Benjamin [Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, MA 02114 (United States); Yu, Fei; Cai, Haidong; Yuan, Xueyu [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Zhang, Xiaoping, E-mail: [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China); Lv, Zhongwei, E-mail: [Department of Nuclear Medicine, Shanghai 10th People' s Hospital, Tongji University School of Medicine, Shanghai 200072 (China)


    Highlights: Black-Right-Pointing-Pointer Hsp90 is over-expressed in human breast cancer. Black-Right-Pointing-Pointer The shRNA-mediated gene silencing of Hsp90 resulted in inhibition of cell growth. Black-Right-Pointing-Pointer Akt and NF-kB were down-regulation after transfection due to Hsp90 silencing. Black-Right-Pointing-Pointer The tumor growth ratio was decline due to Hsp90 silencing. Black-Right-Pointing-Pointer The PCNA expression was down-regulation due to Hsp90 silencing. -- Abstract: Hsp90 interacts with proteins that mediate signaling pathways involved in the regulation of essential processes such as proliferation, cell cycle control, angiogenesis and apoptosis. Hsp90 inhibition is therefore an attractive strategy for blocking abnormal pathways that are crucial for cancer cell growth. In the present study, the role of Hsp90 in human breast cancer MCF-7 cells was examined by stably silencing Hsp90 gene expression with an Hsp90-silencing vector (Hsp90-shRNA). RT-PCR and Western blot analyses showed that Hsp90-shRNA specifically and markedly down-regulated Hsp90 mRNA and protein expression. NF-kB and Akt protein levels were down-regulated in Hsp90-shRNA transfected cells, indicating that Hsp90 knockout caused a reduction of survival factors and induced apoptosis. Treatment with Hsp90-shRNA significantly increased apoptotic cell death and caused cell cycle arrest in the G1/S phase in MCF-7 cells, as shown by flow cytometry. Silencing of Hsp90 also reduced cell viability, as determined by MTT assay. In vivo experiments showed that MCF-7 cells stably transfected with Hsp90-shRNA grew slowly in nude mice as compared with control groups. In summary, the Hsp90-shRNA specifically silenced the Hsp90 gene, and inhibited MCF-7 cell growth in vitro and in vivo. Possible molecular mechanisms underlying the effects of Hsp90-shRNA include the degradation of Hsp90 breast cancer-related client proteins, the inhibition of survival signals and the upregulation of apoptotic pathways. shRNA-mediated interference may have potential therapeutic utility in human breast cancer.

  19. Effects of short-hairpin RNA-inhibited {beta}-catenin expression on the growth of human multiple myeloma cells in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Wenqing, E-mail: [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Yang, Chengwei [Department of Spinal Surgery, Lanzhou General Hospital, Lanzhou Military Area Command, 333 Nanbinhe Road, Lanzhou 730050 (China); Qian, Yu [Department of Orthopaedics, Shaoxing People' s Hospital, 568 Zhongxing North Road, Shaoxing 312000 (China); Fu, Qiang, E-mail: [Department of Orthopaedics, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433 (China)


    Highlights: Black-Right-Pointing-Pointer {beta}-Catenin expression were markedly down-regulated by CTNNB1 shRNA. Black-Right-Pointing-Pointer CTNNB1 shRNA could inhibit the proliferation of RPMI8226 cells. Black-Right-Pointing-Pointer Significantly profound apoptotic cell death in CTNNB1 shRNA cells. Black-Right-Pointing-Pointer In vivo, CTNNB1 silence led to a growth inhibition of myeloma growth. Black-Right-Pointing-Pointer c-myc and {beta}-catenin in the expression cells of cleaved caspase-3 were increased. -- Abstract: Multiple myeloma (MM) is thrombogenic as a consequence of multiple hemostatic effects. Overexpression of {beta}-catenin has been observed in several types of malignant tumors, including MM. However, the relationship between {beta}-catenin expression and MM remains unclear. In the present study, RNA interference was used to inhibit {beta}-catenin expression in RPMI8226 cells. RT-PCR and Western blotting analyses showed that {beta}-catenin mRNA and protein expression were markedly down-regulated by CTNNB1 shRNA. Western blotting showed that the protein levels of cyclin D1 and glutamine synthetase were downregulated and supported the transcriptional regulatory function of {beta}-catenin. The MTT assay showed that CTNNB1 shRNA could have significant inhibitory effects on the proliferation of RPMI8226 cells. The TOPflash reporter assay demonstrated significant downregulation after CTNNB1 shRNA transfection in RPMI8226 cells. Flow cytometric analyses also showed significantly profound apoptosis in CTNNB1 shRNA cells. We found CTNNB1 silence led to growth inhibition of MM growth in vivo. Immunohistochemical analyses showed that c-myc and {beta}-catenin were reduced in CTNNB1 shRNA tumor tissues, but that expression of cleaved caspase-3 was increased. These results show that {beta}-catenin could be a new therapeutic agent that targets the biology of MM cells.

  20. Inhibiting effect of short hairpin RNA on expression of transforming growth factor-β1 in human peritoneal mesothelial cells induced by peritoneal dialysis solution

    Institute of Scientific and Technical Information of China (English)

    LIU Fu-you; LING Guang-hui; LIU Hong; PENG You-ming; LIU Ying-hong; DUAN Shao-bin


    @@ The peritoneum response to peritoneal dialysis can lead to fibrosis. The transforming growth factor-β1 (TGF-β1) plays an important role in regulating tissue repair and remodeling after injury. Excessive synthesis and deposition of matrix proteins by peritoneal mesothelial cells can lead to structural and functional changes in the peritoneal membrane, jeopardizing the long-term efficacy of peritoneal dialysis (PD).

  1. Tumor-targeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chunmao; Ding, Chao; Kong, Minjian [Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009 (China); Dong, Aiqiang, E-mail: [Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009 (China); Qian, Jianfang; Jiang, Daming; Shen, Zhonghua [Department of Cardiothoracic Surgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009 (China)


    Highlights: {yields} We compared lipofection with magnetofection about difference of transfection efficiency on delivery a therapeutic gene in vitro and in vivo. {yields} We investigated the difference of shRNA induced by magnetofection and lipofection into A549 cell and subcutaneous tumor to knockdown IGF-1R overexpressed in A549 cell and A549 tumor. {yields} We investigated in vivo shRNA silenced IGF-1R overexpression 24, 48, and 72 h after shRNA intravenous injection into tumor-bearing mice by way of magnetofection and lipofection. {yields} Our results showed that magnetofection could achieve therapeutic gene targeted delivery into special site, which contributed to targeted gene therapy of lung cancers. -- Abstract: Liposomal magnetofection potentiates gene transfection by applying a magnetic field to concentrate magnetic lipoplexes onto target cells. Magnetic lipoplexes are self-assembling ternary complexes of cationic lipids with plasmid DNA associated with superparamagnetic iron oxide nanoparticles (SPIONs). Type1insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently overexpressed in lung cancer and mediates cancer cell proliferation and tumor growth. In this study, we evaluated the transfection efficiency (percentage of transfected cells) and therapeutic potential (potency of IGF-1R knockdown) of liposomal magnetofection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) in A549 cells and in tumor-bearing mice as compared to lipofection using Lipofectamine 2000. Liposomal magnetofection provided a threefold improvement in transgene expression over lipofection and transfected up to 64.1% of A549 cells in vitro. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1 {+-} 6% and by liposomal magnetofection by 85.1 {+-} 3%. In vivo delivery efficiency of the pGFPshIGF-1R plasmid into the tumor was significantly higher in the liposomal magnetofection group than in the lipofection group. In vivo IGF-1R specific-shRNA by lipofection inhibited IGF-1R protein by an average of 43.8 {+-} 5.3%; that by liposomal magnetofection inhibited IGF-1R protein by 43.4 {+-} 5.7%, 56.3 {+-} 9.6%, and 72.2 {+-} 6.8%, at 24, 48, and 72 h, respectively, after pGFPshIGF-1R injection. Our findings indicate that liposomal magnetofection may be a promising method that allows the targeting of gene therapy to lung cancer.

  2. Cytocompatibility, antibacterial activity and biodegradability of self-assembling beta-hairpin peptide-based hydrogels for tissue regenerative applications (United States)

    Salick, Daphne Ann

    Every year, millions of people suffer from tissue loss or failure. One approach to repair damaged or diseased tissue is through tissue/organ transplantation. However, one of the major problems which exist with this approach is that there are more people in need of a transplant than there are donors. Over the past several decades, scientists and doctors have come together to find a way to overcome this challenge. This collaboration has led to the development of biomimetic scaffolds, which closely mimic the desired tissue of interest to act as a substitute for the unfunctional tissue, with hopes to improve the quality of life. The Schneider and Pochan labs have developed a biomimetic scaffold using self-assembling beta-hairpin peptides. The self-assembly event can be triggered in response to physiological conditions, which is dictated by the monomer, to form non covalently crosslinked mechanically rigid hydrogels. In vitro studies showed that hydrogels were cytocompatible and may not elicit a pro-inflammatory response from murine macrophages. These material properties show promise for the use of these hydrogels in tissue engineering. When implanting a material into a host, a major concern is the introduction of infection. Infection, if not prevented or halted, results in poor tissue integration and function, ultimately leading to implant removal from the host. Interestingly, the beta-hairpin hydrogels were shown to exhibit antibacterial properties against pathogens commonly found in hospital environments. This inherently antibacterial hydrogel is advantageous because it may help decrease or diminish bacterial contamination when implanted in vivo, which may help to increase the success of implants. Also, a unique and exciting feature of these peptide-based hydrogels is their ability to shear-thin and self-heal. Hydrogels can be directly formed in a syringe and be subsequently delivered to a tissue defect in a minimally invasive manner where they will recover to their

  3. Double hairpin elements and tandem repeats in the non-coding region of Adenoides eludens chloroplast gene minicircles. (United States)

    Nelson, Martha J; Green, Beverley R


    Dinoflagellate plastid genomes are unique in having a reduced number of genes, most of which are found on unigenic minicircles of 2-3 kb. Although the dinoflagellate Adenoides eludens has larger minicircles of about 5 kb, they still carry only one gene. In addition, digenic circles of about 10 kb were detected and mapped by PCR. The non-coding regions of both unigenic and digenic circles share a number of common features including a pair of conserved cores in opposite orientation, four large families of tandem repeats and a number of double hairpin elements (DHEs). They most closely resemble the non-coding regions of the Symbiodinium psbA minicircles, but are much longer, less conserved and have an even greater variety of DHEs and tandem repeats. The presence of so many recombinogenic elements suggests models for the origin of minicircles from a multigenic ancestral chloroplast genome, and raises the possibility of recombination-directed replication rather than defined replication origins in the minicircles.

  4. Catalytic Hairpin Assembly Actuated DNA Nanotweezer for Logic Gate Building and Sensitive Enzyme-Free Biosensing of MicroRNAs. (United States)

    Li, Dandan; Cheng, Wei; Li, Yujian; Xu, YongJie; Li, Xinmin; Yin, Yibing; Ju, Huangxian; Ding, Shijia


    A target-switched DNA nanotweezer is designed for AND logic gate operation and enzyme-free detection of microRNAs (miRNAs) by catalytic hairpin assembly (CHA) and proximity-dependent DNAzyme formation. The double crossover motif-based nanotweezer consists of an arched structure as the set strand for target inputs and two split G-rich DNAs at the termini of two arms for signal output. Upon a CHA, a small amount of binary target inputs can switch numerous open nanotweezers to a closed state, which leads to the formation of proximity-dependent DNAzyme in the presence of hemin to produce a highly sensitive biosensing system. The binary target inputs can be used for successful building of AND logic gate, which is validated by polyacrylamide gel electrophoresis, surface plasmon resonance and the biosensing signal. The developed biosensing system shows a linear response of the output chemiluminescence signal to input binary miRNAs with a detection limit of 30 fM. It can be used for miRNAs analysis in complex sample matrix. This system provides a simple and reusable platform for logic gate operation and enzyme-free, highly sensitive, and specific multianalysis of miRNAs.

  5. 微带发夹式带通滤波器设计%Design of Microstrip Hairpin Bandpass Filter

    Institute of Scientific and Technical Information of China (English)

    袁玉先; 孙国泉; 钟仙凤


    Mathematical models are established according to the basic theory of lowpass filter prototype, and the microstrip hairpin bandpass filter of seventh order in the X-band is designed using the serenade software, with a central frequency of 11 GHz and bandwidth of 800 MHz. The got-up microstrip circuit is tested, and the testing re- suits show that the filter meets the demand of design.%依据低通滤波器原型基本理论,进行数学建模,并利用Serenade软件设计一种X波段7阶微带发夹型带通滤波器,其中心频率为11GHz,带宽800MHz。对制作的微带电路进行实际测试,测试结果表明,该滤波器能够满足设计要求。

  6. Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection. (United States)

    Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann


    A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.

  7. Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection (United States)

    Chen, Huixin; Parimelalagan, Mariya; Takei, Fumie; Hapuarachchi, Hapuarachchige Chanditha; Koay, Evelyn Siew-Chuan; Ng, Lee Ching; Ho, Phui San; Nakatani, Kazuhiko; Chu, Justin Jang Hann


    A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP—primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a ‘turn-on’ system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world. PMID:27571201

  8. A novel fluorescent aptasensor based on hairpin structure of complementary strand of aptamer and nanoparticles as a signal amplification approach for ultrasensitive detection of cocaine. (United States)

    Emrani, Ahmad Sarreshtehdar; Danesh, Noor Mohammad; Ramezani, Mohammad; Taghdisi, Seyed Mohammad; Abnous, Khalil


    Cocaine is one of the most commonly misused stimulant which could influence the central nervous system. In this study, a fluorescent aptamer-based sensor (aptasensor) was designed for sensitive and selective detection of cocaine, based on hairpin structure of complementary strand of aptamer (CS), target-induced release of aptamer (Apt) from CS and two kinds of nanoparticles, including silica nanoparticles (SNPs) coated with streptavidin and gold nanoparticles (AuNPs). The designed aptasensor acquires characteristics of AuNPs such as unique optical properties and large surface area, SNPs as amplifiers of fluorescence intensity, higher affinity of Apt toward its target relative to its CS, and finally the hairpin structure of CS that brings the fluorophore (FAM) to close proximity to the surface of SNPs. In the absence of cocaine, FAM is in close proximity to the surface of AuNPs, resulting in a weak fluorescence emission. In the presence of target, FAM comes to close proximity to the surface of SNPs because of the formation of hairpin structure of CS, leading to a very strong fluorescence emission. The fabricated fluorescent aptasensor exhibited a good selectivity toward cocaine with a limit of detection (LOD) as low as 209 pM. Moreover, the designed aptasensor was successfully utilized to detect cocaine in serum with a LOD as low as 293 pM.

  9. A signal-on electrochemical DNA biosensor based on potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition mediated labeling of hairpin-like oligonucleotide with electroactive probe. (United States)

    Hu, Qiong; Kong, Jinming; Li, Yajie; Zhang, Xueji


    A novel electrochemical biosensor was developed for the signal-on detection of sequence-specific DNA by exploiting potential-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (φCuAAC) as an efficient approach for the labeling of hairpin-like oligonucleotide (hairpin) with electroactive probe. The hairpins, dually labeled with thiol and azide at either terminal, were firstly self-assembled on gold electrode and served as the capture probes for the specific recognition of target DNA. Upon hybridization with target DNA, the surface-confined hairpins were unfolded, liberating the azide-containing terminals away from electrode surface. Subsequently, the unfolded hairpins were conveniently and efficiently labeled with ethynylferrocene (EFC) via the φCuAAC. The quantitatively labeled EFC was finally measured via differential pulse voltammetry (DPV) for the signal-on electrochemical detection of sequence-specific DNA. The biosensor presented a good linear response over the range from 1pM to 1nM with a detection limit of 0.62pM. Results also revealed that it was highly specific and held a good detection capability in serum samples. Furthermore, the ability to chemoselectively label hairpin-like oligonucleotide with signal reporter by electrical addressing, together with the simplicity and efficiency of the φCuAAC, makes it compatible with microfluidic devices and microelectrode arrays to achieve the miniaturized and multiplexed detections.

  10. Hairpin RNA derived from viral NIa gene confers immunity to wheat streak mosaic virus infection in transgenic wheat plants. (United States)

    Fahim, Muhammad; Ayala-Navarrete, Ligia; Millar, Anthony A; Larkin, Philip J


    Wheat streak mosaic virus (WSMV), vectored by Wheat curl mite, has been of great economic importance in the Great Plains of the United States and Canada. Recently, the virus has been identified in Australia, where it has spread quickly to all major wheat growing areas. The difficulties in finding adequate natural resistance in wheat prompted us to develop transgenic resistance based on RNA interference (RNAi). An RNAi construct was designed to target the nuclear inclusion protein 'a' (NIa) gene of WSMV. Wheat was stably cotransformed with two plasmids: pStargate-NIa expressing hairpin RNA (hpRNA) including WSMV sequence and pCMneoSTLS2 with the nptII selectable marker. When T(1) progeny were assayed against WSMV, ten of sixteen families showed complete resistance in transgenic segregants. The resistance was classified as immunity by four criteria: no disease symptoms were produced; ELISA readings were as in uninoculated plants; viral sequences could not be detected by RT-PCR from leaf extracts; and leaf extracts failed to give infections in susceptible plants when used in test-inoculation experiments. Southern blot hybridization analysis indicated hpRNA transgene integrated into the wheat genome. Moreover, accumulation of small RNAs derived from the hpRNA transgene sequence positively correlated with immunity. We also showed that the selectable marker gene nptII segregated independently of the hpRNA transgene in some transgenics, and therefore demonstrated that it is possible using these techniques, to produce marker-free WSMV immune transgenic plants. This is the first report of immunity in wheat to WSMV using a spliceable intron hpRNA strategy.

  11. Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1. (United States)

    Zhang, Yunlong; Zhang, Xiuping; Qu, Shaogang


    Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure-function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)3 (CuPh) and cadmium ion (Cd(2+)) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + L-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.

  12. Metschnikowia Species Share a Pool of Diverse rRNA Genes Differing in Regions That Determine Hairpin-Loop Structures and Evolve by Reticulation.

    Directory of Open Access Journals (Sweden)

    Matthias Sipiczki

    Full Text Available Modern taxonomy of yeasts is mainly based on phylogenetic analysis of conserved DNA and protein sequences. By far the most frequently used sequences are those of the repeats of the chromosomal rDNA array. It is generally accepted that the rDNA repeats of a genome have identical sequences due to the phenomenon of sequence homogenisation and can thus be used for identification and barcoding of species. Here we show that the rDNA arrays of the type strains of Metschnikowia andauensis and M. fructicola are not homogenised. Both have arrays consisting of diverse repeats that differ from each other in the D1/D2 domains by up to 18 and 25 substitutions. The variable sites are concentrated in two regions that correspond to back-folding stretches of hairpin loops in the predicted secondary structure of the RNA molecules. The substitutions do not alter significantly the overall hairpin-loop structure due to wobble base pairing at sites of C-T transitions and compensatory mutations in the complementary strand of the hairpin stem. The phylogenetic and network analyses of the cloned sequences revealed that the repeats had not evolved in a vertical tree-like way but reticulation might have shaped the rDNA arrays of both strains. The neighbour-net analysis of all cloned sequences of the type strains and the database sequences of different strains further showed that these species share a continuous pool of diverse repeats that appear to evolve by reticulate evolution.

  13. Ultrasensitive electrochemical sensing platform for microRNA based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification. (United States)

    Shuai, Hong-Lei; Huang, Ke-Jing; Xing, Ling-Li; Chen, Ying-Xu


    An ultrasensitive electrochemical biosensor for microRNA (miRNA) is developed based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification. WO3-Gr is prepared by a simple hydrothermal method and then coupled with gold nanoparticles to act as a sensing platform. The thiol-terminated capture probe H1 is immobilized on electrode through Au-S interaction. In the presence of target miRNA, H1 opens its hairpin structure by hybridization with target miRNA. This hybridization can be displaced from the structure by another stable biotinylated hairpin DNA (H2), and target miRNA is released back to the sample solution for next cycle. Thus, a large amount of H1-H2 duplex is produced after the cyclic process. At this point, a lot of signal indicators streptavidin-conjugated alkaline phosphatase (SA-ALP) are immobilized on the electrode by the specific binding of avidin-biotin. Then, thousands of ascorbic acid, which is the enzymatic product of ALP, induces the electrochemical-chemical-chemical redox cycling to produce a strongly electrochemical response in the presence of ferrocene methanol and tris (2-carboxyethyl) phosphine. Under the optimal experimental conditions, the established biosensor can detect target miRNA down to 0.05fM (S/N=3) with a linear range from 0.1fM to 100pM, and discriminate target miRNA from mismatched miRNA with a high selectivity.

  14. Design of a hairpin polyamide, ZT65B, for targeting the inverted CCAAT box (ICB) site in the multidrug resistant (MDR1) gene. (United States)

    Buchmueller, Karen L; Taherbhai, Zarmeen; Howard, Cameron M; Bailey, Suzanna L; Nguyen, Binh; O'Hare, Caroline; Hochhauser, Daniel; Hartley, John A; Wilson, W David; Lee, Moses


    A novel hairpin polyamide, ZT65B, containing a 3-methylpicolinate moiety was designed to target the inverted CCAAT box (ICB) of the human multidrug resistance 1 gene (MDR1) promoter. Binding of nuclear factor-Y (NF-Y) to the ICB site upregulates MDR1 gene expression and is, therefore, a good target for anticancer therapeutic agents. However, it is important to distinguish amongst different promoter ICB sites so that only specific genes will be affected. All ICB sites have the same sequence but they differ in the sequence of the flanking base pairs, which can be exploited in the design of sequence-specific polyamides. To test this hypothesis, ten ICB-containing DNA hairpins were designed with different flanking base pairs; the sequences ICBa and ICBb were similar to the 3'-ICB site of MDR1 (TGGCT). Thermal-denaturation studies showed that ZT65B effectively targeted ICBa and ICBb (DeltaTM=6.5 and 7.0 degrees C) in preference to the other DNA hairpins (ICBc (5.0 degrees C). DNase I-footprinting assays were carried out with the topoisomerase IIalpha-promoter sequence, which contains five ICB sites; of these, ICB1 and ICB5 are similar to the ICB site of MDR1. ZT65B was found to selectively bind ICB1 and ICB5; footprints were not observed with ICB2, ICB3, or ICB4. A strong, positive induced ligand band at 325 nm in CD studies confirmed that ZT65B binds in the DNA minor groove. The selectivity of ZT65B binding to hairpins that contained the MDR1 ICB site compared to one that did not (ICBd) was confirmed by surface-plasmon studies, and equilibrium constants of 5x10(6)-1x10(7) and 4.6x10(5) M-1 were obtained with ICB1, ICB5,and ICB2 respectively. ZT65B and the previously published JH37 (J. A. Henry, et al. Biochemistry 2004, 43, 12 249-12 257) serve as prototypes for the design of novel polyamides. These can be used to specifically target the subset of ubiquitous gene elements known as ICBs, and thereby affect the expression of one or a few proteins.

  15. Injectable hydrogel as cell carriers: Mechanism of beta-hairpin peptide hydrogel shear thinning, immediate recovery and effects on encapsulated cell payload (United States)

    Yan, Congqi

    To facilitate future biomedical treatment with localized delivery and higher therapy efficacy, much research effort has been devoted recently to the development of hydrogel biomaterials to transport a therapy to in vivo target sites via simple syringe or catheter injection. Most injectable hydrogel materials are free flowing precursor solutions ex vivo that become crosslinked into hydrogels once injected in vivo in response to exposure to environmental stimuli. However, properties of the final hydrogel formed in vivo are unpredictable due to possible leakage, dilution or change of injected gel precursor solution. As an alternate, more recent strategy for injectable hydrogel therapies, beta-hairpin peptide-based hydrogels are a class of injectable hydrogel solids with significant potential use in injectable therapies. These physical hydrogels can shear-thin and consequently flow as a low-viscosity material under a sufficient shear stress but immediately recover back into a solid upon removal of the stress, allowing them to be injected as preformed gel solids. The shear-thinning and immediate self-healing properties of self-assembled beta-hairpin peptide hydrogels enable a direct delivery of gel-encapsulated cells via benign injection to tissue defect sites with well-defined final gel properties in vivo. In this dissertation, mechanisms of gel shear-thinning and immediate recovery were elucidated by investigating gel behavior during and after flow via mechanical and structural characterizations. All studied beta-hairpin hydrogels shear-thin during flow (gel network fracture into large hydrogel domains) and instantly recover after cessation of flow (gel domains are percolated which immediately reforms the solid hydrogel). Importantly, hydrogel flow behavior was further studied in a capillary geometry that mimicked the actual situation of syringe injection. It was observed that all beta-hairpin peptide hydrogels investigated displayed a promising flow profile for

  16. Comparison of specific binding sites for Escherichia coli RNA polymerase with naturally occurring hairpin regions in single-stranded DNA of coliphage M13. [Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Niyogi, S.K.; Mitra, S.


    Escherichia coli RNA polymerase binds specifically to the single-stranded circular DNA of coliphage M13 in the presence of a saturating concentration of the bacterial DNA binding protein presumably as an essential step in the synthesis of the RNA primer required for synthesizing the complementary DNA strand in parental replicative-form DNA. The RNA polymerase-protected DNA regions were isolated after extensive digestion with pancreatic DNase, S1 endonuclease of Aspergillus oryzae, and exonuclease I of E. coli. The physicochemical properties of the RNA polymerase-protected segments (called PI and PII) were compared with those of the naturally occurring hairpin regions.

  17. Investigating the position of the hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis and inhibitor binding. (United States)

    Aitha, Mahesh; Moller, Abraham J; Sahu, Indra D; Horitani, Masaki; Tierney, David L; Crowder, Michael W


    In an effort to examine the relative position of a hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis, rapid freeze quench double electron electron resonance (RFQ-DEER) spectroscopy was used. A doubly-labeled mutant of NDM-1, which had one spin label on the invariant loop at position 69 and another label at position 235, was prepared and characterized. The reaction of the doubly spin labeled mutant with chromacef was freeze quenched at 500μs and 10ms. DEER results showed that the average distance between labels decreased by 4Å in the 500μs quenched sample and by 2Å in the 10ms quenched sample, as compared to the distance in the unreacted enzyme, although the peaks corresponding to distance distributions were very broad. DEER spectra with the doubly spin labeled enzyme with two inhibitors showed that the distance between the loop residue at position 69 and the spin label at position 235 does not change upon inhibitor binding. This study suggests that the hairpin loop in NDM-1 moves over the metal ion during the catalysis and then moves back to its original position after hydrolysis, which is consistent with a previous hypothesis based on NMR solution studies on a related metallo-β-lactamase. This study also demonstrates that this loop motion occurs in the millisecond time domain.

  18. Design of a beta-hairpin peptide-intercalator conjugate for simultaneous recognition of single stranded and double stranded regions of RNA. (United States)

    Cline, Lauren L; Waters, Marcey L


    Designing receptors that bind RNA is a challenging endeavor because of the unique and sometimes complex structure of RNA. However these structural features provide regions for ligands to bind using different types of interactions. To increase specificity and binding affinity to RNA, divalent systems have been designed which incorporate more than one binding motif into one molecule. Using this approach, we have designed a two part heteroconjugate, WKWK-Int, which contains a beta-hairpin peptide covalently linked to an RNA intercalator. This heteroconjugate was designed to bind duplex RNA through intercalation and simultaneously interact with a single stranded bulge region using the side chains of the beta-hairpin peptide. We have used fluorescence anisotropy experiments to show that the heteroconjugate has an increased binding affinity over either one of the individual ligands. Additionally, RNase footprinting experiments show that the structure of the peptide is necessary for the protection of one particular base in the RNA bulge region. When tested against other RNA molecules containing a stem-bulge structure, the designed heteroconjugate was found to be specific for this RNA sequence. This work provides evidence that the covalent linkage of two weak RNA ligands can greatly increase the binding affinity and also provide specificity to the binding event.

  19. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon. (United States)

    Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng


    K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Childhood Short Stature


    Ray, J.


    Childhood short stature comprises Varity of endocrinal, systemic, Skeletal & genetic disorders of pediatrics and is not just confined for endocrinal disorder only. A systemic approach often reduces the need for test which is often expensive &unnecessary. Use growth chart & asses bone age during evaluation. Short & heavy child are generally due to Endocrine causes, Short & thin are due to systemic disease, Short with normal velocity are may be due to Constitutional delay in growth &puberty or ...

  1. Short-circuit logic

    NARCIS (Netherlands)

    Bergstra, J.A.; Ponse, A.


    Short-circuit evaluation denotes the semantics of propositional connectives in which the second argument is only evaluated if the first argument does not suffice to determine the value of the expression. In programming, short-circuit evaluation is widely used. A short-circuit logic is a variant of p

  2. Early-time, beta-hairpin peptide self-assembly and hydrogelation: Structure, kinetics, and shear-recovery (United States)

    Yucel, Tuna

    Recently, there has been growing interest in the supramolecular self-assembly and hydrogelation of peptides for potential biomaterials applications. However, there has been limited work on the physicochemical characterization of these systems that will be crucial for the development of these materials for future applications. The main objective of this dissertation was to provide a solid understanding of the self-assembly kinetics, hydrogelation pathways and the physical origins of the shear-recovery behavior of a self-assembled, peptidic hydrogel system. The MAX1 peptide, (VK)4-VDPPT-(KV)4-NH 2 is unfolded, and completely soluble in acidic to neutral aqueous solution. Increasing the pH or ionic strength of the solution triggers intramolecular peptide folding into beta-hairpins and concomitant intermolecular self-assembly into bilayered nanofibrils. Combined static and dynamic light scattering experiments revealed a direct transition from the initial, monomeric state to self-assembled nanofibrils, without an intermediate self-assembly step. The energy barrier associated with MAX1 self-assembly suggested that the self-assembly process involved intramolecular peptide folding events and cluster reorganization to facilitate intermolecular self-assembly. The assembly kinetics could be modeled using Smoluchowski's equation which indicated an essentially diffusion-limited assembly process. The analysis of early-stage dependence of apparent mass on size and the concentration dependence of assembly kinetics gave different fractal dimension values: The former indicated that the nanofibrils behaved locally as rigid rods, while the latter analysis suggested an increase in the apparent fractal dimension at larger length scales. Cryogenic transmission electron microscopy indicated that the increase in the apparent fractal dimension could be attributed to the formation of branched clusters of well-defined (uniform, 3 nm cross section), semi-flexible, beta-sheet-rich nanofibrils

  3. Hierarchical structures based on self-assembling beta-hairpin peptides and their application as biomaterials and hybrid materials (United States)

    Altunbas, Aysegul

    an effective vehicle for the localized delivery of curcumin over sustained periods of time in vitro. The curcumin-hydrogel is prepared in-situ where curcumin encapsulation within the hydrogel network is accomplished concurrently with peptide self-assembly. Physical characterization methods and in vitro biological studies were used to demonstrate the effectiveness of curcumin-loaded beta-hairpin hydrogels as injectable agents for localized curcumin delivery. Notably, rheological characterization of the curcumin loaded hydrogel before and after shear flow have indicated solid-like properties even at high curcumin payloads. In vitro experiments with a medulloblastoma cell line confirm that the encapsulation of the curcumin within the hydrogel does not have an adverse effect on its bioactivity. Most importantly, the rate of curcumin release and its consequent therapeutic efficacy can be conveniently modulated by changing the morphological characteristics of the peptide hydrogel network. Lastly, MAX8 hydrogel cytocompatibility and biocompatibility was assessed with the future aim of utilizing this hydrogel as a scaffold in liver regeneration studies in rats. MAX8 hydrogel cytotoxity was evaluated using MC3T3-E1 and MG63 cell lines. Encapsulation, syringe delivery and subsequent viability of MG63 cells in hydrogels was also assessed to study the feasibility of using hydrogel/cell constructs as minimally invasive cell delivery vehicles. Biocompatibility was evaluated by monitoring inflammatory response induced by the MAX8 hydrogel via a subcutaneous mice model. Biocompatibility of MAX8 hydrogels at sites other than the subcutaneous region was also investigated using a cylindrical punch resection model in rat liver. The preliminary biocompatibility studies provide an elemental understanding of MAX8 hydrogel behavior in vivo.

  4. On J ade Hairpin and the West Wing Act%论《玉簪记》的“西厢行径”

    Institute of Scientific and Technical Information of China (English)



    In Jade Hairpin,Gao Lian put forward something about The West Win g Act through the mouths of the main characters.Most of the plot of The West Win g Act written by Gao Lian was drawn from The West Cham b er ,and also associated with the same strain spiritually.However,Gao Lian went against The West Cham b er in structure and character images with The West Win g Act ,which enabled The West Win g Act to be entirely different from The West Cham b er ,thus making J ade Hairpin a great success.In the subsequent generations,there appeared many adapted and imitated works and their success or failure were somewhat related to the treatment of The West Win g Act ."Jade Hairpin" and many subsequent creative works inspire us:inherit,but not copy,and one should have his own unique creation,which is the right approach to take the way of theWest Win g Acts .%《玉簪记》中,高濂借剧中主要人物之口提出“西厢行径”的说法。高濂笔下的“西厢行径”,表现为在情节上多所取材《西厢记》,在精神上也与其一脉相承。然而高濂又在结构和人物形象方面反《西厢记》而行,使《玉簪记》形成别具一格的“西厢行径”,从而取得了成功。后世对《西厢记》的改编和模仿之作甚多,它们的成功与失败也与对“西厢行径”的处理有千丝万缕的联系。《玉簪记》及其以后的众多创作启示我们:继承但不照搬,有自我独创之处,才是走“西厢行径”的明路。

  5. [Short lingual frenulum]. (United States)

    Klockars, Tuomas


    Short lingual frenulum is a common structural abnormality with unknown mechanisms of origination. The frequency of occurrence among newborn infants can be as high as 4%. Short lingual frenulum may cause problems, if it restricts the movements of the tongue. Therapeutic indications for short lingual frenulum in newborn infants are mainly breastfeeding problems, whereas in children of preschool age and older the indication is defective pronunciation.

  6. Long and short GRB

    CERN Document Server

    Katz, J I


    We report evidence from the 3B Catalogue that short (T_90 10 s) GRB represent different populations and processes: Their spectral behavior is qualitatively different, with short bursts harder in the BATSE range, but chiefly long bursts detected at higher photon energies; \\langle V/V_max \\rangle = 0.385 \\pm 0.019 for short GRB but \\langle V/V_max \\rangle = 0.282 \\pm 0.014 for long GRB, differing by 0.103 \\pm 0.024. Long GRB may be the consequence of accretion-induced collapse, but this mechanism fails for short GRB, for which we suggest colliding neutron stars.

  7. Construction the hairpin RNA recombinant plasmids targeting human Pokemon gene%靶向人Pokemon基因的shRNA重组质粒的构建

    Institute of Scientific and Technical Information of China (English)

    Wenmiao Cao; Tao Zhang; Buhai Wang; Xizhi Zhang; Jinghua Ren; Jie Yao


    Objective:The aim of this study was to establish the foundation for studying the role of pokemon gene in tumorigenesis and development by constructing recombinant plasmids that can express small interfering RNA (siRNA) targeting human Pokemon gene. Methods: Hairpin siRNA templates targeting Pokemon gene were synthesized and cloned into plasmid vector psiRNA-H1neo. Three vectors derived siRNAs (psiRNA1, 2, 3) and one mocking psiRNAc (as control) were constructed.The recombinant Pokemon siRNA plasmids were constructed and identified using restrictive enzyme analysis and DNA sequencing. Results: Restrictive enzyme analysis and DNA sequencing revealed the successful construction of siRNA expression plasmids. Conclusion: Constructing siRNA templates targeting Pokemon gene may provide us with practical tools for further study the role of Pokemon gene in the development of some diseases and gene therapy of tumor.

  8. Single-atom imino substitutions at A9 and A10 reveal distinct effects on the fold and function of the hairpin ribozyme catalytic core. (United States)

    Spitale, Robert C; Volpini, Rosaria; Mungillo, Michael V; Krucinska, Jolanta; Cristalli, Gloria; Wedekind, Joseph E


    The hairpin ribozyme cleaves a phosphodiester bond within a cognate substrate. Structural and biochemical data indicate the conserved A9 and A10 bases reside close to the scissile bond but make distinct contributions to catalysis. To investigate these residues, we replaced the imino moiety of each base with N1-deazaadenosine. This single-atom change resulted in an 8-fold loss in k(obs) for A9 and displacement of the base from the active site; no effects were observed for A10. We propose that the imino moiety of A9 promotes a key water-mediated contact that favors transition-state formation, which suggests an enhanced chemical repertoire for RNA.

  9. Carbon nanotube enhanced label-free detection of microRNAs based on hairpin probe triggered solid-phase rolling-circle amplification (United States)

    Tian, Qianqian; Wang, Ying; Deng, Ruijie; Lin, Lei; Liu, Yang; Li, Jinghong


    The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification (RCA). Traditionally, RCA, widely applied for signal enhancement in the construction of a variety of biosensors, has an intrinsic limitation of ultrasensitive detection, as it is difficult to separate the enzymes, templates, and padlock DNAs from the RCA products in the homogeneous solution. We purposely designed a solid-phase RCA strategy, using CNTs as the solid substrate, integrated with a hairpin structured probe to recognize target miRNA. In the presence of miRNA the stem-loop structure will be unfolded, triggering the CNT based RCA process. Due to the efficient blocking effect originating from the polymeric RCA products, the label-free assay of miRNA exhibits an ultrasensitive detection limit of 1.2 fM. Furthermore, the protocol possesses excellent specificity for resolving lung cancer-related let-7 family members which have only one-nucleotide variations. The high sensitivity and selectivity give the method great potential for applications in online diagnostics and in situ detection in long-term development.The detection of microRNAs (miRNAs) is imperative for gaining a better understanding of the functions of these biomarkers and has great potential for the early diagnosis of human disease. High sensitivity and selectivity for miRNA detection brings new challenges. Herein, an ultrasensitive protocol for electrochemical detection of miRNA is designed through carbon nanotube (CNT) enhanced label-free detection based on hairpin probe triggered solid-phase rolling-circle amplification

  10. Enzyme-free and sensitive electrochemical determination of the FLT3 gene based on a dual signal amplified strategy: Controlled nanomaterial multilayers and a target-catalyzed hairpin assembly. (United States)

    Sun, Yingying; Ren, Qunxiang; Liu, Bo; Qin, Yan; Zhao, Shuang


    An isothermal, enzyme-free and sensitive electrochemical DNA sensor was developed for the detection of the FLT3 gene in acute myeloid leukemia (AML). First, aminated multi-walled carbon nanotubes (AMWNTs) and gold nanoparticles (AuNPs) were alternately self-assembled on a gold electrode using a layer-by-layer strategy. Then, the hairpin DNA probe 1 (H1), with a thiol group at the 3' end and a ferrocenyl moiety (Fc) at the 5' end, was immobilized on the AMWNTs/AuNPs multilayer films through Au-S bonding. When the target DNA (TD) appeared, it hybridized with and opened the hairpin structure of H1, and Fc was forced away from the electrode surface, leading to a significant decrease in the current peak of square wave voltammetry. Subsequently, the hairpin DNA probe 2 (H2) bound to H1, freeing the TD to trigger another reaction cycle. The combination of this target-catalyzed hairpin assembly and the LBL assembly of nanomaterials achieved a detection limit of 0.1 pM with a wide linear range of 0.1-1000 pM. The sensor discriminated between mismatched DNA and the target DNA with high selectivity. This dual signal amplification strategy is relatively simple and inexpensive because it does not need any enzymes or sophisticated equipment and successfully assayed the FLT3 gene from real samples.

  11. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin. (United States)

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E


    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.

  12. Childhood Short Stature

    Directory of Open Access Journals (Sweden)



    Full Text Available Childhood short stature comprises Varity of endocrinal, systemic, Skeletal & genetic disorders of pediatrics and is not just confined for endocrinal disorder only. A systemic approach often reduces the need for test which is often expensive &unnecessary. Use growth chart & asses bone age during evaluation. Short & heavy child are generally due to Endocrine causes, Short & thin are due to systemic disease, Short with normal velocity are may be due to Constitutional delay in growth &puberty or Familial short stature, differentiation can be done by Bone Age. In Girls Turner syndrome has to be kept in mind. Purpose of evaluation to find out the child who does not need treatment, who cannot be treated & the child who can be benefited from treatment.

  13. Idiopathic short stature

    Directory of Open Access Journals (Sweden)

    Vlaški Jovan


    Full Text Available Growth is a complex process and the basic characteristic of child- hood growth monitoring provides insight into the physiological and pathological events in the body. Statistically, the short stature means departure from the values of height for age and sex (in a particular environment, which is below -2 standard deviation score, or less than -2 standard deviation, i.e. below the third percentile. Advances in molecular genetics have contributed to the improvement of diagnostics in endocrinology. Analysis of patients’ genotypes should not be performed before taking a classical history, detailed clinical examination and appropriate tests. In patients with idiopathic short stature specific causes are excluded, such as growth hormone deficiency, Turner syndrome, short stature due to low birth weight, intrauterine growth retardation, small for gestational age, dysmorphology syndromes and chronic childhood diseases. The exclusion of abovementioned conditions leaves a large number of children with short stature whose etiology includes patients with genetic short stature or familial short stature and those who are low in relation to genetic potential, and who could also have some unrecognized endocrine defect. Idiopathic short stature represents a short stature of unknown cause of heterogeneous etiology, and is characterized by a normal response of growth hormone during stimulation tests (>10 ng/ml or 20 mJ/l, without other disorders, of normal body mass and length at birth. In idiopathic short stature standard deviation score rates <-2.25 (-2 to -3 or <1.2 percentile. These are also criteria for the initiation of growth hormone therapy. In children with short stature there is also the presence of psychological and social suffering. Goals of treatment with growth hormone involve achieving normal height and normal growth rate during childhood.

  14. Congenital short pancreas

    Institute of Scientific and Technical Information of China (English)

    DU Juan; XU Guo-qiang; XU Ping; JIN En-yun; LIU Qiong; LI You-ming


    @@ Congenital short pancreas, also known as partial agenesis or hypoplasia of the dorsal pancreas1 is a rare congenital abnormality consisting of the parenchyma and ductal system restricted to the head with some residual dorsal tapering and arborizing ducts communicating with the minor papill.2 Complete pancreatic agenesis is fatal, and only nine possible examples of partial agenesis have been previously reported in adults in the literature.3-10 Three of them were polysplenia syndrome associated with short pancreas,and only six patients with congenital short pancreas with normal situs. Here we present a new case associated with steatorrhoea.

  15. Imaging in short stature

    Directory of Open Access Journals (Sweden)

    Vikas Chaudhary


    Full Text Available Short stature can be a sign of disease, disability, and social stigma causing psychological stress. It is important to have an early diagnosis and treatment. Short stature may result from skeletal dysplasias, endocrine disorders, may be familial, or may be the result of malnutrition and chronic illnesses. A team effort of the healthcare professionals like pediatricians, endocrinologists, radiologists, and pathologists is required to diagnose, treat and monitor various pathological conditions associated with growth abnormality. In this review, we have discussed the role of imaging in diagnosing and characterizing various pathological conditions associated with short stature.

  16. Minimizing Shortness of Breath (United States)

    ... is also placed on proper use of the abdominal muscles to better control episodes of shortness of breath, ... Treatment & Programs Health Insights Doctors & Departments Research & Science Education & Training Make a Donation Make an Appointment Contact ...

  17. Short-cut math

    CERN Document Server

    Kelly, Gerard W


    Clear, concise compendium of about 150 time-saving math short-cuts features faster, easier ways to add, subtract, multiply, and divide. Each problem includes an explanation of the method. No special math ability needed.

  18. Shortness of Breath (United States)

    ... shortness of breath with physical exertion beyond your customary activity such as when climbing stairs. Allergic Reactions ... 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524- ...

  19. Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection

    Directory of Open Access Journals (Sweden)

    Spena Angelo


    Full Text Available Abstract Background Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS, is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana. Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection. Results In the ihprolC-PP197 gene (intron hair pin rolC PPV 197, a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330 was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80% transgenic plants are virus free and symptomless. Some plants (20% contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23–25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation

  20. The Kissing-Loop T-Shaped Structure Translational Enhancer of Pea Enation Mosaic Virus Can Bind Simultaneously to Ribosomes and a 5′ Proximal Hairpin (United States)

    Gao, Feng; Gulay, Suna P.; Kasprzak, Wojciech; Dinman, Jonathan D.


    The Pea Enation Mosaic Virus (PEMV) 3′ translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5′-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5′ and 3′ PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3′ translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2′-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5′ hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5′ end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation. PMID:23986599

  1. Long-Short Portfolio

    Directory of Open Access Journals (Sweden)

    Leo Shubert


    Full Text Available Long-short strategies are one of the most successful tools, applied by hedge funds manager. One under-evaluated stock is bought (long position and an over-evaluated stock is sold (short position at the same time. After a short term, when the values of the stocks are as expected, profit can be realized by a closing transaction. The possibility to find first obvious over- and under-evaluated stocks depends on the number of participants in this markets. While the hedge funds strategies become more popular, the chance to achieve profit by this strategies is shrinking.Therefore two models to generate long-short portfolios are proposed. By this approaches a portfolio A for the long- and a portfolio B for the short position were computed. The difference of the values of A and B is designed to oscillate from negative to positive and reverse. This behavior of oscillating or mean reverting stock prices was stated by e.g. E. Fama and K. R. French (1988. Mean reversion of portfolios can offer the possibility of statistical arbitrage. The proposed linear models were tested by stocks of the Tokyo stock exchange. The results seem to be applicable and show an additional advantage of low systematic risk.

  2. Idiopathic short stature. (United States)

    Pasquino, A M; Albanese, A; Bozzola, M; Butler, G E; Buzi, F; Cherubini, V; Chiarelli, F; Cavallo, L; Drop, S L; Stanhope, R; Kelnar, C J


    Idiopathic short stature (ISS) is a term used to describe the status of children with short stature that cannot be attributed to a specific cause. Many children diagnosed as having ISS have partial GH insensitivity, which can result from disturbances at various points of the GH-IGF-I axis. Several clinical studies on spontaneous growth in ISS showed that adult height was almost in the range of target height. GH treatment led to adult height not significantly higher than the pretreatment predicted adult height in most reports. No metabolic side effects have been observed, even when the dose was higher than in GH deficiency. Manipulation of puberty with gonadotrophin releasing hormone analogues reported by a few authors in a small number of children has shown conflicting results. Long-term psychological benefits of GH therapy for short normal children have not been demonstrated to date.

  3. Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication. (United States)

    Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean


    The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

  4. "Picking willow farewell" in "The legend of purple hairpin"%论《紫钗记》之“折柳送别”

    Institute of Scientific and Technical Information of China (English)



    "Picking willow farewell" is the important content in "The legend of purple hairpin". Resign as colorful, twitters melancholy, long acting on the stage. "Picking willow farewell" in ancient China has a long history tradition, Huo Xiao-yu’s willow in Yangguan has obvious differences in the image from the Liu Meng-mei’s in the peony pavilion.%《折柳阳关》是《紫钗记》中的重要关目。一直作为辞采华美、情致惆怅的生旦戏在舞台上长演不衰。“折柳送别”在中国古代有着悠久的历史的传统习俗,霍小玉阳关送别所折之柳与《牡丹亭》中柳梦梅手执之柳在意象上有着明显的差异。

  5. Sequence-specific DNA alkylation and transcriptional inhibition by long-chain hairpin pyrrole-imidazole polyamide-chlorambucil conjugates targeting CAG/CTG trinucleotide repeats. (United States)

    Asamitsu, Sefan; Kawamoto, Yusuke; Hashiya, Fumitaka; Hashiya, Kaori; Yamamoto, Makoto; Kizaki, Seiichiro; Bando, Toshikazu; Sugiyama, Hiroshi


    Introducing novel building blocks to solid-phase peptide synthesis, we readily synthesized long-chain hairpin pyrrole-imidazole (PI) polyamide-chlorambucil conjugates 3 and 4 via the introduction of an amino group into a GABA (γ-turn) contained in 3, to target CAG/CTG repeat sequences, which are associated with various hereditary disorders. A high-resolution denaturing polyacrylamide sequencing gel revealed sequence-specific alkylation both strands at the N3 of adenines or guanines in CAG/CTG repeats by conjugates 3 and 4, with 11bp recognition. In vitro transcription assays using conjugate 4 revealed that specific alkylation inhibited the progression of RNA polymerase at the alkylating sites. Chiral substitution of the γ-turn with an amino group resulted in higher binding affinity observed in SPR assays. These assays suggest that conjugates 4 with 11bp recognition has the potential to cause specific DNA damage and transcriptional inhibition at the alkylating sites. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. The beta hairpin structure within ribosomal protein S5 mediates interplay between domains II and IV and regulates HCV IRES function. (United States)

    Bhat, Prasanna; Shwetha, Shivaprasad; Sharma, Divya Khandige; Joseph, Agnel Praveen; Srinivasan, Narayanaswamy; Das, Saumitra


    Translation initiation in Hepatitis C Virus (HCV) is mediated by Internal Ribosome Entry Site (IRES), which is independent of cap-structure and uses a limited number of canonical initiation factors. During translation initiation IRES-40S complex formation depends on high affinity interaction of IRES with ribosomal proteins. Earlier, it has been shown that ribosomal protein S5 (RPS5) interacts with HCV IRES. Here, we have extensively characterized the HCV IRES-RPS5 interaction and demonstrated its role in IRES function. Computational modelling and RNA-protein interaction studies demonstrated that the beta hairpin structure within RPS5 is critically required for the binding with domains II and IV. Mutations disrupting IRES-RPS5 interaction drastically reduced the 80S complex formation and the corresponding IRES activity. Computational analysis and UV cross-linking experiments using various IRES-mutants revealed interplay between domains II and IV mediated by RPS5. In addition, present study demonstrated that RPS5 interaction is unique to HCV IRES and is not involved in 40S-3' UTR interaction. Further, partial silencing of RPS5 resulted in preferential inhibition of HCV RNA translation. However, global translation was marginally affected by partial silencing of RPS5. Taken together, results provide novel molecular insights into IRES-RPS5 interaction and unravel its functional significance in mediating internal initiation of translation.

  7. Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana. (United States)

    Ghag, Siddhesh B; Shekhawat, Upendra K S; Ganapathi, Thumballi R


    Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is among the most destructive diseases of banana (Musa spp.). Because no credible control measures are available, development of resistant cultivars through genetic engineering is the only option. We investigated whether intron hairpin RNA (ihpRNA)-mediated expression of small interfering RNAs (siRNAs) targeted against vital fungal genes (velvet and Fusarium transcription factor 1) in transgenic banana could achieve effective resistance against Foc. Partial sequences of these two genes were assembled as ihpRNAs in suitable binary vectors (ihpRNA-VEL and ihpRNA-FTF1) and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium-mediated genetic transformation. Eleven transformed lines derived from ihpRNA-VEL and twelve lines derived from ihpRNA-FTF1 were found to be free of external and internal symptoms of Foc after 6-week-long greenhouse bioassays. The five selected transgenic lines for each construct continued to resist Foc at 8 months postinoculation. Presence of specific siRNAs derived from the two ihpRNAs in transgenic banana plants was confirmed by Northern blotting and Illumina sequencing of small RNAs derived from the transgenic banana plants. The present study represents an important effort in proving that host-induced post-transcriptional ihpRNA-mediated gene silencing of vital fungal genes can confer efficient resistance against debilitating pathogens in crop plants.

  8. Resistance of non-transgenic papaya plants to papaya ringspot virus (PRSV) mediated by intron-containing hairpin dsRNAs expressed in bacteria. (United States)

    Shen, W; Yang, G; Chen, Y; Yan, P; Tuo, D; Li, X; Zhou, P


    RNA-mediated virus resistance based on natural antiviral RNA silencing has been exploited as a powerful tool for engineering virus resistance in plants. In this study, a conserved 3'-region (positions 9839-10117, 279 nt) of the capsid protein (CP) gene of papaya ringspot virus (PRSV), designated CP279, was used to generate an intron-containing hairpin RNA (ihpRNA) construct by one-step, zero-background ligation-independent cloning (OZ-LIC). The RNaseIII-deficient Escherichia coli strain M-JM109lacY was identified as the best choice for producing large quantities of specific ihpRNA-CP279. Resistance analyses and ELISA data verified that most papaya plants mechanically co-inoculated with TRIzol-extracted ihpRNA-CP279 and PRSV were resistant to PRSV, and resistance was maintained throughout the test period (>2 months post-inoculation). In contrast, a 1-2 day interval between sequential inoculation of PRSV and ihpRNA-CP279 did not result in complete protection against PRSV infection, but delayed the appearance of viral symptoms by 3 to 4 days. These findings indicate that direct mechanical inoculation of papaya plants with bacterially-expressed ihpRNA-CP279 targeting the PRSV CP gene can interfere with virus infection. This work lays a foundation for developing a non-transgenic approach to control PRSV by directly spraying plants with ihpRNA or crude bacterial extract preparations.

  9. A regenerative ratiometric electrochemical biosensor for selective detecting Hg²⁺ based on Y-shaped/hairpin DNA transformation. (United States)

    Jia, Jing; Chen, Hong Guo; Feng, Ji; Lei, Jing Lei; Luo, Hong Qun; Li, Nian Bing


    Inspired by dual-signaling ratiometric mechanism which could reduce the influence of the environmental change, a novel, convenient, and reliable method for the detection of mercury ions (Hg(2+)) based on Y-shaped DNA (Y-DNA) was developed. Firstly, the Y-DNA was formed via the simple annealing way of using two different redox probes simultaneously, omitting the multiple operation steps on the electrode. The Y-DNA was immobilized on the gold electrode surface and then an obvious ferrocene (Fc) signal and a weak methylene blue (MB) signal were observed. Upon addition of Hg(2+), the Y-DNA structure was transformed to hairpin structure based on the formation of T-Hg(2+)-T complex. During the transformation, the redox MB gets close to and the redox Fc gets far away from the electrode surface, respectively. This special design allows a reliable Hg(2+) detection with a detection range from 1 nM to 5 μM and a low detection limit down to 0.094 nM. Furthermore, this biosensor exhibits good selectivity and repeatability, and can be easily regenerated by using L-cysteine. This study offers a simple and effective method for designing ratiometric biosensors for detecting other ions and biomolecules.


    Directory of Open Access Journals (Sweden)

    Z Moinfar


    Full Text Available Short rib polydactyly syndrome (SRPS is a very rare congenital anomaly that is classified into four subtypes. It is an autosomal recessive inherited disease. We report a case of this syndrome without a previous family history of congenital defects.

  11. Short-rotation plantations (United States)

    Philip E. Pope; Jeffery O. Dawson


    Short-rotation plantations offer several advantages over longer, more traditional rotations. They enhance the natural productivity of better sites and of tree species with rapid juvenile growth. Returns on investment are realized in a shorter period and the risk of loss is reduced compared with long term investments. Production of wood and fiber can be maximized by...

  12. Congenital Short QT Syndrome

    Directory of Open Access Journals (Sweden)

    Charles Antzelevitch


    Full Text Available Long QT intervals in the ECG have long been associated with sudden cardiac death. The congenital long QT syndrome was first described in individuals with structurally normal hearts in 1957.1 Little was known about the significance of a short QT interval. In 1993, after analyzing 6693 consecutive Holter recordings Algra et al concluded that an increased risk of sudden death was present not only in patients with long QT interval, but also in patients with short QT interval (<400 ms.2 Because this was a retrospective analysis, further evaluation of the data was not possible. It was not until 2000 that a short-QT syndrome (SQTS was proposed as a new inherited clinical syndrome by Gussak et al.3 The initial report was of two siblings and their mother all of whom displayed persistently short QT interval. The youngest was a 17 year old female presenting with several episodes of paroxysmal atrial fibrillation requiring electrical cardioversion.3 Her QT interval measured 280 msec at a heart rate of 69. Her 21 year old brother displayed a QT interval of 272 msec at a heart rate of 58, whereas the 51 year old mother showed a QT of 260 msec at a heart rate of 74. The authors also noted similar ECG findings in another unrelated 37 year old patient associated with sudden cardiac death.

  13. {sup 13}C structuring shifts for the analysis of model {beta}-hairpins and {beta}-sheets in proteins: diagnostic shifts appear only at the cross-strand H-bonded residues

    Energy Technology Data Exchange (ETDEWEB)

    Shu, Irene; Scian, Michele; Stewart, James M.; Kier, Brandon L.; Andersen, Niels H., E-mail: [University of Washington, Department of Chemistry (United States)


    The present studies have shown that {sup 13}C=O, {sup 13}C{sup {alpha}} and {sup 13}C{sup {beta}} of H-bonded strand residues in {beta}-hairpins provide additional probes for quantitating the extent of folding in {beta}-hairpins and other {beta}-sheet models. Large differences in the structuring shifts (CSDs) of these {sup 13}C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three {sup 13}C probes. This prompts us to suggest that efforts to determine the extent of hairpin folding from {sup 13}C shifts should be based exclusively on the observation at the cross-strand H-bonded sites. Furthermore, the statistics suggest the {sup 13}C Prime and {sup 13}C{sup {beta}}CSDs will provide the best differentiation with 100 %-folded CSD values approaching -2.6 and +3 ppm, respectively, for the H-bonded sites. These conclusions can be extended to edge-strands of protein {beta}-sheets. Our survey of reported {sup 13}C shifts in {beta}-proteins indicates that some of the currently employed random coil values need to be adjusted, particularly for ionization-induced effects.

  14. The solution structure of double helical arabino nucleic acids (ANA and 2'F-ANA): effect of arabinoses in duplex-hairpin interconversion. (United States)

    Martín-Pintado, Nerea; Yahyaee-Anzahaee, Maryam; Campos-Olivas, Ramón; Noronha, Anne M; Wilds, Christopher J; Damha, Masad J; González, Carlos


    We report here the first structure of double helical arabino nucleic acid (ANA), the C2'-stereoisomer of RNA, and the 2'-fluoro-ANA analogue (2'F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2'-deoxy-2'F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities. One structure is a monomeric hairpin in which the stem is formed by base paired 2'F-ANA nucleotides and the loop by unpaired ANA nucleotides. The second structure is a bimolecular duplex, with all the nucleotides (2'F-ANA and ANA) forming Watson-Crick base pairs. The duplex structure is canonical B-form, with all arabinoses adopting a pure C2'-endo conformation. In the ANA:ANA segment, steric interactions involving the 2'-OH substituent provoke slight changes in the glycosidic angles and, therefore, in the ANA:ANA base pair geometry. These distortions are not present in the 2'F-ANA:2'F-ANA regions of the duplex, where the -OH substituent is replaced by a smaller fluorine atom. 2'F-ANA nucleotides adopt the C2'-endo sugar pucker and fit very well into the geometry of B-form duplex, allowing for favourable 2'F···H8 interactions. This interaction shares many features of pseudo-hydrogen bonds previously observed in 2'F-ANA:RNA hybrids and in single 2'F-ANA nucleotides.

  15. Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant (United States)

    Akbar, Sehrish; Wang, Ming-Bo; Liu, Qing


    Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane. PMID:28255554

  16. Proton-Coupled Electron Transfer and a Tyrosine-Histidine Pair in a Photosystem II-Inspired β-Hairpin Maquette: Kinetics on the Picosecond Time Scale. (United States)

    Pagba, Cynthia V; McCaslin, Tyler G; Chi, San-Hui; Perry, Joseph W; Barry, Bridgette A


    Photosystem II (PSII) and ribonucleotide reductase employ oxidation and reduction of the tyrosine aromatic ring in radical transport pathways. Tyrosine-based reactions involve either proton-coupled electron transfer (PCET) or electron transfer (ET) alone, depending on the pH and the pKa of tyrosine's phenolic oxygen. In PSII, a subset of the PCET reactions are mediated by a tyrosine-histidine redox-driven proton relay, YD-His189. Peptide A is a PSII-inspired β-hairpin, which contains a single tyrosine (Y5) and histidine (H14). Previous electrochemical characterization indicated that Peptide A conducts a net PCET reaction between Y5 and H14, which have a cross-strand π-π interaction. The kinetic impact of H14 has not yet been explored. Here, we address this question through time-resolved absorption spectroscopy and 280-nm photolysis, which generates a neutral tyrosyl radical. The formation and decay of the neutral tyrosyl radical at 410 nm were monitored in Peptide A and its variant, Peptide C, in which H14 is replaced by cyclohexylalanine (Cha14). Significantly, both electron transfer (ET, pL 11, L = lyonium) and PCET (pL 9) were accelerated in Peptide A and C, compared to model tyrosinate or tyrosine at the same pL. Increased electronic coupling, mediated by the peptide backbone, can account for this rate acceleration. Deuterium exchange gave no significant solvent isotope effect in the peptides. At pL 9, but not at pL 11, the reaction rate decreased when H14 was mutated to Cha14. This decrease in rate is attributed to an increase in reorganization energy in the Cha14 mutant. The Y5-H14 mechanism in Peptide A is reminiscent of proton- and electron-transfer events involving YD-H189 in PSII. These results document a mechanism by which proton donors and acceptors can regulate the rate of PCET reactions.

  17. Mungbean yellow mosaic virus (MYMV) AC4 suppresses post-transcriptional gene silencing and an AC4 hairpin RNA gene reduces MYMV DNA accumulation in transgenic tobacco. (United States)

    Sunitha, Sukumaran; Shanmugapriya, Gnanasekaran; Balamani, Veluthambi; Veluthambi, Karuppannan


    Mungbean yellow mosaic virus (MYMV) is a legume-infecting geminivirus that causes yellow mosaic disease in blackgram, mungbean, soybean, Frenchbean and mothbean. AC4/C4, which is nested completely within the Rep gene, is less conserved among geminiviruses. Much less is known about its role in viral pathogenesis other than its known role in the suppression of host-mediated gene silencing. Transient expression of MYMV AC4 by agroinfiltration suppressed post-transcriptional gene silencing in Nicotiana benthamiana 16c expressing green fluorescence protein, at a level comparable to MYMV TrAP expression. AC4 full-length gene and an inverted repeat of AC4 (comprising the full-length AC4 sequence in sense and antisense orientations with an intervening intron) which makes a hairpin RNA (hpRNA) upon transcription were introduced into tobacco by Agrobacterium-mediated leaf disc transformation. Leaf discs of the transgenic plants were agroinoculated with partial dimers of MYMV and used to study the effect of the AC4-sense and AC4 hpRNA genes on MYMV DNA accumulation. Leaf discs of two transgenic plants that express the AC4-sense gene displayed an increase in MYMV DNA accumulation. Leaf discs of six transgenic plants containing the AC4 hpRNA gene accumulated small-interfering RNAs (siRNAs) specific to AC4, and upon agroinoculation with MYMV they exhibited a severe reduction in the accumulation of MYMV DNA. Thus, the MYMV AC4 hpRNA gene has emerged as a good candidate to engineer resistance against MYMV in susceptible plants.

  18. Increased electrocatalyzed performance through hairpin oligonucleotide aptamer-functionalized gold nanorods labels and graphene-streptavidin nanomatrix: Highly selective and sensitive electrochemical biosensor of carcinoembryonic antigen. (United States)

    Wen, Wei; Huang, Jing-Yi; Bao, Ting; Zhou, Jun; Xia, Hong-Xing; Zhang, Xiu-Hua; Wang, Sheng-Fu; Zhao, Yuan-Di


    We report a triplex signal amplification strategy for sensitive biosensing of cancer biomarker by taking advantage of hairpin-shaped oligonucleotide-functionalized gold nanorods (HO-GNRs), graphene and the avidin-biotin reation. The strategy expands electrochemical detection of carcinoembryonic antigen (CEA) by using an aptamer as biosensor's recognition element and HO-GNRs as signal enhancer. To construct this biosensor, the GNR was used as a carrier of horseradish peroxidase (HRP) and HO aptamer with a biotin at the 3'-end and a thiol at the 5'-end, which amplified the electrochemical response because of a large molar ratio of HRP to HO. In the presence of target CEA, the binding reactions of CEA with the loop portions of the HOs caused HOs' loop-stem structure opened and exposed the biotins, and then HRP-GNRs-HO conjugates were captured on graphene and streptavidin modified electrodes via the reaction between the exposed biotins and preimmobilized streptavidins. The accumulation of HRP effectively catalyzed the hydrogen peroxide-mediated oxidation of o-phenylenediamine to generate an electrochemical reduction current for CEA detection. Under optimal conditions, the electrochemical biosensor exhibited a wide dynamic range of 5pgmL(-1) and 50ngmL(-1) toward CEA standards with a low detection limit of 1.5pgmL(-1) (signal-to-noise ratio of 3). The proposed biosensor accurately detected CEA concentration in 8 human serum samples from patients with lung diseases, showing excellent correlations with standard chemiluminescence immunoassay. Furthermore, these results of target DNA detection made it abundantly clear that the proposed strategy can also be extended for detection of other relative biomarkers using different functional DNA structures, which shows great prospects in single-nucleotide polymorphisms analysis, biomedical sensing and application for accurate clinical diseases diagnostic.

  19. Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

    Institute of Scientific and Technical Information of China (English)

    CHENG Chun-zhen[1,2,5; YANG Jia-wei[3; YAN Hu-bin[4; BEI Xue-jun[1,2; ZHANG Yong-yan[1,2; LU Zhi-ming[1; ZHONG Guang-yan[2,5


    The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat- egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag- ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Following transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested following inoculation with CT14A and/ or TR-L5"I4, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently challenged with CT14A. These results showed that expressing p20 hpRNAwas sufficient to confer sour orange with CTV resistance/tolerance.

  20. Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains

    Institute of Scientific and Technical Information of China (English)

    CHENG Chun-zhen; YANG Jia-wei; YAN Hu-bin; BEI Xue-jun; ZHANG Yong-yan; LU Zhi-ming; ZHONG Guang-yan


    The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strat-egies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA frag-ments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Fol owing transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested fol owing inoculation with CT14A and/or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently chal enged with CT14A. These results showed that expressing p20 hpRNA was sufifcient to confer sour orange with CTV resistance/tolerance.

  1. Short Text Classification: A Survey

    Directory of Open Access Journals (Sweden)

    Ge Song


    Full Text Available With the recent explosive growth of e-commerce and online communication, a new genre of text, short text, has been extensively applied in many areas. So many researches focus on short text mining. It is a challenge to classify the short text owing to its natural characters, such as sparseness, large-scale, immediacy, non-standardization. It is difficult for traditional methods to deal with short text classification mainly because too limited words in short text cannot represent the feature space and the relationship between words and documents. Several researches and reviews on text classification are shown in recent times. However, only a few of researches focus on short text classification. This paper discusses the characters of short text and the difficulty of short text classification. Then we introduce the existing popular works on short text classifiers and models, including short text classification using sematic analysis, semi-supervised short text classification, ensemble short text classification, and real-time classification. The evaluations of short text classification are analyzed in our paper. Finally we summarize the existing classification technology and prospect for development trend of short text classification

  2. Short QT syndrome

    Directory of Open Access Journals (Sweden)

    Fiorenzo Gaita


    Full Text Available The short QT syndrome (SQTS is a recently described genetic arrhythmogenic disorder, characterized by abnormally short QT intervals on surface electrocardiogram (ECG and a high incidence of sudden death (SD during life, including the first months of life. The inheritance of SQTS is autosomal dominant, with genetic heterogeneity. Gain-of-function mutations in 3 genes encoding potassium channels have been associated to the disease: KCNH2 encoding IKr (SQT1, KCNQ1 encoding IKs (SQT2, and KCNJ2 encoding IK1 (SQT3. Loss-of-function mutations in 3 genes encoding the cardiac L-type calcium channel, CACNA1C, CACNB2b and CACNA2D1 may underlie a mixed phenotype of Brugada pattern ECG (or non-specific repolarization changes in case of CACNA2D1 and shorter than normal QT intervals. Clinical presentation is often severe, as cardiac arrest represents the first clinical presentation in most subjects. Moreover, often a noticeable family history of cardiac SD is present. Atrial fibrillation may be observed, also in young individuals. At electrophysiological study, short atrial and ventricular refractory periods are found, and atrial and ventricular fibrillation are easily induced by programmed electrical stimulation. The outcome of patients with SQTS becomes relatively safe when they are identified and treated. Currently, the suggested therapeutic strategy is an implantable cardioverter- defibrillator (ICD in patients with personal history of aborted SD or syncope. In asymptomatic adult patients from highly symptomatic families and in newborn children pharmacological treatment with hydroquinidine, which has been shown to prolong the QT interval and reduce the inducibility of ventricular arrhythmias, may be proposed.

  3. 靶向VEGF发夹状RNA对宫颈癌HeLa细胞抑制作用的研究%Inhibitory effect of small hairpin RNA targeting at VEGF on cervical carcinoma cell line HeLa

    Institute of Scientific and Technical Information of China (English)

    庞然然; 邢丽娜


    OBJECTIVE; To study apoptosis of the tumour cells by RNA interference (RNAi) technology to depress human vascular endothelial growth factor (VEGF) gene expression in HeLa cells,and provide a theoretical basis for the treatment of human cervical carcinoma. METHODS: Short hairpin RNA targeting at VEGF gene was designed and synthesized, which was integrated into plasmid expression vector (PGPU6) with green fluorescent protein (GFP),then trans-fected into HeLa cells by lipofectamine. The expression of GFP and calculate transfection efficiency was observed by fluorescence microscope. The VEGF mRNA expression in HeLa cells was detected by RT-PCR respectively. Apoptosis was measured by flow cytometry. RESULTS: PGPU6/GFP/Neo-shRNA,a shRNA plasmid expression vector.was successfully transfected into HeLa cells. After 48 hours, The VEGF mRNA expression in HeLa cells transfected with PGPU6-shVEGFl was decreased to 75. 0%. Compared with HeLa group and HeLa-shNC group, the apoptosis of HeLa cells in HeLa-shVEGFl group was remarkably increased (P<0. 01). CONCLUSIONS: The PGPU6-shRNA expression vector with GFP constructed can detect the gene transfection more conveniently and do not affect the transcription of U6 promoter. VEGF gene silenced can obviously increase apoptosis of HeLa cells, which may be the new target and efficient way for tumor therapy in the future.%目的:运用RNAi技术下调血管内皮生长因子(VEGF)在HeLa细胞中的表达,观察其对肿瘤细胞凋亡的影响,为人宫颈癌治疗提供理论依据.方法:设计并构建针对VEGF的携带绿色荧光蛋白( GFP)发夹状RNA (shRNA)质粒表达载体(PGPU6/GFP/Neo-shRNA),脂质体法转染HeLa细胞;荧光显微镜观察GFP的表达,并计算转染效率;RT-PCR检测HeLa细胞VEGF的表达,筛选出靶序列;再用流式细胞仪法检测细胞凋亡.结果:构建的PGPU6/GFP/Neo载体成功转入HeLa细胞;转染48 h后,HeLa-shVEGF1组HeLa细胞VEGFmRNA表达的抑制率为75.0%;与HeLa

  4. Scottish Short Stone Rows (United States)

    Ruggles, Clive L. N.

    Short stone rows received a good deal of attention during the 1980s and 1990s, at a time when archaeoastronomy in prehistoric Britain and Ireland was moving beyond reassessments of Alexander Thom's "megalithic observatories" by identifying coherent groups of similar monuments with clear orientation trends. Many such rows are found in western Scotland, with the main concentration in Argyll and the island of Mull. Systematic analyses of their orientations produced credible evidence of an awareness of the 18.6-year lunar node cycle, within a "primary-secondary" pattern whereby isolated rows were oriented close to moonrise or moonset at the southern major standstill limit, while others oriented in this way were accompanied by a second row oriented in a declination range that could be interpreted either as lunar or solar. A detailed investigation of the landscape situation of the sites in northern Mull, accompanied by excavations at two of the sites, suggested that they were deliberately placed in locations where critical moonsets would be seen against prominent distant landscape features, but where the distant horizon in most or all other directions was hidden from view. A lack of independent archaeological evidence may help to explain why archaeoastronomical investigations at short stone rows have never progressed beyond "data-driven" studies of orientations and landscape situation. Nonetheless, the work that was done at these sites raised important general methodological issues, and pioneered techniques, that remain relevant across archaeoastronomy today.

  5. Short bowel syndrome.

    LENUS (Irish Health Repository)

    Donohoe, Claire L


    The short bowel syndrome (SBS) is a state of malabsorption following intestinal resection where there is less than 200 cm of intestinal length. The management of short bowel syndrome can be challenging and is best managed by a specialised multidisciplinary team. A good understanding of the pathophysiological consequences of resection of different portions of the small intestine is necessary to anticipate and prevent, where possible, consequences of SBS. Nutrient absorption and fluid and electrolyte management in the initial stages are critical to stabilisation of the patient and to facilitate the process of adaptation. Pharmacological adjuncts to promote adaptation are in the early stages of development. Primary restoration of bowel continuity, if possible, is the principle mode of surgical treatment. Surgical procedures to increase the surface area of the small intestine or improve its function may be of benefit in experienced hands, particularly in the paediatric population. Intestinal transplant is indicated at present for patients who have failed to tolerate long-term parenteral nutrition but with increasing experience, there may be a potentially expanded role for its use in the future.

  6. The short toric polynomial

    CERN Document Server

    Hetyei, Gábor


    We introduce the short toric polynomial associated to a graded Eulerian poset. This polynomial contains the same information as the two toric polynomials introduced by Stanley, but allows different algebraic manipulations. The intertwined recurrence defining Stanley's toric polynomials may be replaced by a single recurrence, in which the degree of the discarded terms is independent of the rank. A short toric variant of the formula by Bayer and Ehrenborg, expressing the toric $h$-vector in terms of the $cd$-index, may be stated in a rank-independent form, and it may be shown using weighted lattice path enumeration and the reflection principle. We use our techniques to derive a formula expressing the toric $h$-vector of a dual simplicial Eulerian poset in terms of its $f$-vector. This formula implies Gessel's formula for the toric $h$-vector of a cube, and may be used to prove that the nonnegativity of the toric $h$-vector of a simple polytope is a consequence of the Generalized Lower Bound Theorem holding for ...

  7. 靶向CCR7基因shRNA表达载体的构建及鉴定%Construction and identification of pGC-silencer-CRM30 CCR7 short hairpin RNA expression vectors

    Institute of Scientific and Technical Information of China (English)

    孙仁虎; 李疆; 崔静; 吕清; 刘兴华; 王国斌


    目的 设计并构建靶向 CCR7 基因 shRNA真核表达质粒,并检测其干扰效果.方法 以 CCR7 为靶基因设计具有短发夹结构的模板寡核苷酸,退火形成互补双链结构,再克隆至pGC-silencer-CRM30 载体,构建的3条重组短发夹shRNA表达载体分别命名为pGC-silencer-CRM30-CCR7 A、-CCR7 B 和-CCR7 C.采用测序法鉴定寡核苷酸序列.将构建好的真核表达载体转染人结肠癌SW480 细胞,荧光显微镜观察转染效果,RT-PCR法检测CCR7干扰效率.结果 重组质粒测序结果 与Genebank中的CCR7 cDNA序列相符,转染SW480细胞后,荧光显微镜下可观察到绿色荧光蛋白,RT-PCR结果 表明,pGC-silencer-CRM30-CCR7 C干扰效果最强.结论 靶向CCR7 基因shRNA表达载体构建无误,为进一步探讨趋化因子受体CCR7在胃肠道恶性肿瘤生物学行为中的作用奠定了基础.

  8. Short Interest and Stock Returns


    Paul Asquith; Pathak, Parag A.; Jay R. Ritter


    Using a longer time period and both NYSE-Amex and Nasdaq stocks, this paper examines short interest and stock returns in more detail than any previous study and finds that many documented patterns are not robust. While equally weighted high short interest portfolios generally underperform, value weighted portfolios do not. In addition, there is a negative correlation between market returns and short interest over our whole period. Finally, inferences from short time periods, such as 1988-1994...

  9. Short fiber reinforced thermoplastic blends

    NARCIS (Netherlands)

    Malchev, P.G.


    The present thesis investigates the potential of short fiber reinforced thermoplastic blends, a combination of an immiscible polymer blend and a short fiber reinforced composite, to integrate the easy processing solutions available for short fiber reinforced composites with the high mechanical perfo

  10. Short fiber reinforced thermoplastic blends

    NARCIS (Netherlands)

    Malchev, P.G.


    The present thesis investigates the potential of short fiber reinforced thermoplastic blends, a combination of an immiscible polymer blend and a short fiber reinforced composite, to integrate the easy processing solutions available for short fiber reinforced composites with the high mechanical perfo

  11. Short duration gamma ray bursts

    Indian Academy of Sciences (India)

    Patrick Das Gupta


    After a short review of gamma ray bursts (GRBs), we discuss the physical implications of strong statistical correlations seen among some of the parameters of short duration bursts (90 < 2 s). Finally, we conclude with a brief sketch of a new unified model for long and short GRBs.

  12. Short apraxia screening test. (United States)

    Leiguarda, Ramon; Clarens, Florencia; Amengual, Alejandra; Drucaroff, Lucas; Hallett, Mark


    Limb apraxia comprises many different and common disorders, which are largely unrecognized essentially because there is no easy-to-use screening test sensitive enough to identify all types of limb praxis deficits. We evaluated 70 right-handed patients with limb apraxia due to a single focal lesion of the left hemisphere and 40 normal controls, using a new apraxia screening test. The test covered 12 items including: intransitive gestures, transitive gestures elicited under verbal, visual, and tactile modalities, imitation of meaningful and meaningless postures and movements, and a multiple object test. Interrater reliability was maximum for a cutoff of >2 positive items identifying apraxia on the short battery (Cohen's kappa .918, p 3 items (Cohen's kappa .768, p 2 was higher, indicating greater apraxia diagnosis agreement between raters at this cutoff value. The screening test proved to have high specificity and sensitivity to diagnose every type of upper limb praxis deficit, thus showing advantages over previously published tests.

  13. Oxytocin: a short review

    Directory of Open Access Journals (Sweden)

    P. F. Levay


    Full Text Available Oxytocin is traditionally associated with parturition and lactation. The similarity in oxytocin plasma levels in males and females implies a wider physiological role for the hormone. Oxytocin would now appear to be involved not only in milk ejection, but also in the production of milk. The hormone has further been shown to play a paracrine role in menstruation and to be of importance for normal fertilisation. Several endocrine modulatory as well as neurotransmitter effects have also been reported for oxytocin. The discovery of the role of oxytocin in central nervous system processes such as pain, anxiety, memory and learning has stimuluted a search for possible therapeutic applications of oxytocin in cases such as chronic pain and Alzheimer’s disease. A short review is presented of some of the biochemical and physiological aspects underlying the functions and possible therapeutic applications of oxytocin.

  14. Short QT syndrome. (United States)

    Schimpf, Rainer; Wolpert, Christian; Gaita, Fiorenzo; Giustetto, Carla; Borggrefe, Martin


    The short QT syndrome constitutes a new clinical entity that is associated with a high incidence of sudden cardiac death, syncope, and/or atrial fibrillation even in young patients and newborns. Patients with this congenital electrical abnormality are characterized by rate-corrected QT intervalsdelayed-rectifier current I(Kr) have been identified in the first two reported families with familial sudden cardiac death. Recently, two further gain-of-function mutations in the KCNQ1 gene encoding the alpha-subunit of the KvLQT1 (I(Ks)) channel and in the KCNJ2 gene encoding the strong inwardly rectifying channel protein Kir2.1 confirmed a genetically heterogeneous disease. The possible substrate for the development of ventricular tachyarrhythmias may be a significant transmural dispersion of the repolarisation due to a heterogeneous abbreviation of the action potential duration. The implantable cardioverter defibrillator is the therapy of choice in patients with syncope and a positive family history of sudden cardiac death. However, ICD therapy in patients with a short QT syndrome has an increased risk for inappropriate shock therapies due to possible T wave oversensing. The impact of sotalol, ibutilide, flecainide, and quinidine on QT prolongation has been evaluated, but only quinidine effectively suppressed gain-of-function in I(Kr) with prolongation of the QT interval. In patients with a mutation in HERG, it rendered ventricular tachycardias/ventricular fibrillation non-inducible and restored the QT interval/heart rate relationship towards a normal range. It may serve as an adjunct to ICD therapy or as a possible alternative treatment, especially for children and newborns.

  15. A STAT3-inhibitory hairpin decoy oligodeoxynucleotide discriminates between STAT1 and STAT3 and induces death in a human colon carcinoma cell line

    Directory of Open Access Journals (Sweden)

    Souissi Inès


    Full Text Available Abstract Background The Signal Transducer and Activator of Transcription 3 (STAT3 is activated in tumor cells, and STAT3-inhibitors are able to induce the death of those cells. Decoy oligodeoxynucleotides (dODNs, which bind to the DNA Binding Domain (DBD of STAT3, are efficient inhibitors. However, they also inhibit STAT1, whose activity is essential not only to resistance to pathogens, but also to cell growth inhibition and programmed cell death processes. The aim of this study was to design STAT3-specific dODNs which do not affect STAT1-mediated processes. Results New dODNs with a hairpin (hpdODNs were designed. Modifications were introduced, based on the comparison of STAT3- and STAT1-DBD interactions with DNA using 3D structural analyses. The designed hpdODNs were tested for their ability to inhibit STAT3 but not STAT1 by determining: i cell death in the active STAT3-dependent SW480 colon carcinoma cell line, ii absence of inhibition of interferon (IFN γ-dependent cell death, iii expression of STAT1 targets, and iv nuclear location of STAT3 and STAT1. One hpdODN was found to efficiently induce the death of SW480 cells without interfering with IFNγ-activated STAT1. This hpdODN was found in a complex with STAT3 but not with STAT1 using an original in-cell pull-down assay; this hpdODN also did not inhibit IFNγ-induced STAT1 phosphorylation, nor did it inhibit the expression of the STAT1-target IRF1. Furthermore, it prevented the nuclear transfer of STAT3 but not that of IFNγ-activated STAT1. Conclusions Comparative analyses at the atomic level revealed slight differences in STAT3 and STAT1 DBDs' interaction with their DNA target. These were sufficient to design a new discriminating hpdODN that inhibits STAT3 and not STAT1, thereby inducing tumor cell death without interfering with STAT1-dependent processes. Preferential interaction with STAT3 depends on oligodeoxynucleotide sequence modifications but might also result from DNA shape changes

  16. Diseño e implementación de un analizador de biosecuencias para el análisis de mutaciones producidas por elongación de la horquilla 'hairpin'




    [ES] La complementariedad de bases de Watson y Crick representa una gran fuente de estudio en biología estructural y las estructuras ahorquilladas (hairpin structures) son uno de los tipos de secuencia que más aplicaciones puede reportar. En particular, la inducción iterada de horquilla en una secuencia y su posterior elongación es una técnica que está siendo implementada para la amplificación e identificación de secuencias de DNA o RNA que se encuentren menos representadas ...

  17. Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus. (United States)

    Soler, Nuria; Plomer, Montserrat; Fagoaga, Carmen; Moreno, Pedro; Navarro, Luis; Flores, Ricardo; Peña, Leandro


    Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.

  18. Newnes short wave listening handbook

    CERN Document Server

    Pritchard, Joe


    Newnes Short Wave Listening Handbook is a guide for starting up in short wave listening (SWL). The book is comprised of 15 chapters that discuss the basics and fundamental concepts of short wave radio listening. The coverage of the text includes electrical principles; types of signals that can be heard in the radio spectrum; and using computers in SWL. The book also covers SWL equipment, such as receivers, converters, and circuits. The text will be of great use to individuals who want to get into short wave listening.

  19. Short-circuit impedance measurement

    DEFF Research Database (Denmark)

    Pedersen, Knud Ole Helgesen; Nielsen, Arne Hejde; Poulsen, Niels Kjølstad


    Methods for estimating the short-circuit impedance in the power grid are investigated for various voltage levels and situations. The short-circuit impedance is measured, preferably from naturally occurring load changes in the grid, and it is shown that such a measurement system faces different...

  20. Short implants in oral rehabilitation

    Directory of Open Access Journals (Sweden)

    Emmanuel Panobianco Chizolini


    Full Text Available Introduction and objective: The placement of short dental implants is used as an alternative treatment modality to bone grafting procedures. The aim of this study was to discuss, through a literature review, the features, indications and biomechanical aspects of short implants, as well as to report the clinical factors that influence on their indication. Literature review and conclusion: It was found that short implants osseointegration can be compromised by risk factors that must be controlled to achieve treatment success. In conclusion, the main indication of short implants is to avoid an invasive surgery at atrophic areas of maxilla and mandible. Furthermore, implant design associated with surface treatment are factors that compensate its short length.

  1. 湍流边界层流向-展向平面中发卡涡的涡迹特征%Hairpin vortex signature in the streamwise-spanwise plane in a turbulent boundary layer

    Institute of Scientific and Technical Information of China (English)

    刘璐璐; 张军; 翟树成; 张国平


    The analysis of detailed flow structure of a turbulent boundary layer is of great significance for establishing the link among the detailed flow structure characteristics, flow noise and wall friction, which can help lay the foundation for deeper research of the drag and noise reduction mechanism. PIV (particle image velocimetry) technique is applied in this paper to study the hairpin vortex signatures in the streamwise-spanwise plane in turbulent boundary layer flow on a flat plate. The experiment successfully identifies low-speed streaks from PIV instantaneous velocity field and the distributions of spanwise spacing of low-speed streaks along with wall-normal distance and Reynolds number are obtained through statistical analysis in the logarithmic layer. Meanwhile, the counter-rotating vortices surrounding the long low-speed streaks in the streamwise-spanwise plane are extracted through vortex identification criterion, which reveals the relationship between hairpin packets and long low-speed streaks. Besides, this paper performs a research about the distribution of spanwise spacing of hairpin vortex legs, considering various wall-normal distances and the effect of Reynolds numbers.%研究湍流边界层精细流动结构特征,有助于建立流动结构特征与壁面摩阻、流噪声之间的关联,从而为湍流边界层减阻降噪机理的深层次研究奠定基础。该文应用PIV(粒子图像测速)技术,对平板湍流边界层中发卡涡在流向-展向平面的涡迹特征进行研究。利用PIV瞬时速度场,捕捉到明显的低速条带,通过统计分析获得对数层范围低速条带间距随壁面法向距离的变化以及雷诺数的影响规律。同时,通过涡识别准则,提取在流向-展向平面内长低速条带两侧的反向漩涡带,揭示了发卡涡包与长低速条带的内在关系。此外,还研究了流向-展向平面发卡涡涡腿间距随壁面法向距离的变化规律以及雷诺数的影响。

  2. Short bowel syndrome in adults

    DEFF Research Database (Denmark)

    Matarese, Laura E; Jeppesen, Palle B; O'Keefe, Stephen J D


    Short bowel syndrome (SBS) is a heterogeneous disorder with broad variation in disease severity arising from different types of intestinal resection. The spectrum of malabsorption ranges from intestinal insufficiency to intestinal failure. Individualized patient strategies involving modifications...

  3. The classification on short message

    Institute of Scientific and Technical Information of China (English)


    This paper discusses the importance of the classification of short message, and details some key technologies related. Through implementing a fundamental prototype, some basic models and technical references are provided.

  4. Cyclization of short DNA fragments (United States)

    Lam, Pui-Man; Zhen, Yi


    From the per unit length free energy for DNA under tension, we have calculated an effective contour length dependent persistence length for short DNA. This effective persistence length results from the enhanced fluctuations in short DNA. It decreases for shorter DNA, making shorter DNA more flexible. The results of the J-factor calculated using this effective persistence length are in good agreement with experimental data.

  5. BoS: a large and diverse family of short interspersed elements (SINEs) in Brassica oleracea. (United States)

    Zhang, Xiaoyu; Wessler, Susan R


    Short interspersed elements (SINEs) are nonautonomous non-LTR retrotransposons that populate eukaryotic genomes. Numerous SINE families have been identified in animals, whereas only a few have been described in plants. Here we describe a new family of SINEs, named BoS, that is widespread in Brassicaceae and present at approximately 2000 copies in Brassica oleracea. In addition to sharing a modular structure and target site preference with previously described SINEs, BoS elements have several unusual features. First, the head regions of BoS RNAs can adopt a distinct hairpin-like secondary structure. Second, with 15 distinct subfamilies, BoS represents one of the most diverse SINE families described to date. Third, several of the subfamilies have a mosaic structure that has arisen through the exchange of sequences between existing subfamilies, possibly during retrotransposition. Analysis of BoS subfamilies indicate that they were active during various time periods through the evolution of Brassicaceae and that active elements may still reside in some Brassica species. As such, BoS elements may be a valuable tool as phylogenetic makers for resolving outstanding issues in the evolution of species in the Brassicaceae family.

  6. A label-free and enzyme-free ultra-sensitive transcription factors biosensor using DNA-templated copper nanoparticles as fluorescent indicator and hairpin DNA cascade reaction as signal amplifier. (United States)

    Sha, Liang; Zhang, Xiaojun; Wang, Guangfeng


    Detection and quantification of specific protein with ultralow concentration play a crucial role in biotechnological applications and biomedical diagnostics. In this paper, a label-free and enzyme-free amplified fluorescent biosensor has been developed for transcription factors detection based on AT-rich double-stranded DNA-templated copper nanoparticles (ds DNA/Cu NPs) and hairpin DNA cascade reaction. This strategy was demonstrated by using nuclear factor-kappa B p50 (NF-κB p50) and specific recognition sequences as a model case. In this assay, a triplex consists of double-stranded DNA containing NF-κB p50 specifically binding sequences and a special design single-stranded DNA (Trigger) which is able to activate the hairpin DNA cascade amplifier (HDCA). In the presence of NF-κB p50, the triplex became unstable since the target bound to the recognition sequences with strong affinity. The selective binding event confirmed that the Trigger was successfully released from the triplex and initiated HDCA to yield the product which could effectively template the formation of fluorescent Cu NPs. The experimental results revealed that the advanced strategy was ultra-sensitive for detecting NF-κB p50 in the concentration range from 0.1 to 1000 pM with a detection limit of 0.096 pM. In addition, the relative standard deviation was 4.08% in 3 repetitive assays of 500 pM NF-κB p50, which indicated that the reproducibility of this strategy was acceptable. Besides desirable sensitivity, the developed biosensor also showed high selectivity, cost-effective, and simplified operations. In addition, the proposed biosensing platform is versatile. By conjugating with various specific recognition units, it could hold considerable potential to sensitive and selective detect various DNA-binding proteins and might find wide applications in biomedical fields.

  7. Dechirper Wakefields for Short Bunches

    CERN Document Server

    Bane, Karl


    In previous work [1] general expressions, valid for arbitrary bunch lengths, were derived for the wakefields of corrugated structures with flat geometry, such as is used in the RadiaBeam/LCLS dechirper. However, the bunch at the end of linac-based X-ray FELs--like the LCLS--is extremely short, and for short bunches the wakes can be considerably simplified. In this work, we first derive analytical approximations to the short-range wakes. These are generalized wakes, in the sense that their validity is not limited to a small neighborhood of the symmetry axis, but rather extends to arbitrary transverse offsets of driving and test particles. The validity of these short-bunch wakes holds not only for the corrugated structure, but rather for any flat structure whose beam-cavity interaction can be described by a surface impedance. We use these wakes to obtain, for a short bunch passing through a dechirper: estimates of the energy loss as function of gap, the transverse kick as function of beam offset, the slice ener...

  8. The novel as short story

    Directory of Open Access Journals (Sweden)

    Kirk Schlueter


    Full Text Available In recent history, the novel has been thought of and defined primarily as a long prose narrative. However, this has not been the case historically, as the original meaning of "novel" was for "a piece of news" or "a short story or novella." Returning to this original definition, I propose a new way of viewing the work known contemporarily as the novel as a collection, or sequence, of united short stories rather than a single indivisible work, with the component short stories or novellas comprising the sequence renamed as "novels." A brief examination of several classic works traditionally considered novels serves to illustrate how this change in definition will affect reading.

  9. Infantile Short Bowel Syndrome: short and long term evaluation

    NARCIS (Netherlands)

    J.F. Olieman (Joanne)


    textabstractInfantile short bowel syndrome is a condition which is characterized by malabsorption of nutrients, as a result of congenital intestinal shortening or massive small bowel resection. Survival rates have improved over the years, but morbidity remains high and clinical management of these p

  10. Chaos a very short introduction

    CERN Document Server

    Smith, Leonard


    Chaos: A Very Short Introduction shows that we all have an intuitive understanding of chaotic systems. It uses accessible maths and physics (replacing complex equations with simple examples like pendulums, railway lines, and tossing coins) to explain the theory, and points to numerous examples in philosophy and literature (Edgar Allen Poe, Chang-Tzu, and Arthur Conan Doyle) that illuminate the problems. The beauty of fractal patterns and their relation to chaos, as well as the history of chaos, and its uses in the real world and implications for the philosophy of science are all discussed in this Very Short Introduction.

  11. Almost primes in short intervals

    Institute of Scientific and Technical Information of China (English)


    In this paper,we prove that the short interval(x-x101/232,x] contains at least an almost prime P2 for sufficiently large x,where P2 denotes an integer having at most two prime factors counted with multiplicity.

  12. Short Bowel Syndrome: clinical management


    Loris Pironi; Mariacristina Guidetti; Elisabetta Lanzoni; Caterina Pazzeschi; Catia Corsini


    The management of a case of intestinal failure due to Short Bowel Syndrome (SBS) is described. Patients’ care needs an expert multidisciplinary approach. Published data have demonstrated that the lack of a specialist staff is a risk factor for patients’ death. The creation of networks linking non-specialist doctors with dedicated centers is recommended.

  13. Review of short wavelength lasers

    Energy Technology Data Exchange (ETDEWEB)

    Hagelstein, P.L.


    There has recently been a substantial amount of research devoted to the development of short wavelength amplifiers and lasers. A number of experimental results have been published wherein the observation of significant gain has been claimed on transitions in the EUV and soft x-ray regimes. The present review is intended to discuss the main approaches to the creation of population inversions and laser media in the short wavelength regime, and hopefully aid workers in the field by helping to provide access to a growing literature. The approaches to pumping EUV and soft x-ray lasers are discussed according to inversion mechanism. The approaches may be divided into roughly seven categories, including collisional excitation pumping, recombination pumping, direct photoionization and photoexcitation pumping, metastable state storage plus optical pumping, charge exchange pumping, and finally, the extension of free electron laser techniques into the EUV and soft x-ray regimes. 250 references.

  14. Complexity a very short introduction

    CERN Document Server

    Holland, John H


    The importance of complexity is well-captured by Hawking's comment: "Complexity is the science of the 21st century". From the movement of flocks of birds to the Internet, environmental sustainability, and market regulation, the study and understanding of complex non-linear systems has become highly influential over the last 30 years. In this Very Short Introduction, one of the leading figures in the field, John Holland, introduces the key elements and conceptual framework of complexity. From complex physical systems such as fluid flow and the difficulties of predicting weather, to complex adaptive systems such as the highly diverse and interdependent ecosystems of rainforests, he combines simple, well-known examples - Adam Smith's pin factory, Darwin's comet orchid, and Simon's 'watchmaker' - with an account of the approaches, involving agents and urn models, taken by complexity theory. ABOUT THE SERIES: The Very Short Introductions series from Oxford University Press contains hundreds of titles in almost eve...

  15. Optimal short-sighted rules

    Directory of Open Access Journals (Sweden)

    Sacha eBourgeois-Gironde


    Full Text Available The aim of this paper is to assess the relevance of methodological transfers from behavioral ecology to experimental economics with respect to the elicitation of intertemporal preferences. More precisely our discussion will stem from the analysis of Stephens and Anderson’s (2001 seminal article. In their study with blue jays they document that foraging behavior typically implements short sighted choice rules which are beneficial in the long-run. Such long term profitability of short-sighted behavior cannot be evidenced when using a self-control paradigm (one which contrasts in a binary way sooner smaller and later larger payoffs but becomes apparent when ecological patch-paradigms (replicating economic situations in which the main trade-off consists in staying on a food patch or leaving for another patch are implemented. We transfer this methodology in view of contrasting foraging strategies and self-control in human intertemporal choices.

  16. Short Introduction to Quantum Computation (United States)


    Proceedings of the Air Force Office of Scientific Research Computational Mathematics Meeting 1996 Revision 2 Short Introduction to Quantum...useful for nanoscale computing and quantum computing. KEY WORDS: quantum computing, nano-scale computing, Moore’s law 1 Introduction It is likely that...memory) Digital Devices magnetostrictive delay line Intel 1103 integrated circuit IBM 3340 disk drive Smallest DRAM cell reported on at ISSC Current

  17. Short Course in the Microbiome

    Directory of Open Access Journals (Sweden)

    Kimberly Falana


    provides us with ideas as to how we can manipulate our microbiome to prevent certain diseases. However, given the variety of applications, there are scientific challenges, though there are very promising areas in reference to the clinical benefits of understanding more about our micro‐ biome, whether in our gut or on our skin: the outlook is bright. A summary of the short course is presented as a meeting dispatch.

  18. Short-range communication system (United States)

    Alhorn, Dean C. (Inventor); Howard, David E. (Inventor); Smith, Dennis A. (Inventor)


    A short-range communication system includes an antenna, a transmitter, and a receiver. The antenna is an electrical conductor formed as a planar coil with rings thereof being uniformly spaced. The transmitter is spaced apart from the plane of the coil by a gap. An amplitude-modulated and asynchronous signal indicative of a data stream of known peak amplitude is transmitted into the gap. The receiver detects the coil's resonance and decodes same to recover the data stream.


    Institute of Scientific and Technical Information of China (English)

    Qi Haoliang; Li Mu; Gao Jianfeng; Li Sheng


    The major problem of the most current approaches of information models lies in that individual words provide unreliable evidence about the content of the texts. When the document is short, e.g. only the abstract is available, the word-use variability problem will have substantial impact on the Information Retrieval (IR) performance. To solve the problem, a new technology to short document retrieval named Reference Document Model (RDM) is put forward in this letter. RDM gets the statistical semantic of the query/document by pseudo feedback both for the query and document from reference documents. The contributions of this model are three-fold: (1) Pseudo feedback both for the query and the document; (2) Building the query model and the document model from reference documents; (3) Flexible indexing units, which can be any linguistic elements such as documents, paragraphs, sentences, n-grams, term or character. For short document retrieval, RDM achieves significant improvements over the classical probabilistic models on the task of ad hoc retrieval on Text REtrieval Conference (TREC) test sets. Results also show that the shorter the document, the better the RDM performance.

  20. The structure of the XPF-ssDNA complex underscores the distinct roles of the XPF and ERCC1 helix- hairpin-helix domains in ss/ds DNA recognition. (United States)

    Das, Devashish; Folkers, Gert E; van Dijk, Marc; Jaspers, Nicolaas G J; Hoeijmakers, Jan H J; Kaptein, Robert; Boelens, Rolf


    Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction.

  1. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  2. Stars a very short introduction

    CERN Document Server

    King, Andrew


    Stars: A Very Short Introduction looks at how stars live, producing all the chemical elements beyond helium, and how they die, leaving remnants such as black holes. Every atom of our bodies has been part of a star. Our very own star, the Sun, is crucial to the development and sustainability of life on Earth. Understanding stars is key to understanding the galaxies they inhabit, the existence of planets, and the history of our entire Universe. This VSI explores the science of stars, the mechanisms that allow them to form, the processes that allow them to shine, and the results of their death.

  3. Planets a very short introduction

    CERN Document Server

    Rothery, David A


    Planets: A Very Short Introduction demonstrates the excitement, uncertainties, and challenges faced by planetary scientists, and provides an overview of our Solar System and its origins, nature, and evolution. Terrestrial planets, giant planets, dwarf planets and various other objects such as satellites (moons), asteroids, trans-Neptunian objects, and exoplanets are discussed. Our knowledge about planets has advanced over the centuries, and has expanded at a rapidly growing rate in recent years. Controversial issues are outlined, such as What qualifies as a planet? What conditions are required for a planetary body to be potentially inhabited by life? Why does Pluto no longer have planet status? And Is there life on other planets?

  4. Magnetism a very short introduction

    CERN Document Server

    Blundell, Stephen J


    Magnetism: A Very Short Introduction explains the mysteries and importance of magnetism. For centuries magnetism has been used for various exploits: as a great healer, a navigation aid through compasses, and through motors, generators, and turbines it has given us power. Our understanding of electricity and magnetism, from the work of Galvani, Ampère, Faraday, and Tesla is explored, and how Maxwell and Faraday's work led to the unification of electricity and magnetism is explained. With a discussion of the relationship between magnetism and relativity, quantum magnetism, and its impact on computers and information storage, how magnetism has changed our fundamental understanding of the Universe is shown.

  5. Moons a very short introduction

    CERN Document Server

    Rothery, David A


    Moons: A Very Short Introduction introduces the reader to the varied and fascinating moons of our Solar System. Beginning with the early discoveries of Galileo and others, it describes their variety of mostly mythological names, and the early use of Jupiter’s moons to establish position at sea and to estimate the speed of light. It discusses the structure, formation, and profound influence of our Moon, those of the other planets, and ends with the recent discovery of moons orbiting asteroids, whilst looking forward to the possibility of discovering microbial life beyond Earth and of finding moons of exoplanets in planetary systems far beyond our own.

  6. Statistics a very short introduction

    CERN Document Server

    Hand, David J


    Statistics has evolved into an exciting discipline which uses deep theory and powerful software to shed light on the world around us: from clinical trials in medicine, to economics, sociology, and countless other subjects vital to understanding modern life. This Very Short Introduction explores and explains how statistics works today. - ;Modern statistics is very different from the dry and dusty discipline of the popular imagination. In its place is an exciting subject which uses deep theory and powerful software tools to shed light and enable understanding. And it sheds this light on all aspe

  7. Superconductivity a very short introduction

    CERN Document Server

    Blundell, Stephen


    Superconductivity is one of the most exciting areas of research in physics today. Outlining the history of its discovery, and the race to understand its many mysterious and counter-intuitive phenomena, this Very Short Introduction explains in accessible terms the theories that have been developed, and how they have influenced other areas of science, including the Higgs boson of particle physics and ideas about the early Universe. It is an engaging and informative accountof a fascinating scientific detective story, and an intelligible insight into some deep and beautiful ideas of physics

  8. Short Conjugators in Solvable Groups

    CERN Document Server

    Sale, Andrew W


    We give an upper bound on the size of short conjugators in certain solvable groups. Diestel-Leader graphs, which are a horocyclic product of trees, are discussed briefly and used to study the lamplighter groups. The other solvable groups we look at can be recognised in a similar vein, as groups which act on a horocyclic product of well known spaces. These include the Baumslag-Solitar groups BS(1,q) and semidirect products of Z^n with Z^k. Results can also be applied to the conjugacy of parabolic elements in Hilbert modular groups and to elements in 3-manifold groups.

  9. Gravity a very short introduction

    CERN Document Server

    Clifton, Timothy


    Gravity is one of the four fundamental interactions that exist in nature and is essential for understanding the behaviour of the Universe, and all astrophysical bodies within it. Yet it remains puzzling. Gravity: A Very Short Introduction looks at the development of our understanding of gravity since the early observations of Kepler, Newtonian theory, and Einstein’s theory of gravity. It also discusses the recent detection of waves of gravitational radiation that were predicted by Einstein. This VSI concludes by considering the testing and application of General Relativity in astrophysics and cosmology, and looks at dark energy and efforts such as string theory to combine gravity with quantum mechanics.

  10. Sound a very short introduction

    CERN Document Server

    Goldsmith, Mike


    Sound is integral to how we experience the world, in the form of noise as well as music. But what is sound? What is the physical basis of pitch and harmony? And how are sound waves exploited in musical instruments? Sound: A Very Short Introduction looks at the science of sound and the behaviour of sound waves with their different frequencies. It also explores sound in different contexts, covering the audible and inaudible, sound underground and underwater, acoustic and electronic sound, and hearing in humans and animals. It concludes with the problem of sound out of place—noise and its reduction.

  11. Nothing a very short introduction

    CERN Document Server

    Close, Frank


    What is 'nothing'? What remains when you take all the matter away? Can empty space - a void - exist? This Very Short Introduction explores the science and the history of the elusive void: from Aristotle who insisted that the vacuum was impossible, via the theories of Newton and Einstein, to our very latest discoveries and why they can tell us extraordinary things about the cosmos. Frank Close tells the story of how scientists have explored the elusive void, and the rich discoveries that they have made there. He takes the reader on a lively and accessible history through ancient ideas and cultural superstitions to the frontiers of current research

  12. Fractals a very short introduction

    CERN Document Server

    Falconer, Kenneth


    Many are familiar with the beauty and ubiquity of fractal forms within nature. Unlike the study of smooth forms such as spheres, fractal geometry describes more familiar shapes and patterns, such as the complex contours of coastlines, the outlines of clouds, and the branching of trees. In this Very Short Introduction, Kenneth Falconer looks at the roots of the 'fractal revolution' that occurred in mathematics in the 20th century, presents the 'new geometry' of fractals, explains the basic concepts, and explores the wide range of applications in science, and in aspects of economics.This is esse

  13. Galaxies a very short introduction

    CERN Document Server

    Gribbin, John


    Galaxies: A Very Short Introduction explores the building blocks of the Universe. Standing like islands in space, each is made up of many hundreds of millions of stars in which the chemical elements are made, around which planets form, and where on at least one of those planets intelligent life has emerged. Our own galaxy, the Milky Way, is just one of several hundred million other galaxies. Yet it was only in the 1920s that we realised that there is more to the Universe. Since then, many exciting discoveries have been made about our own galaxy and about those beyond.

  14. [Short history of mixed states]. (United States)

    Cermolacce, M; Belzeaux, R; Corréard, N; Dassa, D; Dubois, M; Micoulaud-Franchi, J-A; Pringuey, D; Fakra, E; Maurel, M; Azorin, J-M


    The notion of mixed states is classically associated with descriptions and categories inherited from Kraepelin. However, simultaneous descriptions of depressive and manic manifestations can be traced back to ancient times. Semiology and definitions of these clinical associations have evolved across the times. We provide here a short insight on four distinct periods: Greek authors from ancient times, pre-Kraepelinian psychiatry (18th and 19th centuries), Kraepelin's conceptualization, and contemporary psychiatry (20th and 21st centuries). Copyright © 2013 Sociedade Brasileira de Farmacognosia. Published by Elsevier Masson SAS.. All rights reserved.

  15. Relativity a very short introduction

    CERN Document Server

    Stannard, F Russell


    Relativity: A Very Short Introduction makes the theory of relativity understandable and accessible. 100 years ago, Einstein's theory of relativity shattered the world of physics. Our comforting Newtonian ideas of space and time were replaced by bizarre and counterintuitive conclusions: if you move at high speed, time slows down, space squashes up and you get heavier; travel fast enough and you could weigh as much as a jumbo jet, be squashed thinner than a CD without feeling a thing - and live forever. And that was just the special theory. With the general theory came even stranger ideas of curved space-time, which changed our understanding of gravity and the cosmos.

  16. Radioactivity a very short introduction

    CERN Document Server

    Tuniz, Claudio


    Radioactivity: A Very Short Introduction explains radioactivity and discusses its fundamental role in nature. Radioactivity remains misunderstood and feared perhaps because nuclear radiation cannot be detected by human senses, and can undoubtedly do great harm if appropriate precautions are not taken. Radioactivity in the stars and in the Earth and its wide range of applications in biomedicine, science, industry, agriculture are described, as well as the mechanisms of nuclear fission and fusion, and the harnessing of nuclear power. The issues surrounding safety and security and the increasing concerns about nuclear terrorism are also considered.

  17. Isotopes a very short introduction

    CERN Document Server

    Ellam, Rob


    An isotope is a variant form of a chemical element, containing a different number of neutrons in its nucleus. Most elements exist as several isotopes. Many are stable while others are radioactive, and some may only exist fleetingly before decaying into other elements. In this Very Short Introduction, Rob Ellam explains how isotopes have proved enormously important across all the sciences and in archaeology. Radioactive isotopes may be familiar from their use in nuclear weapons, nuclear power, and in medicine, as well as in carbon dating. They have been central to establishing the age of the Earth and the origins of the solar system. Combining previous and new research, Ellam provides an overview of the nature of stable and radioactive isotopes, and considers their wide range of modern applications. ABOUT THE SERIES: The Very Short Introductions series from Oxford University Press contains hundreds of titles in almost every subject area. These pocket-sized books are the perfect way to get ahead in a new subjec...

  18. Short rotation Wood Crops Program

    Energy Technology Data Exchange (ETDEWEB)

    Wright, L.L.; Ehrenshaft, A.R.


    This report synthesizes the technical progress of research projects in the Short Rotation Woody Crops Program for the year ending September 30, 1989. The primary goal of this research program, sponsored by the US Department of Energy's Biofuels and Municipal Waste Technology Division, is the development of a viable technology for producing renewable feedstocks for conversion to biofuels. One of the more significant accomplishments was the documentation that short-rotation woody crops total delivered costs could be $40/Mg or less under optimistic but attainable conditions. By taking advantage of federal subsidies such as those offered under the Conservation Reserve Program, wood energy feedstock costs could be lower. Genetic improvement studies are broadening species performance within geographic regions and under less-than-optimum site conditions. Advances in physiological research are identifying key characteristics of species productivity and response to nutrient applications. Recent developments utilizing biotechnology have achieved success in cell and tissue culture, somaclonal variation, and gene-insertion studies. Productivity gains have been realized with advanced cultural studies of spacing, coppice, and mixed-species trials. 8 figs., 20 tabs.

  19. Exact Solutions to Short Pulse Equation

    Institute of Scientific and Technical Information of China (English)

    FU Zun-Tao; ZHENG Ming-Hua; LIU Shi-Kuo


    In this paper, dependent and independent variable transformations are introduced to solve the short pulse equation. It is shown that different kinds of solutions can be obtained to the short pulse equation.

  20. Prognostic indicators influencing short term outcomes among ...

    African Journals Online (AJOL)

    Prognostic indicators influencing short term outcomes among operated head injury patients ... AFRICAN JOURNALS ONLINE (AJOL) · Journals · Advanced Search ... indicators for short term outcomes in operated head injury patients at KCMC.

  1. WPC's Short Range Forecast Coded Bulletin (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Short Range Forecast Coded Bulletin. The Short Range Forecast Coded Bulletin describes the expected locations of high and low pressure centers, surface frontal...

  2. Inductively Shorted Bicone Fed Tapered Dipole Antenna (United States)


    Switching from a balun feed to a coaxial cable feed showed expected similar impedances for the shorted antenna with number 14 wire shorts. Switching ...possibility is to add broadband ferrite core chokes placed about the shorts 70 near their ends 50, 52, 54, 56, 58, 60 ,62, 64 to prevent antenna...currents from flowing onto the shorts. Some problems with this method are that ferrites have loss and limited bandwidth, and the resulting floating

  3. A Short Story with Long Results (United States)

    Jackman, Norma F.


    After reading a short story, a sixth grade class became involved in an avid discussion, a debate, and dramatic play - all based on the short story. A vast amount of learning had taken place through the study of a single short story. (Author/DR)

  4. Copernicus a very short introduction

    CERN Document Server

    Gingerich, Owen


    Nicolaus Copernicus (1473–1543) was the astronomer whose shocking vision of a sun-centered universe turned out to be the essential blueprint for a physical understanding of celestial motions. Copernicus: A Very Short Introduction offers a fascinating portrayal of the man who launched the modern vision of the universe. It sets Copernicus in the context of a rapidly changing world, where the recent invention of printing with movable type not only made sources more readily available to him, but also fueled Martin Luther’s transformation of the religious landscape. Copernicus’s heliocentric revolution is revealed as an aesthetic achievement not dictated by observational “proofs,” but another new way of looking at the ancient cosmos.

  5. Infinity a very short introduction

    CERN Document Server

    Stewart, Ian


    Infinity is an intriguing topic, with connections to religion, philosophy, metaphysics, logic, and physics as well as mathematics. Its history goes back to ancient times, with especially important contributions from Euclid, Aristotle, Eudoxus, and Archimedes. The infinitely large (infinite) is intimately related to the infinitely small (infinitesimal). Cosmologists consider sweeping questions about whether space and time are infinite. Philosophers and mathematicians ranging from Zeno to Russell have posed numerous paradoxes about infinity and infinitesimals. Many vital areas of mathematics rest upon some version of infinity. The most obvious, and the first context in which major new techniques depended on formulating infinite processes, is calculus. But there are many others, for example Fourier analysis and fractals. In this Very Short Introduction, Ian Stewart discusses infinity in mathematics while also drawing in the various other aspects of infinity and explaining some of the major problems and insights ...

  6. Astrophysics a very short introduction

    CERN Document Server

    Binney, James


    Astrophysics is the physics of the stars, and more widely the physics of the Universe. It enables us to understand the structure and evolution of planetary systems, stars, galaxies, interstellar gas, and the cosmos as a whole. In this Very Short Introduction, the leading astrophysicist James Binney shows how the field of astrophysics has expanded rapidly in the past century, with vast quantities of data gathered by telescopes exploiting all parts of the electromagnetic spectrum, combined with the rapid advance of computing power, which has allowed increasingly effective mathematical modelling. He illustrates how the application of fundamental principles of physics - the consideration of energy and mass, and momentum - and the two pillars of relativity and quantum mechanics, has provided insights into phenomena ranging from rapidly spinning millisecond pulsars to the collision of giant spiral galaxies. This is a clear, rigorous introduction to astrophysics for those keen to cut their teeth on a conceptual trea...

  7. A Short Term Analogue Memory

    DEFF Research Database (Denmark)

    Shah, Peter Jivan


    A short term analogue memory is described. It is based on a well-known sample-hold topology in which leakage currents have been minimized partly by circuit design and partly by layout techniques. Measurements on a test chip implemented in a standard 2.4 micron analogue CMOS process show a droop...... rate of 0.075mV per second with a 1pF hold capacitor. This is equivalent to a retention time of approximately 1½ minute with 10 bits accuracy, assuming a full scale of +/-3.5V. It is expected that this can be improved by more than an order of magnitude by improving the layout of the hold capacitor...


    Directory of Open Access Journals (Sweden)

    Radu BORES


    Full Text Available This paper aims at providing insight into some of the implication of short selling for markets, investors as well as regulators. Findings show that capital flows are adversely affected by strict regulation and bans of short sales, while market liquidity, and bid-ask spread can be improved by allowing short selling. Additionally portfolios that incorporate short selling strategies can have lower volatility in returns. The informational content of short sales can provide important feedback for informed investors and lead to better price discovery.

  9. The Short Storytelling of Anton Chekhov


    ALPAY, Imge


    A short story is a subgenre of the story.  Due to it’s shortness compared to story it is named as short story. The first examples of the short story are given by American writers like Washington Irving, Edgar Allen Poe by the beginning of 19th century. Since then the short story has been considered as literary genre in itself. As for World literature, in Russian literature, this literature genre has made progress especially with Chekhov-type of story, besides from Pushkin, Gogol, Gorki. One c...

  10. A molecularly cloned, live-attenuated japanese encephalitis vaccine SA14-14-2 virus: a conserved single amino acid in the ij Hairpin of the Viral E glycoprotein determines neurovirulence in mice. (United States)

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Yun, Gil-Nam; Lee, Eun-Young; Li, Long; Kuhn, Richard J; Rossmann, Michael G; Morrey, John D; Lee, Young-Min


    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV represents the JE serogroup, which also includes West Nile, Murray Valley encephalitis, and St. Louis encephalitis viruses. Within this serogroup, JEV is a vaccine-preventable pathogen, but the molecular basis of its neurovirulence remains unknown. Here, we constructed an infectious cDNA of the most widely used live-attenuated JE vaccine, SA14-14-2, and rescued from the cDNA a molecularly cloned virus, SA14-14-2MCV, which displayed in vitro growth properties and in vivo attenuation phenotypes identical to those of its parent, SA14-14-2. To elucidate the molecular mechanism of neurovirulence, we selected three independent, highly neurovirulent variants (LD50, 1.5×105 PFU) by serial intracerebral passage in mice. Complete genome sequence comparison revealed a total of eight point mutations, with a common single G1708→A substitution replacing a Gly with Glu at position 244 of the viral E glycoprotein. Using our infectious SA14-14-2 cDNA technology, we showed that this single Gly-to-Glu change at E-244 is sufficient to confer lethal neurovirulence in mice, including rapid development of viral spread and tissue inflammation in the central nervous system. Comprehensive site-directed mutagenesis of E-244, coupled with homology-based structure modeling, demonstrated a novel essential regulatory role in JEV neurovirulence for E-244, within the ij hairpin of the E dimerization domain. In both mouse and human neuronal cells, we further showed that the E-244 mutation altered JEV infectivity in vitro, in direct correlation with the level of neurovirulence in vivo, but had no significant impact on viral RNA replication. Our results provide a crucial step toward developing novel therapeutic and preventive strategies against JEV and possibly other encephalitic flaviviruses.

  11. Engineering Complex Microbial Phenotypes with Continuous Genetic Integration and Plasmid Based Multi-Gene Library (United States)


    the chromosome. Repeat this step for continu ous integration. 4’h Round: Oisru1Hing the Sp gene to insert he te ro logous DNA with Tet rn...provide a selective advantage. Briefly, the collection of cells will be stressed until the stationary phase of cultures, and will then be transferred into

  12. A novel plasmid-based microarray screen identifies suppressors of rrp6Delta in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Torben Heick


    RNP protein Nab6p and the tRNA transporter Los1p, which could not have been identified in a traditional plate screen; both genes are toxic when overexpressed in rrp6Δ strains at 37°C. Nab6p binds poly(A)+ RNA, and the functions of Nab6p and Los1p suggest that mRNA metabolism and/or protein synthesis...

  13. Engineering Complex Microbial Phenotypes with Continuous Genetic Integration and Plasmid Based Multi-gene Library (United States)


    various fragments from L plant arum. Cells were grown in LB medium with 5% (V/V) ethanol and starting optical density of 0.3. Survival final titer is observed. Expression of the HSP plasmid appears to slow both growth and production throughout the entire fermentation . However...this may be an effect of overexpression of the HSPs during the early portions of the fermentation where there are low concentrations of ethanol and

  14. Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development. (United States)

    Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan


    In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Intestino Corto Short bowel syndrome

    Directory of Open Access Journals (Sweden)

    María Matilde Socarrás Suárez


    Full Text Available El intestino corto está asociado a pérdida o disfunción del intestino delgado por resección del mismo, que causa diarreas, tránsito intestinal acelerado, malabsorción intestinal, y eventualmente la pérdida de peso y el desgaste muscular. El objetivo de este trabajo fue actualizar el conocimiento acerca de este síndrome. Se realiza una revisión del tema de intestino corto donde se refiere a su definición, causas fundamentales frecuentes e infrecuentes en el niño y en el adulto, cómo se adapta el intestino a la resección de diferentes extensiones, las funciones del íleon terminal. Se hacen una valoración clínica inicial, con el interrogatorio médico, revisión minuciosa de la historia clínica para cuantificar la capacidad de absorción. Se habla de los síntomas y signos de deficiencia nutricional. Se explican las estrategias del tratamiento, que tienen 3 etapas de evolución clínica. Se concluye que se indica la dietoterapia adecuada según el estado nutricional del paciente y la resección intestinal realizada, evitando las complicaciones para lograr una calidad máxima de vidaShort bowel is associated with loss or dysfunction of the small bowel due to its resection, which causes diarrheas, accelerated intestinal transit, intestinal malabsorption and, eventually, weight loss ansd muscular waste. The objective of this paper was to update knowledge about this syndrome. A review of the short intestine topic is made, making reference to its definition, common and uncommon main cuases in the child and adult, how the bowel adapts itslef to resection of different extensions, and the functions of the terminal ileum. An initial clinical assessment is made with the medical questionnaire and a detailed review of the medical history to quantify the absorption capacity. The symptoms and signs of nutritional deficiency are dealt with. The strategies of the treatment consisting of 3 stages of clinical evolution are explained. It is concluded

  16. Light a very short introduction

    CERN Document Server

    Walmsley, Ian


    Light enables us to see the world around us. Our sense of sight provides us with direct information about space and time, the physical arrangement of the world, and how it changes. This almost universal shared sensation of vision has led to a fascination with the nature and properties of light across the ages. But the light we see is just a small part of the whole spectrum of electromagnetic radiation, ranging from radio waves to gamma rays. In this Very Short Introduction Ian Walmsley discusses early attempts to explain light, and the development of apparently opposing particulate and wave theories by scientists such as Isaac Newton and Christiaan Huygens. He shows how light was recognized as an electromagnetic wave in the 19th century, and the development of the quantum mechanics view of wave-particle duality in the 20th century. He also describes the many applications of light, domestic and scientific, such as microwaves, DVDs, and lasers. We now use the whole range of electromagnetic radiation to peer bot...

  17. Gravity a very short introduction

    CERN Document Server

    Clifton, Timothy


    Gravity is one of the four fundamental interactions that exist in nature. It also has the distinction of being the oldest, weakest, and most difficult force to quantize. Understanding gravity is not only essential for understanding the motion of objects on Earth, but also the motion of all celestial objects, and even the expansion of the Universe itself. It was the study of gravity that led Einstein to his profound realizations about the nature of space and time. Gravity is not only universal, it is also essential for understanding the behavior of the Universe, and all astrophysical bodies within it. In this Very Short Introduction Timothy Clifton looks at the development of our understanding of gravity since the early observations of Kepler and Newtonian theory. He discusses Einstein's theory of gravity, which now supplants Newton's, showing how it allows us to understand why the frequency of light changes as it passes through a gravitational field, why GPS satellites need their clocks corrected as they orbi...

  18. Onboard Short Term Plan Viewer (United States)

    Hall, Tim; LeBlanc, Troy; Ulman, Brian; McDonald, Aaron; Gramm, Paul; Chang, Li-Min; Keerthi, Suman; Kivlovitz, Dov; Hadlock, Jason


    Onboard Short Term Plan Viewer (OSTPV) is a computer program for electronic display of mission plans and timelines, both aboard the International Space Station (ISS) and in ISS ground control stations located in several countries. OSTPV was specifically designed both (1) for use within the limited ISS computing environment and (2) to be compatible with computers used in ground control stations. OSTPV supplants a prior system in which, aboard the ISS, timelines were printed on paper and incorporated into files that also contained other paper documents. Hence, the introduction of OSTPV has both reduced the consumption of resources and saved time in updating plans and timelines. OSTPV accepts, as input, the mission timeline output of a legacy, print-oriented, UNIX-based program called "Consolidated Planning System" and converts the timeline information for display in an interactive, dynamic, Windows Web-based graphical user interface that is used by both the ISS crew and ground control teams in real time. OSTPV enables the ISS crew to electronically indicate execution of timeline steps, launch electronic procedures, and efficiently report to ground control teams on the statuses of ISS activities, all by use of laptop computers aboard the ISS.

  19. Long short-term memory. (United States)

    Hochreiter, S; Schmidhuber, J


    Learning to store information over extended time intervals by recurrent backpropagation takes a very long time, mostly because of insufficient, decaying error backflow. We briefly review Hochreiter's (1991) analysis of this problem, then address it by introducing a novel, efficient, gradient-based method called long short-term memory (LSTM). Truncating the gradient where this does not do harm, LSTM can learn to bridge minimal time lags in excess of 1000 discrete-time steps by enforcing constant error flow through constant error carousels within special units. Multiplicative gate units learn to open and close access to the constant error flow. LSTM is local in space and time; its computational complexity per time step and weight is O(1). Our experiments with artificial data involve local, distributed, real-valued, and noisy pattern representations. In comparisons with real-time recurrent learning, back propagation through time, recurrent cascade correlation, Elman nets, and neural sequence chunking, LSTM leads to many more successful runs, and learns much faster. LSTM also solves complex, artificial long-time-lag tasks that have never been solved by previous recurrent network algorithms.

  20. gender differences in short message language use

    Institute of Scientific and Technical Information of China (English)



    “language and gender” is one of the main research fields in sociolinguistics.with the introduction of short message service,another communication method appears.due to the problem of privacy aggression,few people have done researches on short gender differences in short message language use are discussed here and at last reasons for the differences will also be generalized.

  1. Short Tandem Repeat DNA Internet Database (United States)

    SRD 130 Short Tandem Repeat DNA Internet Database (Web, free access)   Short Tandem Repeat DNA Internet Database is intended to benefit research and application of short tandem repeat DNA markers for human identity testing. Facts and sequence information on each STR system, population data, commonly used multiplex STR systems, PCR primers and conditions, and a review of various technologies for analysis of STR alleles have been included.

  2. Short-term intercultural psychotherapy: ethnographic inquiry. (United States)

    Seeley, Karen M


    This article examines the challenges specific to short-term intercultural treatments and recently developed approaches to intercultural treatments based on notions of cultural knowledge and cultural competence. The article introduces alternative approaches to short-term intercultural treatments based on ethnographic inquiry adapted for clinical practice. Such approaches allow clinicians conducting short-term intercultural treatments to foreground clients' indigenous conceptions of selfhood, mind, relationship, and emotional disturbance, and thus to more fully grasp their internal, interpersonal, and external worlds. This article demonstrates the uses of clinically adapted ethnographic inquiry in three short-term intercultural cases.

  3. Chaos: A Very Short Introduction

    Energy Technology Data Exchange (ETDEWEB)

    Klages, R [School of Mathematical Sciences, Mile End Road, London, E1 4NS (United Kingdom)


    field. I feel that occasionally the book is also getting a bit too intricate for the complete layman, and experts may not agree on all details of the more conceptual discussions. Altogether I thoroughly enjoyed reading this book. It was a happy companion while travelling and a nice bedtime literature. It is furthermore an excellent reminder of the 'big picture' underlying nonlinear science as it applies to the real world. I will gladly recommend this book as background literature for students in my introductory course on dynamical systems. However, the book will be of interest to anyone who is looking for a very short account on fundamental problems and principles in modern nonlinear science. (book review)

  4. Assembling the LHC short straight sections

    CERN Multimedia

    Maximilien Brice


    The building where the short straight sections are being assembled, was often called ‘Lego Land’ by the workers because of the wide variety of sets of magnets and cryostats. Short straight sections contain magnets for manipulating the beam inside cryostats with liquid helium to keep the magnets at a cool 1.9 K (-271.3°C).

  5. Evaluation of the Child with Short Stature


    Holmes, S. Bruce


    The author reviews the various causes of short stature in childhood. The different patterns of growth seen with each of these conditions are emphasized, as an understanding of these patterns is essential to diagnosis. Short stature is a frequent finding in childhood, with endocrine causes being relatively rare.

  6. Worst Asymmetrical Short-Circuit Current

    DEFF Research Database (Denmark)

    Arana Aristi, Iván; Holmstrøm, O; Grastrup, L


    In a typical power plant, the production scenario and the short-circuit time were found for the worst asymmetrical short-circuit current. Then, a sensitivity analysis on the missing generator values was realized in order to minimize the uncertainty of the results. Afterward the worst asymmetrical...

  7. Short range radio research in Twente

    NARCIS (Netherlands)

    Meijerink, Arjan


    The research and education by the Telecommunication Engineering Group at the University of Twente is dedicated to physical layer topics in communications. Three research tracks have prominence: Short Range Radio, Microwave Photonics, and Electromagnetic Compatibility. Arjan is active in the Short Ra

  8. Accelerating universes from short-range interactions

    Energy Technology Data Exchange (ETDEWEB)

    Diez-Tejedor, Alberto [Departamento de Fisica Teorica, Universidad del Pais Vasco, Apdo. 644, 48080, Bilbao (Spain)]. E-mail:; Feinstein, Alexander [Departamento de Fisica Teorica, Universidad del Pais Vasco, Apdo. 644, 48080, Bilbao (Spain)]. E-mail:


    We show that short-range interactions between the fundamental particles in the universe can drive a period of accelerated expansion. This description fits the early universe. In the present day universe, if one postulates short-range interactions or a sort of 'shielded gravity', the picture may repeat.

  9. Accelerating Universes from Short-Range Interactions

    CERN Document Server

    Diez-Tejedor, A; Diez-Tejedor, Alberto; Feinstein, Alexander


    We show that short-range interactions between the fundamental particles in the universe can drive a period of accelerated expansion. This description fits the early universe. In the present day universe, if one postulates short-range interactions or a sort of "shielded gravity", the picture may repeat.

  10. Did You Really Read That Short Story? (United States)

    Rogers, C. D.

    Two studies carried out by James Squire and Jesse Stuart explored processes used in reading the short story, ways of exploring student's processes, possibilities within the short story as a literary genre, and methods teachers can use. Squire's study showed that introspection and retrospection are useful in studying students' reading processes. In…

  11. Short-term managerial contracts facilitate cartels

    NARCIS (Netherlands)

    Han, M.A.


    This paper shows how a series of commonly observed short-term CEO employment contracts improves cartel stability compared to a long-term contract. When a manager’s short-term appointment is renewed if and only if the firm hits a certain profit target, then (a) defection from collusion results in sup

  12. Ultra-Short Pulse Tracking by Using Wavelength Dispersion for a Short-Time Optical Buffer

    Institute of Scientific and Technical Information of China (English)

    Tsuyoshi; Konishi; Hideaki; Furukawa; Kousuke; Asano; Kazuyoshi; Itoh


    To synchronize a control signal with a packet signal in response to changing timing jitter, we investigate ultra-short pulse tracking by using wavelength dispersion for a short-time optical buffer in an optical router.

  13. Short-Distance Structure of Nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Douglas Higinbotham, Eliazer Piasetzky, Stephen Wood


    One of Jefferson Lab's original missions was to further our understanding of the short-distance structure of nuclei. In particular, to understand what happens when two or more nucleons within a nucleus have strongly overlapping wave-functions; a phenomena commonly referred to as short-range correlations. Herein, we review the results of the (e,e'), (e,e'p) and (e,e'pN) reactions that have been used at Jefferson Lab to probe this short-distance structure as well as provide an outlook for future experiments.

  14. High Power Short Wavelength Laser Development (United States)


    Unlimited güä^äsjäsiiiüüü X NRTC-77-43R P I High Power Short Wavelength Laser Development November 1977 D. B. Cohn and W. B. Lacina...NO NRTC-77-43R, «. TITLE fana »uetjjitj BEFORE COMPLETING FORM CIPIENT’S CATALOO NUMBER KIGH.POWER SHORT WAVELENGTH LASER DEVELOPMENT , 7...fWhtn Data Enterte NRTC-77-43R HIGH POWER SHORT WAVELENGTH LASER DEVELOPMENT ARPA Order Number: Program Code Number: Contract Number: Principal

  15. Case report: Congenital short bowel syndrome

    Directory of Open Access Journals (Sweden)

    Palle Lalitha


    Full Text Available Congenital short bowel syndrome (SBS is a relatively rare condition as compared to acquired SBS. It is associated with significant mortality and morbidity. Infants usually present with failure to thrive, recurrent vomiting, and diarrhea. It is important to suspect and diagnose this condition promptly, as early initiation of parenteral nutrition or surgery, if necessary, may result in a favorable outcome. We discuss a case of an infant aged 26 days, who presented with failure to thrive, recurrent vomiting, and weight loss. A contrast study of the gastrointestinal tract revealed a short small bowel, with malrotation. The infant was started on parenteral nutrition, but succumbed shortly thereafter to severe disseminated sepsis.

  16. A multidisciplinary approach to short bowel syndrome. (United States)

    Denegri, Andrea; Paparo, Francesco; Denegri, Roberto; Revelli, Matteo; Frascio, Marco; Rollandi, Gian Andrea; Fornaro, Rosario


    Short bowel syndrome is a complex clinical picture, characterized by signs and symptoms of malabsorption and subsequent malnutrition, which often occurs after extensive bowel resections. Short bowel syndrome's treatment must begin together with the planning of the first surgery, especially for disease that may need multiple interventions. Patients with short bowel should be individually managed because they all are different in diagnosis, length of the remaining bowel and in psychosocial characteristics. For all these reasons, a multidisciplinary approach between the various specialists is therefore needed.

  17. Optimal parametric modelling of measured short waves

    Digital Repository Service at National Institute of Oceanography (India)

    Mandal, S.

    The spectral analysis of measured short waves can efficiently be carried out by the fast Fourier transform technique. Even though many present techniques can be used for the simulation of time series waves, these may not provide accurate...

  18. Giant pubertal prolactinoma: Complete resolution following short ...

    African Journals Online (AJOL)


    Apr 6, 2016 ... disappeared in a short time with cabergoline treatment. Key words: ... stature, and delayed development of his secondary sex characteristics. .... Leong KS, Foy PM, Swift AC, Atkin SL, Hadden DR, MacFarlane IA. CSF.

  19. Minimalism in the modern short story

    Directory of Open Access Journals (Sweden)

    A Razi


    Full Text Available Short story has recently become the focus of attention in the late decades in Iran. The expanding value of writing short story is actually a reasonable outcome of the dominance of minimalism- a movement which is based upon simplicity and shortness. Minimalist writers, leaving out redundant features of narration, mainly focus on essentialities through applying a variety of techniques such as cuttings from the interesting moments of real life, evading introduction, applying inter-referents, choice of words, short stanzas and sentences and so on. Looking upon critic’s opinion about such a tendency over the past and present, this article will come up with a brief explanation of the properties of such stories. Finally a sample story “candles will never go dead” will be analyzed and discussed in the lights of such techniques.

  20. GASSST: global alignment short sequence search tool

    National Research Council Canada - National Science Library

    Rizk, Guillaume; Lavenier, Dominique


    .... Our goal with our new aligner GASSST (Global Alignment Short Sequence Search Tool) is thus 2-fold-achieving high performance with no restrictions on the number of indels with a design that is still effective on long reads...

  1. Analyzing Short-Term Disability Benefits. (United States)

    Houff, James N.; Wiatrowski, William J.


    The Bureau of Labour Statistics has combined data on sick leave and sickness and accident insurance. Results show that short-term disability benefits vary by length of service and between the private and public sectors. (Author)

  2. Microbial production of short chain diols. (United States)

    Jiang, Yudong; Liu, Wei; Zou, Huibin; Cheng, Tao; Tian, Ning; Xian, Mo


    Short chain diols (propanediols, butanediols, pentanediols) have been widely used in bulk and fine chemical industries as fuels, solvents, polymer monomers and pharmaceutical precursors. The chemical production of short chain diols from fossil resources has been developed and optimized for decades. Consideration of the exhausting fossil resources and the increasing environment issues, the bio-based process to produce short chain diols is attracting interests. Currently, a variety of biotechnologies have been developed for the microbial production of the short chain diols from renewable feed-stocks. In order to efficiently produce bio-diols, the techniques like metabolically engineering the production strains, optimization of the fermentation processes, and integration of a reasonable downstream recovery processes have been thoroughly investigated. In this review, we summarized the recent development in the whole process of bio-diols production including substrate, microorganism, metabolic pathway, fermentation process and downstream process.

  3. Methods for Allocating Ambiguous Short-reads

    National Research Council Canada - National Science Library

    Taub, Margaret; Lipson, Doron; Speed, Terence P


    With the rise in prominence of biological research using new short-read DNA sequencing technologies comes the need for new techniques for aligning and assigning these reads to their genomic location of origin...

  4. Short Communication: Visual assessment, proximate composition ...

    African Journals Online (AJOL)

    Short Communication: Visual assessment, proximate composition and cost analysis of three ... Abstract. With increased search for least-cost alternative feed resource in animal ... In terms of odour, all three test mixtures were strongly pungent.

  5. Short communication report Final for Jerome

    African Journals Online (AJOL)

    Dr. Ahmed

    Short communication report. AN ANALYSIS OF HUMAN ... Nigerian Institute for Trypanosomiasis Research. PMB 2077 ... The purpose of this paper is to evaluate the trends of the disease in Nigeria .... Springer-Verlag, Paris,. 215-233. Ford, J.

  6. Intestinal adaptation in short bowel syndrome

    NARCIS (Netherlands)

    Welters, C.F.M.; Dejong, C.H.C.; Deutz, N.E.P.; Heineman, E.


    Intestinal adaptation in short bowel syndrome. Welters CF, Dejong CH, Deutz NE, Heineman E. Department of Surgery, Academic Hospital and University of Maastricht, The Netherlands. Regaining enteral autonomy after extensive small bowel resection is dependent on intestinal adaptation. This adaptationa

  7. Prediction of β-hairpin Motif in Enzyme Based on Values of Matrix Scoring and Averaged Chemical Shifts%基于矩阵打分值和化学位移值预测酶蛋白质中β-发夹模体

    Institute of Scientific and Technical Information of China (English)

    宋航宇; 胡秀珍


    Enzyme is a kind of protein that acts with catalytic functions in living organisms ,thus the study of the structure and function of enzyme is essential .β-hairpin is a specific super -secondary structure motif which carries abundant folding information .The prediction of β-hairpin motifs in en-zyme protein is helpful to study the structure and function of enzyme .In this paper ,we constructed a non-redundant β-hairpin database ,w hich have 2 ,818β-hairpins and 1 ,098 non-β-hairpins motif in 1 ,080 enzyme protein chains .The hydropaths composed of position and adjacent to dipeptide hydro-paths composition of position extracted as parameter for matrix scoring algorithm ,the prediction re-sults were unsatisfactory .Then ,we selected the scores of hydropathy composition of position ,the scores of adjacent to dipeptide hydropathy composition of position and averaged chemical shift as the in-put parameters for Support Vector Machine algorithm ,the overall accuracy and Matthew ’s correlation coefficient reached 81 .8% and 0 .636 respectively by 5-fold cross-validation .%酶是一类具有催化功能的蛋白质,对其结构和功能的研究是非常有必要的。特殊模体β-发夹是一种简单的超二级结构,它们包含了丰富的折叠信息,对酶中β-发夹的预测有助于酶结构和功能的预测。本文建立了非冗余的β-发夹模体数据集,包含1080条酶蛋白质链,2818个β-发夹模体,1098个非β-发夹。提取位点亲疏水组分及位点亲疏水紧邻关联组分作为参数,运用矩阵打分算法得到的预测结果不理想。将位点亲疏水、位点亲疏水紧邻关联组分为参数得到的打分值和平均化学位移值共同作为特征参数,输入支持向量机算法对模体进行预测,5交叉检验预测总精度是81.8%,相关系数达到了0.636。

  8. Anthrlin short contact therapy in psoriasis


    Kar P; Jha P; Snehi P


    Forty cases of psoriasis were treated with dithranol daily for 20 minutes as short contact therapy. The results were compared with another 20 psoriasis patients using dithranol paste as per Ingram technique. Short contact therapy resulted in complete regression in 24 (60%) patients, 90% regression of lesions in 5 (12.5%) patients and deterioration in 2 (5%) cases. The average rate of clearance of lesions was between 10-30 days. Psoriatic lesions disappeared without...

  9. Modern treatment of short bowel syndrome

    DEFF Research Database (Denmark)

    Jeppesen, Palle B


    Recently, the US Food and Drug Administration and the European Medicines Agency approved the glucagon-like peptide 2 analogue, teduglutide, for the treatment of short bowel syndrome (SBS), and this review describes the physiological basis for its clinical use.......Recently, the US Food and Drug Administration and the European Medicines Agency approved the glucagon-like peptide 2 analogue, teduglutide, for the treatment of short bowel syndrome (SBS), and this review describes the physiological basis for its clinical use....

  10. Short wave diathermy for small spontaneous pneumothorax



    BACKGROUND: The treatment of small spontaneous pneumothorax can involve observation, tube thoracostomy, and surgery. This study evaluated the use of short wave diathermy as a method of accelerating the resolution of small pneumothoraces. METHODS: Twenty two patients with pneumothoraces of less than 30% by volume were randomly allocated to receive short wave diathermy for 25 minutes each day (n = 11) or observation with bed rest (n = 11). Chest radiographs were taken until the pneumothor...

  11. Short Time Quantum Propagator and Bohmian Trajectories


    De Gosson, Maurice,; Hiley, Basil


    We begin by giving correct expressions for the short-time action; following the work of one of us and Makri--Miller. We use these estimates to derive a correct expression modulo modulo delta(t)sqr for the quantum propagator and we show that the quantum potential is negligible modulo delta(t)sqr for a point source. We finally prove that this implies that the quantum motion is classical for very short times.

  12. Short Implants: New Horizon in Implant Dentistry. (United States)

    Jain, Neha; Gulati, Manisha; Garg, Meenu; Pathak, Chetan


    The choice of implant length is an essential factor in deciding the survival rates of these implants and the overall success of the prosthesis. Placing an implant in the posterior part of the maxilla and mandible has always been very critical due to poor bone quality and quantity. Long implants can be placed in association with complex surgical procedures such as sinus lift and bone augmentation. These techniques are associated with higher cost, increased treatment time and greater morbidity. Hence, there is need for a less invasive treatment option in areas of poor bone quantity and quality. Data related to survival rates of short implants, their design and prosthetic considerations has been compiled and structured in this manuscript with emphasis on the indications, advantages of short implants and critical biomechanical factors to be taken into consideration when choosing to place them. Studies have shown that comparable success rates can be achieved with short implants as those with long implants by decreasing the lateral forces to the prosthesis, eliminating cantilevers, increasing implant surface area and improving implant to abutment connection. Short implants can be considered as an effective treatment alternative in resorbed ridges. Short implants can be considered as a viable treatment option in atrophic ridge cases in order to avoid complex surgical procedures required to place long implants. With improvement in the implant surface geometry and surface texture, there is an increase in the bone implant contact area which provides a good primary stability during osseo-integration.

  13. Short-term memory across eye blinks. (United States)

    Irwin, David E


    The effect of eye blinks on short-term memory was examined in two experiments. On each trial, participants viewed an initial display of coloured, oriented lines, then after a retention interval they viewed a test display that was either identical or different by one feature. Participants kept their eyes open throughout the retention interval on some blocks of trials, whereas on others they made a single eye blink. Accuracy was measured as a function of the number of items in the display to determine the capacity of short-term memory on blink and no-blink trials. In separate blocks of trials participants were instructed to remember colour only, orientation only, or both colour and orientation. Eye blinks reduced short-term memory capacity by approximately 0.6-0.8 items for both feature and conjunction stimuli. A third, control, experiment showed that a button press during the retention interval had no effect on short-term memory capacity, indicating that the effect of an eye blink was not due to general motoric dual-task interference. Eye blinks might instead reduce short-term memory capacity by interfering with attention-based rehearsal processes.

  14. Automated DC Substation Short-Current Evaluation

    Directory of Open Access Journals (Sweden)

    Petr Dohnal


    Full Text Available Technical praxis has no easy-to-use standardized method for a DC short-current evaluation. For a correct rating of rectifying blocks (diodes and input and output current-carrying cables is need to know short-current parameters. The current flow in the substation is derived by commutation of rectifying blocks, which define current of transformers and DC part. For evaluating exists a semi empiric formula, that has a delimited relevance to small power range up to 50 kWatts of output power. New way of evaluation of short current is based on simulation of simplified substation model using the capabilities of DYNAST simulation program, or other dynamic equation solver based on numerical method, like Matlab.

  15. Short pulse laser systems for biomedical applications

    CERN Document Server

    Mitra, Kunal


    This book presents practical information on the clinical applications of short pulse laser systems and the techniques for optimizing these applications in a manner that will be relevant to a broad audience, including engineering and medical students as well as researchers, clinicians, and technicians. Short pulse laser systems are useful for both subsurface tissue imaging and laser induced thermal therapy (LITT), which hold great promise in cancer diagnostics and treatment. Such laser systems may be used alone or in combination with optically active nanoparticles specifically administered to the tissues of interest for enhanced contrast in imaging and precise heating during LITT. Mathematical and computational models of short pulse laser-tissue interactions that consider the transient radiative transport equation coupled with a bio-heat equation considering the initial transients of laser heating were developed to analyze the laser-tissue interaction during imaging and therapy. Experiments were first performe...

  16. Distinguishing short and long Fermi GRBs

    CERN Document Server

    Tarnopolski, Mariusz


    Two classes of GRBs, short and long, have been determined without any doubts, and are usually ascribed to different progenitors, yet these classes overlap for a variety of descriptive parameters. A subsample of 46 long and 22 short $Fermi$ GRBs with estimated Hurst Exponents (HEs), complemented by minimum variability time-scales (MVTS) and durations ($T_{90}$) is used to perform a supervised Machine Learning (ML) and Monte Carlo (MC) simulation using a Support Vector Machine (SVM) algorithm. It is found that while $T_{90}$ itself performs very well in distinguishing short and long GRBs, the overall success ratio is higher when the training set is complemented by MVTS and HE. These results may allow to introduce a new (non-linear) parameter that might provide less ambiguous classification of GRBs.

  17. Anthrlin short contact therapy in psoriasis

    Directory of Open Access Journals (Sweden)

    Kar P


    Full Text Available Forty cases of psoriasis were treated with dithranol daily for 20 minutes as short contact therapy. The results were compared with another 20 psoriasis patients using dithranol paste as per Ingram technique. Short contact therapy resulted in complete regression in 24 (60% patients, 90% regression of lesions in 5 (12.5% patients and deterioration in 2 (5% cases. The average rate of clearance of lesions was between 10-30 days. Psoriatic lesions disappeared without leaving spotty hyperpigmentation. Dithranol paste as used in the Ingram technique showed complete clearing in 11 (55% patients, 90% clearing in 5 (25% cases and deterioration in 4 (20% patients. Ninety percent cases developed spotty hyperpigmentation and 50% patients experienced marked to fiery red erythema during the course of treatment. Short contact therapy gave minimal side effects and good cosmetic results.

  18. Short-sale Constraints and Credit Runs

    DEFF Research Database (Denmark)

    Venter, Gyuri

    of prices to some agents who learn about the quality of an investment opportunity from market prices and have additional private information. Then I apply this observation when modeling a run on an investment bank by its short-term creditors, who are endowed with dispersed information and also learn from...... the price of an asset the bank holds. I show that short-selling constraints in the financial market lead to the revival of self-fulfilling beliefs about the beliefs and actions of others, and create multiple equilibria. In the equilibrium where agents rely more on public information (i.e., the price......), creditors with high private signals are more lenient to roll over debt, and a bank with lower asset quality remains solvent. This leads to higher allocative efficiency in the real economy. My result thus implies that the decrease in average informativeness due to short-sale constraints can be more than...

  19. Nutritive support in short Bowel syndrome (sbs

    Directory of Open Access Journals (Sweden)

    Simić Dušica


    Full Text Available Short bowel syndrome most commonly result after bowel resection for necrosis of the bowel. It may be caused by arterial or venous thrombosis, volvolus and in children, necrotizing enterocolitis. The other causes are Crohn,s disease intestinal atresia. The factors influencing the risk on short bowel syndrome are the remaining length of the small bowel, the age of onset, the length of the colon, the presence or absence of the ileo-coecal valve and the time after resection. Besides nutritional deficiencies there some other consequences of extensive resections of the small intestine (gastric acid hypersecretion, d-lactic acidosis, nephrolithiasis, cholelithiasis, which must be diagnosed, treated, and if possible, prevented. With current therapy most patients with short bowel have normal body mass index and good quality of life.

  20. A Secure Short Message Communication Protocol

    Institute of Scientific and Technical Information of China (English)

    Chao-Wen Chang; Heng Pan; Hong-Yong Jia


    According to the security requirement of the short message service (SMS) industry application, a secure short message communication protocol is proposed. This is an application level protocol constructed on the standard SMS communication protocol using public key authentication and key agreement without the need of wireless public key infrastructure (WPKI). Secure short message transmission and dynamic key agreement between mobile terminals and the accessing gateway are realized. The security of the proposed protocol is validated through the BAN logic. Compared with the standard SMS protocol, the effective payload rate of our protocol can reach 91.4%, and subscriber identity module (SIM) tool kit (STK) applications based on our protocol suit well for all kinds of mobile terminals in practical application.

  1. Symbolic Values in Steinbeck's Short Stories

    Institute of Scientific and Technical Information of China (English)



    The essay will analyze the symbols created by Steinbeck and disclose the meaning of the most widely used symbols John Steinbeck resorted toin some of his short stories. These interpretations of the symbols may reveal to readers an utterly different system of values. The author will explore those stories that are relevant from this point of view. This paper will highlight,emphasize and interpret different symbols presented in his stories too. And the author will give a possible way of tackling of Steinbeck short stories.

  2. Women stereotypes in Shi Zhecun's short stories. (United States)

    Rosenmeier, Christopher


    This article analyses the representation of women in two 1933 short story collections by Shi Zhecun: An Evening of Spring Rain and Exemplary Conduct of Virtuous Women. It discusses how the New Woman image was a site of contestation in Republican China, and argues that Shi Zhecun’s short stories contain four basic stereotypes: the enigmatic woman, the estranged wife, the prostitute, and the inhibited woman. Using these narratives of women and how they were perceived by men, Shi Zhecun deconstructed the New Woman image by subverting the various ways modernity was projected onto women.

  3. Estimating the short-circuit impedance

    DEFF Research Database (Denmark)

    Nielsen, Arne Hejde; Pedersen, Knud Ole Helgesen; Poulsen, Niels Kjølstad


    and current are derived each period, and the short-circuit impedance is estimated from variations in these components created by load changes in the grid. Due to the noisy and dynamic grid with high harmonic distortion it is necessary to threat the calculated values statistical. This is done recursively...... through a RLS-algorithm. The algorithms have been tested and implemented on a PC at a 132 kV substation supplying a rolling mill. Knowing the short-circuit impedance gives the rolling mill an opportunity to adjust the arc furnace operation to keep flicker below a certain level. Therefore, the PC performs...

  4. Short dental implants: a systematic review. (United States)

    Annibali, S; Cristalli, M P; Dell'Aquila, D; Bignozzi, I; La Monaca, G; Pilloni, A


    Growing evidence has suggested the utility of short dental implants for oral reconstructive procedures in clinical situations of limited vertical bone height. The aim of this review was to systematically evaluate clinical studies of implants type of edentulism, and prosthetic restoration. Two randomized controlled trials and 14 observational studies were selected and analyzed for data extraction. In total, 6193 short-implants were investigated from 3848 participants. The observational period was 3.2 ± 1.7 yrs (mean ± SD). The cumulative survival rate (CSR) was 99.1% (95%CI: 98.8-99.4). The biological success rate was 98.8% (95%CI: 97.8-99.8), and the biomechanical success rate was 99.9% (95%CI: 99.4-100.0). A higher CSR was reported for rough-surfaced implants. The provision of short implant-supported prostheses in patients with atrophic alveolar ridges appears to be a successful treatment option in the short term; however, more scientific evidence is needed for the long term.

  5. [Short pregnancy interval and reproductive disorders

    NARCIS (Netherlands)

    Jongbloet, P.H.; Zielhuis, G.A.; Pasker-de Jong, P.C.M.


    The cause of the 'borderline personality disorder' of Vincent van Gogh has been discussed in social-psychiatric terms related to so-called 'substitute children', born after the loss of a previous child. A biological-organic genesis, i.e. the very short birth interval of precisely one year between Va

  6. Estimating the short-circuit impedance

    DEFF Research Database (Denmark)

    Nielsen, Arne Hejde; Pedersen, Knud Ole Helgesen; Poulsen, Niels Kjølstad


    through a RLS-algorithm. The algorithms have been tested and implemented on a PC at a 132 kV substation supplying a rolling mill. Knowing the short-circuit impedance gives the rolling mill an opportunity to adjust the arc furnace operation to keep flicker below a certain level. Therefore, the PC performs...

  7. Short single axioms for boolean algebra.

    Energy Technology Data Exchange (ETDEWEB)

    McCune, W.; Veroff, R.; Fitelson, B.; Harris, K.; Feist, A.; Wos, L.; Mathematics and Computer Science; Univ. of New Mexico; Univ. of Wisconsin at Madison; Duke Univ.


    We present short single equational axioms for Boolean algebra in terms of disjunction and negation and in terms of the Sheffer stroke. Previously known single axioms for these theories are much longer than the ones we present. We show that there is no shorter axiom in terms of the Sheffer stroke. Automated deduction techniques were used in several parts of the work.

  8. A Short Sheffer axiom for Boolean algebra.

    Energy Technology Data Exchange (ETDEWEB)

    Veroff, R.; McCune, W.


    A short Sheffer stroke identity is shown to be a single axiom for Boolean algebra. The axiom has length 15 and 3 variables. The proof shows that it is equivalent to Sheffer's original 3-basis for the theory. Automated deduction techniques were used to find the proof. The shortest single axiom previously known to us has length 105 and 6 variables.

  9. Short WSRT HI observations of spiral galaxies

    NARCIS (Netherlands)

    Rhee, MH; vanAlbada, TS


    We have obtained short HI observations of 60 late type spiral galaxies with the Westerbork Synthesis Radio Telescope (WSRT). Several HI properties are presented, including the radial surface density distribution of HI and a position-velocity map. When possible these are compared to those measured fr

  10. Teaching Hemingway's "The Short Happy Life." (United States)

    Stacy, Gerald


    Considers many ways to teach Hemingway's "The Short Happy Life of Francis Macomber." Explores the ironic implications of Macomber's experience and compares it with the experience of Sammy in another initiation story, John Updike's "A&P." Describes how he leads the discussion about this story, and ends the discussion by…

  11. Time series tapering for short data samples

    DEFF Research Database (Denmark)

    Kaimal, J.C.; Kristensen, L.


    We explore the effect of applying tapered windows on atmospheric data to eliminate overestimation inherent in spectra computed from short time series. Some windows are more effective than others in correcting this distortion. The Hamming window gave the best results with experimental data...

  12. Visual Short-Term Memory Complexity

    DEFF Research Database (Denmark)

    Sørensen, Thomas Alrik

    Several recent studies have explored the nature and limits of visual short-term memory (VSTM) (e.g. Luck & Vogel, 1997). A general VSTM capacity limit of about 3 to 4 letters has been found, thus confirming results from earlier studies (e.g. Cattell, 1885; Sperling, 1960). However, Alvarez...

  13. Short rotation forestry harvesting - systems and costs (United States)

    Bruce R. Hartsough; Bryce J. Stokes


    Single stem short rotation plantations in the United States are largely dedicated to pulp production, with fuel as a secondary product. There are very limited plantings for fuel production, and others where the primary purpose is treatment of various wastewater's. All production harvesting of single stem plantations is conducted with conventional forestry...

  14. Short Communication - Aquatic Oil Pollution Impact Indicators ...

    African Journals Online (AJOL)

    Short Communication - Aquatic Oil Pollution Impact Indicators. ... increased biochemical oxygen demand, increased water temperature and acidity of the water ... Journal of Applied Sciences and Environmental Management Vol. ... Journal Quality · for Researchers · for Journals · for Authors · for Policy Makers · about Open ...

  15. Residual stresses of thin, short rectangular plates (United States)

    Andonian, A. T.; Danyluk, S.


    The analysis of the residual stresses in thin, short rectangular plates is presented. The analysis is used in conjunction with a shadow moire interferometry technique by which residual stresses are obtained over a large spatial area from a strain measurement. The technique and analysis are applied to a residual stress measurement of polycrystalline silicon sheet grown by the edge-defined film growth technique.

  16. A Short Introduction to Kolmogorov Complexity

    NARCIS (Netherlands)

    Nannen, Volker


    This is a short introduction to Kolmogorov complexity and information theory. The interested reader is referred to the literature, especially the textbooks [CT91] and [LV97] which cover the elds of information theory and Kolmogorov complexity in depth and with all the necessary rigor.

  17. Quantum interactive proofs with short messages

    CERN Document Server

    Beigi, Salman; Watrous, John


    This paper considers three variants of quantum interactive proof systems in which short (meaning logarithmic-length) messages are exchanged between the prover and verifier. The first variant is one in which the verifier sends a short message to the prover, and the prover responds with an ordinary, or polynomial-length, message; the second variant is one in which any number of messages can be exchanged, but where the combined length of all the messages is logarithmic; and the third variant is one in which the verifier sends polynomially many random bits to the prover, who responds with a short quantum message. We prove that in all of these cases the short messages can be eliminated without changing the power of the model, so the first variant has the expressive power of QMA and the second and third variants have the expressive power of BQP. These facts are proved through the use of quantum state tomography, along with the finite quantum de Finetti theorem for the first variant.

  18. Abdominal Pain (Stomach Pain), Short-Term (United States)

    ... in Children and TeensRead MoreBMI Calculator Abdominal Pain (Stomach Pain), Short-termJust about everyone has had a " ... time or another. But sudden severe abdominal pain (stomach pain), also called acute pain, shouldn't be ...

  19. Woody biomass from short rotation energy crops (United States)

    R.S. Zalesny; M.W. Cunningham; R.B. Hall; J. Mirck; D.L. Rockwood; John Stanturf; T.A. Volk


    Short rotation woody crops (SRWCs) are ideal for woody biomass production and management systems because they are renewable energy feedstocks for biofuels, bioenergy, and bioproducts that can be strategically placed in the landscape to conserve soil and water, recycle nutrients, and sequester carbon. This chapter is a synthesis of the regional implications of producing...

  20. LIFE with Short-Term Memory (United States)

    Alonso-Sanz, Ramón

    This chapter considers an extension to the standard framework of cellular automata in which, cells are endowed with memory of their previous state values. The effect of short-term memory, i.e., memory of only the latest states, in the (formally unaltered) Life rule is described in this work.

  1. Transient nanobubbles in short-time electrolysis

    NARCIS (Netherlands)

    Svetovoy, Vitaly; Sanders, Remco G.P.; Elwenspoek, Michael Curt


    Water electrolysis in a microsystem is observed and analyzed on a short-time scale of ∼10 μs. The very unusual properties of the process are stressed. An extremely high current density is observed because the process is not limited by the diffusion of electroactive species. The high current is

  2. A Short Proof of Krattenthaler Formulas

    Institute of Scientific and Technical Information of China (English)

    MA Xin Rong


    With an effort to investigate a unified approach to the Lagrange inverse Krattenthaler established operator method we finally found a general pair of inverse relations, called the Krattenthaler formulas. The present paper presents a very short proof of this formula via Lagrange interpolation.Further, our method of proof declares that the Krattenthaler result is unique in the light of Lagrange interpolation.

  3. Regional variation in short distance homogamy

    NARCIS (Netherlands)

    Haandrikman, K.; van Wissen, L.J.G.


    A third of all Dutch cohabiters choose a partner from the same municipality, so-called short distance homogamy. This article analyses the regional variation in this phenomenon, and it explains this variation in terms of geographical, socioeconomic, demographic and cultural determinants. Population

  4. Nonperturbative short-range dynamics in TMDs

    Energy Technology Data Exchange (ETDEWEB)

    Weiss, Christian [JLAB


    This presentation covers: deep inelastic processes and transverse momentum distributions; chiral symmetry breaking, including the physical picture, the dynamical model, and parton distributions; partonic structures, including transverse momentum distributions, coordinate space correlator, and short range correlations; and measurements of semi-inclusive deep inelastic scattering, correlations, and multi-parton processes in pp interactions.

  5. Short-Duration Gamma-Ray Bursts

    CERN Document Server

    Berger, Edo


    Gamma-ray bursts (GRBs) display a bimodal duration distribution, with a separation between the short- and long-duration bursts at about 2 sec. The progenitors of long GRBs have been identified as massive stars based on their association with Type Ic core-collapse supernovae, their exclusive location in star-forming galaxies, and their strong correlation with bright ultraviolet regions within their host galaxies. Short GRBs have long been suspected on theoretical grounds to arise from compact object binary mergers (NS-NS or NS-BH). The discovery of short GRB afterglows in 2005, provided the first insight into their energy scale and environments, established a cosmological origin, a mix of host galaxy types, and an absence of associated supernovae. In this review I summarize nearly a decade of short GRB afterglow and host galaxy observations, and use this information to shed light on the nature and properties of their progenitors, the energy scale and collimation of the relativistic outflow, and the properties ...

  6. Compression of Short Text on Embedded Systems

    DEFF Research Database (Denmark)

    Rein, S.; Gühmann, C.; Fitzek, Frank


    The paper details a scheme for lossless compression of a short data series larger than 50 bytes. The method uses arithmetic coding and context modelling with a low-complexity data model. A data model that takes 32 kBytes of RAM already cuts the data size in half. The compression scheme just takes...

  7. Asian Short Fiction. Asian Studies Instructional Module. (United States)

    Waugh, Susan

    This curriculum outline introduces the components of a course which explores the genre of short novels, including works by twentieth-century Japanese and Chinese authors. First, the catalogue course description and required texts are presented, highlighting the instructor's historical introduction to the development of Western, Japanese, and…

  8. Short-termism in the European Union

    DEFF Research Database (Denmark)

    Strand, Therese


    The 2007–08 global financial crisis gave rise to an extensive debate in the United States (US) on short-termism and its influence on agency relationships, incentives, and fiduciary duties. This debate and the consecutive legal creativity of US policymakers display spillover effects and constitute...

  9. Textbook Error: Short Circuiting on Electrochemical Cell (United States)

    Bonicamp, Judith M.; Clark, Roy W.


    Short circuiting an electrochemical cell is an unreported but persistent error in the electrochemistry textbooks. It is suggested that diagrams depicting a cell delivering usable current to a load be postponed, the theory of open-circuit galvanic cells is explained, the voltages from the tables of standard reduction potentials is calculated and…

  10. Short-chain 3-hydroxyacyl-CoA dehydrogenase is a negative regulator of insulin secretion in response to fuel and non-fuel stimuli in INS832/13 β-cells. (United States)

    Pepin, Emilie; Guay, Claudiane; Delghingaro-Augusto, Viviane; Joly, Erik; Madiraju, S R Murthy; Prentki, Marc


    Hyperinsulinemia associated with non-ketotic hypoglycemia is observed in patients with mutated β-oxidation enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase (HADHSC). In the present study, we investigated the mechanism underlying HADHSC-mediated regulation of insulin secretion. Knockdown of HADHSC expression by RNA interference in INS832/13 β-cells was achieved using short hairpin RNA and short interference RNA. Knockdown of HADHSC increased both fuel- (glucose or leucine plus glutamine) and non-fuel (high KCl)-induced insulin secretion. Enhanced glucose-stimulated insulin secretion (GSIS) induced by HADHSC knockdown was independent of changes in cytosolic Ca(2+) and also occurred in the presence of fatty acids. L-Carnitine, used in the formation of acyl-carnitine compounds, increased GSIS in control cells, but was unable to further increase the augmented GSIS in HADHSC-knockdown cells. The pan transaminase inhibitor amino-oxyacetate reversed HADHSC knockdown-mediated increases in GSIS. Oxidation of [1-(14) C]-palmitate and -octanoate was not reduced in HADHSC-knockdown cells. L-3-Hydroxybutyryl-carnitine (tested using its precursor L-3-hydroxybutyrate) and L-3-hydroxyglutarate, which accumulate in blood and urine, respectively, of HADHSC-deficient patients, did not change insulin secretion. Insulin secretion promoted by both fuel and non-fuel stimuli is negatively regulated by HADHSC. Enhanced secretion after HADHSC knockdown is not due to inhibition of fatty acid oxidation causing an accumulation of long-chain fatty acids or their CoA derivatives. L-3-Hydroxybutyrate and L-3-hydroxyglutarate do not mediate enhanced secretion caused by reduced HADHSC activity. Transamination reaction(s) and the formation of short-chain acylcarnitines and CoAs may be implicated in the mechanism whereby HADHSC deficiency results in enhanced insulin secretion and hyperinsulinemia. © 2010 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Blackwell Publishing

  11. MMPI-2 short form proposal: CAUTION. (United States)

    Gass, Carlton S; Gonzalez, Camille


    The Minnesota Multiphasic Personality Inventory-2 (MMPI-2) is widely used in neuropsychology, though its length (567 items) is sometimes prohibitive. This study investigated some psychometric characteristics of the 180-item version of the MMPI-2 () in order to delineate its strengths, limitations, and appropriate scope of clinical application. Limited reliability and poor predictive accuracy were recently reported for many of the MMPI-2 short-form scales in a study that used 205 brain-injured patients. In the present investigation, we used a psychiatric sample (N=186) with normal neurological findings to examine short-form accuracy in predicting basic scale scores, profile code types, identifying high-point scales, and classifying scores as pathological (T>/=65) or normal-range. The results suggest that, even as applied to neurologically normal individuals, the proposed short form of the MMPI-2 is unreliable for predicting clinical code types, identifying the high-point scale, or predicting the scores on most of the basic scales. In contrast, this short form can be used to predict whether the full-scale scores fall within the pathological range (T>/=65). These findings suggest that clinicians might be able to salvage a small amount of information from the shortened (180-item) version of the MMPI-2 when MMPI-2 protocols are incomplete. However, clinicians should not use a standard interpretive approach with this test, and routine clinical application is unwarranted. Future evaluations of short-form validity should provide a more detailed examination of individual protocols, including an analysis of the frequency of accurate prediction of full-form scores.

  12. The silent role of AIF short hairpin RNA interference recombinant vector targeting neuroblastoma cells SHSY5Y%AIF靶向短发夹RNA干扰重组载体对神经母细胞瘤细胞SHSY5Y的沉默作用

    Institute of Scientific and Technical Information of China (English)

    王占强; 李叶丹; 李春宇; 张极星; 王全才; 贾春红; 张鸿


    目的 探讨AIF基因短发夹RNA(shRNA)表达质粒对神经母细胞瘤细胞(SHSY5YAIF)基因的干扰作用.方法 根据siRNA设计原则,构建靶向AIF基因的短发夹RNA干扰质粒(pGPU6/GFP/AIF),使用脂质体法转染人神经母细胞瘤细胞,通过RT-PCR和Wester blot印迹检测神经母细胞瘤细胞AIF基因mRNA和蛋白表达水平.结果 pGPU6/GFP/AIF重组质粒经限制性内切酶酶切及测序证明基因插入正确.质粒成功转染SHSY5Y细胞后,该细胞的AIFmRNA和蛋白表达明显下降(P<0.05).结论 成功构建靶向AIF基因的shRNA表达载体转染神经母细胞瘤细胞,有效抑制AIFmRNA和蛋白表达,为AIF基因靶向治疗提供前期的实验依据.

  13. Expression of lentivirus-mediated short hairpin RNA (shRNA) targeting collagen type Ⅰ in rat mesangial cells%慢病毒介导的Ⅰ型胶原shRNA在大鼠系膜细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    单毅; 周春华


    目的 探讨慢病毒介导的Ⅰ型胶原(Col Ⅰ)短发夹RNA(shRNA)在大鼠系膜细胞的表达情况.方法 利用三质粒合成法将表达质粒合成慢病毒表达载体,分别用感染增强剂(对照组)、空慢病毒载体(pSC-GFP组)和慢病毒表达载体(pSC-GFP/Col Ⅰ组)感染大鼠系膜细胞.采用流式细胞仪检测各组大鼠系膜细胞的转染效率,并采用RT-PCR和Western blotting方法检测各组Col ⅠmRNA及Col Ⅰ蛋白表达情况.结果 pSC-GFP和pSC-GFP/Col Ⅰ均能高效感染大鼠系膜细胞,其中pSC-GFP感染效率为72.42%,pSC-GFP/Col Ⅰ感染效率高达75.42%.RT-PCR检测提示pSC-GFP/Col Ⅰ组细胞Col ⅠmRNA表达量较对照组、pSC-GFP组显著降低(P<0.05),其抑制效率为32.64%±0.12%,而后两组Col ⅠmRNA表达差异无统计学意义.Western blotting检测显示,与对照组、pSC-GFP组比较,pSC-GFP/Col Ⅰ组蛋白表达明显降低(P<0.05),而对照组与pSC-GFP组之间Col Ⅰ蛋白表达差异无统计学意义.结论 慢病毒载体能稳定、高效地将Col Ⅰ shRNA转染系膜细胞,并明显抑制Col Ⅰ的表达,为肾脏纤维化的基因治疗提供了理论依据.

  14. 洋为中用:运用概念整合理论分析中国古典文学作品的尝试——以陆游《钗头凤》为例%The Cognitive Explanatory of Phoenix hairpin by Virtual of the Conceptual Blending Theory

    Institute of Scientific and Technical Information of China (English)

    杨欢; 夏日光


    Conceptual blending theory has expounded the information in two mental spaces which map and integrate with each other,then obtain a new meaning.On the basis of analyzing the current research status of conceptual blending theory on poetry,the paper puts forward that conceptual blending theory has powerful explanatory force on the poem Phoenix hairpin.This paper tries to interpret the emotion and theme in the poem Phoenix hairpin from the perspective of blending network,relation compress,mapping and new-built structure by virtual of the conceptual blending theory.It is of great significance to appreciate the ancient poesy and poetry teaching.%概念整合理论是阐述两个心智空间中的信息相互映射整合、从而得出新创意义的理论。针对概念整合理论在诗词领域的研究现状,本文提出概念整合理论对陆游的《钗头凤》一词主旨的表达具有独特的解释力的假说。文章运用概念整合网络、关系压缩、映射、构建认知模型等方法,为《钗头凤》一词的情感及主旨的领悟打开新的视窗,为该词的欣赏与翻译提供新的思路。

  15. Combination of magnetic tweezers with DNA hairpin as a p otential approach to the study of RecA-mediated homologous recombination%磁镊结合DNA发夹的方法在RecA蛋白介导的同源重组机制研究中的潜在应用∗

    Institute of Scientific and Technical Information of China (English)

    张宇微; 颜燕; 农大官; 徐春华; 李明


    -resolution technique, the smFRET enables us to observe more details of reactions. However, its valid measuring distance is 3–8 nm, which limits the observation range. Here, we propose an approach by combining magnetic tweezers with DNA hairpin, which may solve the problem effectively in the study of HR. In this paper, one end of the DNA molecule with a 270 bp hairpin is immobilized onto the surface of the flow cell, while a magnetic bead is attached to the other end. An external magnetic force is applied to the magnetic bead by placing a permanent magnet above the flow cell. The first 90 bp (from the junction of the hairpin) of the hairpin is homologous to the ssDNA within the ssDNA-RecA filament. Thus, the filament searches for homology along the hairpin, and incorporates into the homologous segment for strand exchange. After that, the displaced strand can be opened by pulling at a force of ∼7 pN, and each opened base pair results in a 0.82 nm increase in DNA extension. By using this approach, we show that 1) RecA-mediated strand exchange proceeds in a stepwise manner and the average speed is∼7.6 nt/s, which is in accordance with previous result;2) the dynamic interaction between the second DNA-binding site (SBS) and the displaced strand can be observed in real-time, and the binding force is calculated accurately through the x-dimensional fluctuations; 3) the processes of strand-exchange in different directions can be observed, and the directions are distinguishable through the reaction patterns. The results suggest that the combination of magnetic tweezers with DNA hairpin is a potential approach to the study of RecA or other recombinases. Therefore, our design can be an important single-molecule approach to the research of HR mechanism.

  16. Short-living Supermassive Magnetar Model for the Early X-ray Flares Following Short GRBs

    Institute of Scientific and Technical Information of China (English)

    Wei-Hong Gao; Yi-Zhong Fan


    We suggest a short-lived supermassive magnetar model to account for the X-ray flares following short γ-ray bursts. In this model the central engine of the short γ-ray bursts is a supermassive millisecond magnetar, formed in coalescence of double neutron stars. The X-ray flares are powered by the dipole radiation of the magnetar. When the magnetar has lost a significant part of its angular momentum, it collapses to a black hole and the X-ray flares cease abruptly.

  17. 19 CFR 207.27 - Short life cycle products. (United States)


    ... 19 Customs Duties 3 2010-04-01 2010-04-01 false Short life cycle products. 207.27 Section 207.27... SUBSIDIZED EXPORTS TO THE UNITED STATES Final Determinations, Short Life Cycle Products § 207.27 Short life... scope of the product category into which to classify the short life cycle merchandise identified by the...

  18. Growing up with short stature : Psychosocial consequences of hormone treatment

    NARCIS (Netherlands)

    Visser-van Balen, J.


    Growing up with short stature. Psychosocial consequences of hormone treatment To enhance height in children with short stature, growth hormone (GH) can be used. In short children without a detectable pathology underlying their short stature, there is no medical rationale for growth hormone treatment

  19. A short course in sustainable product development

    DEFF Research Database (Denmark)

    McAloone, Tim C.


    This short course in sustainable product development models, methods and mindsets is designed to fit into the Unical course on Engineering Design Methods. Three modules (called “seminars”) will guide you through . The demands for sustainable development . Professional methods for analysing and ch...... and changing products’ environmental profiles . A new approach to product service system development, where the physical product becomes an incidental aspect in the final offering to the customer......This short course in sustainable product development models, methods and mindsets is designed to fit into the Unical course on Engineering Design Methods. Three modules (called “seminars”) will guide you through . The demands for sustainable development . Professional methods for analysing...

  20. Familial short fifth metacarpals and insulin resistance

    Energy Technology Data Exchange (ETDEWEB)

    Hyari, Muwafag; Hamamy, Hanan; Barham, Muries; Ajlouni, Kamel [National Center for Diabetes, Endocrinology and Genetics, P.O. Box 13165, Amman (Jordan); Al-Hadidy, Azmy [Jordan University Hospital, Department of Radiology, Amman (Jordan)


    Very few reports on the phenotype of short fifth metacarpals have been published in the medical literature. We report a Jordanian family in which three sisters aged 15, 13 and 8 years revealed bilateral shortening of the fifth fingers and radiological shortening of the fifth metacarpals. The father had unilateral short fifth metacarpal. The elder two sisters, their father as well as their brother and another sister manifested insulin resistance. Spherocytosis was diagnosed in one of the girls and her father. The parents are non-consanguineous. This constellation of findings has not been previously reported and could point to the presence of two disorders segregating in the family or to a novel syndrome with autosomal dominant inheritance and variable expressivity. (orig.)

  1. Short REM latency in impotence without depression. (United States)

    Schmidt, H S; Nofzinger, E A


    In a retrospective study, the presence of depression was studied in a group of 14 impotent patients who were selected on the basis of the similarity between their electroencephalographic (EEG) sleep patterns and those of patients with endogenous depression. Specifically, the value of rapid eye movement (REM) latency plus age less than 100 was used as a selection criterion. Sleep continuity disturbances, increased REM time, and increased REM% were noted in the short REM latency impotent group. On the basis of MMPI and psychiatric history and interview, only one of these impotent patients showed major depression. The authors conclude that impotent patients with a short REM latency are not, as a group, depressed and that the incidence of depression in impotent men should be determined irrespective of EEG sleep findings.

  2. Short Message Service using SMS Gateway

    Directory of Open Access Journals (Sweden)

    Veena K.Katankar


    Full Text Available Short message service (SMS will play a very vital role in the future business areas whose are popularly known as mobile banking, organizational marketing system etc. For this future, SMS could make a mobile device in a business tool as it has the availability and the effectiveness. This thesis is about software development that is based on short messaging service (SMS system for delivering messages through SMS gateway. Main goal of proposed system is to provide multi level local authentication to the SMS gateway service. This service can be implemented in any multi departmental organization whereSMS service is used for notification system and marketing purpose. Proposed system has web interface and the encryption method for providing service.

  3. Compression of Short Text on Embedded Systems

    DEFF Research Database (Denmark)

    Rein, S.; Gühmann, C.; Fitzek, Frank


    The paper details a scheme for lossless compression of a short data series larger than 50 bytes. The method uses arithmetic coding and context modelling with a low-complexity data model. A data model that takes 32 kBytes of RAM already cuts the data size in half. The compression scheme just takes...... a few pages of source code,is scaleablein memory size, and may be useful in sensor or cellular networks to spare bandwidth. As we demonstrate the method allows for battery savings when applied to mobile phones.......The paper details a scheme for lossless compression of a short data series larger than 50 bytes. The method uses arithmetic coding and context modelling with a low-complexity data model. A data model that takes 32 kBytes of RAM already cuts the data size in half. The compression scheme just takes...

  4. Short Telomere Length and Ischemic Heart Disease

    DEFF Research Database (Denmark)

    Scheller Madrid, Alexander; Rode, Line; Nordestgaard, Børge Grønne


    BACKGROUND: Short telomeres are associated with aging and have been associated with a high risk of ischemic heart disease in observational studies; however, the latter association could be due to residual confounding and/or reverse causation. We wanted to test the hypothesis that short telomeres...... are associated with high risk of ischemic heart disease using a Mendelian randomization approach free of reverse causation and of most confounding. METHODS: We genotyped 3 genetic variants in OBFC1 (oligonucleotide/oligosaccharide binding fold containing 1), TERT (telomerase reverse transcriptase), and TERC...... logistic and instrumental variable analysis for genetic estimates. RESULTS: Observationally, a 200-bp-shorter telomere length was associated with a multivariable adjusted hazard ratio for ischemic heart disease of 1.02 (95% CI, 1.01-1.03). Per allele, telomeres were shorter by 67 bp (73-60). In meta...

  5. Textbook Error: Short Circuiting an Electrochemical Cell (United States)

    Bonicamp, Judith M.; Clark, Roy W.


    Sometimes errors in electrochemistry discussions are not in the text but in the figures. Many errors in the electrochemical chapters of general chemistry textbooks have been reported previously, yet we found a serious error in the diagrams in eight, 21st century texts. The figures in them show many of the cells shorted out with a wire. Since a shorted electrochemical cell produces no measurable voltage it is unreasonable to ask students to calculate the voltage produced by such a cell. Depending upon the pedagogical context, these diagrams are either seriously wrong or reflect a poor choice of a load for the cell. We offer suggestions as to what loads might be appropriately shown in textbook diagrams within different electrochemical contexts. We also offer an analogy to electrical potential energy and include a diagram to clarify the interrelationships between electromotive force E , reaction quotient Q , and Gibbs free energy G (an EQG diagram).

  6. Short Time Cycles of Purely Quantum Refrigerators

    CERN Document Server

    Feldmann, Tova


    Four stroke Otto refrigerator cycles with no classical analogue are studied. Extremely short cycle times with respect to the internal time scale of the working medium characterize these refrigerators. Therefore these cycles are termed sudden. The sudden cycles are characterized by the stable limit cycle which is the invariant of the global cycle propagator. During their operation the state of the working medium possesses significant coherence which is not erased in the equilibration segments due to the very short time allocated. This characteristic is reflected in a difference between the energy entropy and the Von Neumann entropy of the working medium. A classification scheme for sudden refrigerators is developed allowing simple approximations for the cooling power and coefficient of performance.

  7. A short-form measure of loneliness. (United States)

    Hays, R D; DiMatteo, M R


    The revised UCLA Loneliness Scale (ULS-20) and a four-item short form (ULS-4) are widely used in personality research (Russell, Peplau, & Cutrona, 1980). In an exploratory factor analysis of the ULS-20, we identified eight items that loaded substantially on the first factor. These items were combined to form an alternative short-form measure, the ULS-8. The results of this study indicate that the ULS-8 is reliable, valid, and a better substitute for the ULS-20 than is the ULS-4. Consistent with the previous research, the loneliness measures (ULS-20, ULS-8, ULS-4) were strongly related to socially undesirable personality characteristics, but loneliness was uncorrelated with the six different health-related behaviors (exercise, meal regularity, alcohol use, hard drug use, smoking, and hours of sleep) assessed in this study.

  8. Ultra-short pulse laser proton acceleration

    Energy Technology Data Exchange (ETDEWEB)

    Zeil, Karl; Kraft, Stephan; Bussmann, Michael; Cowan, Thomas; Kluge, Thomas; Metzkes, Josefine; Richter, Tom; Schramm, Ulrich [Forschungszentrum Dresden-Rossendorf, Dresden (Germany)


    We present a systematic investigation of ultra-short pulse laser acceleration of protons yielding unprecedented maximum proton energies of 17 MeV using the Ti:Sapphire lased high power laser of 100 TW Draco at the Research Centre Dresden-Rossendorf. For plain few micron thick foil targets a linear scaling of the maximum proton energy with laser power is observed and attributed to the short acceleration period close to the target rear surface. Although excellent laser pulse contrast was available slight deformations of the target rear were found to lead to a predictable shift of the direction of the energetic proton emission away from target normal towards the laser direction. The change of the emission characteristics are compared to analytical modelling and 2D PIC simulations.

  9. Ultra-Wideband, Short Pulse Electromagnetics 9

    CERN Document Server

    Rachidi, Farhad; Kaelin, Armin; Sabath, Frank; UWB SP 9


    Ultra-wideband (UWB), short-pulse (SP) electromagnetics are now being used for an increasingly wide variety of applications, including collision avoidance radar, concealed object detection, and communications. Notable progress in UWB and SP technologies has been achieved by investigations of their theoretical bases and improvements in solid-state manufacturing, computers, and digitizers. UWB radar systems are also being used for mine clearing, oil pipeline inspections, archeology, geology, and electronic effects testing. Ultra-wideband Short-Pulse Electromagnetics 9 presents selected papers of deep technical content and high scientific quality from the UWB-SP9 Conference, which was held from July 21-25, 2008, in Lausanne, Switzerland. The wide-ranging coverage includes contributions on electromagnetic theory, time-domain computational techniques, modeling, antennas, pulsed-power, UWB interactions, radar systems, UWB communications, and broadband systems and components. This book serves as a state-of-the-art r...

  10. A short proof of increased parabolic regularity

    Directory of Open Access Journals (Sweden)

    Stephen Pankavich


    Full Text Available We present a short proof of the increased regularity obtained by solutions to uniformly parabolic partial differential equations. Though this setting is fairly introductory, our new method of proof, which uses a priori estimates and an inductive method, can be extended to prove analogous results for problems with time-dependent coefficients, advection-diffusion or reaction diffusion equations, and nonlinear PDEs even when other tools, such as semigroup methods or the use of explicit fundamental solutions, are unavailable.

  11. Long life of a short film. (United States)

    Wadia, R V


    What follows is an account of my personal experiences as an independent Indian film director who had the fortune to make the country's first openly gay film, the short BOMgAY That said, I would like this essay to be accepted as, rather than a critical appraisal of Indian cinema, a humbly autobiographical account of one individual caught in the hectic throes of political (and cinematic) visibility.

  12. A short course in sustainable product development

    DEFF Research Database (Denmark)

    McAloone, Tim C.


    This short course in sustainable product development models, methods and mindsets is designed to fit into the Unical course on Engineering Design Methods. Three modules (called “seminars”) will guide you through . The demands for sustainable development . Professional methods for analysing...... and changing products’ environmental profiles . A new approach to product service system development, where the physical product becomes an incidental aspect in the final offering to the customer...

  13. Hedge Ratios for short and leveraged ETFs

    Directory of Open Access Journals (Sweden)

    Leo Schubert


    Full Text Available Exchange Traded Funds (ETFs exist for stock-, bond- and commodity markets. In most cases the underlying of an ETF is an index. Fund management today uses the active and passive way to construct a portfolio. ETFs can be used for passive portfolio management. Then ETFs with positive leverage factors are preferred. In the frame of active portfolio also the ETFs with negative leverage factors can be applied for the hedge or cross hedge of a portfolio. These hedging possibilities will be analyzed in this paper. Short ETFs exist with different leverage factors. In Europe, the leverage factors 1 (e.g. ShortDAX ETF and 2 (e.g. DJ STOXX 600 Double Short are offered while in the financial markets of the United States factors from 1 to 4 can be found. To investigate the effect of the different leverage factors and other parameters Monte Carlo Simulation was used. The results show e.g. that higher leverage factors achieve higher profits as well as losses. In the case, that a bearish market is supposed, minimizing the variance of the hedge seem not to be until to get better hedging results, due to a very skewed return distribution of the hedge. The risk measure target-shortfall-probability confirms the use of the standard hedge weightings which depend only on the leverage factor. This characteristic remains, when a portfolio has to be hedged instead of the underlying index of the short ETF. For portfolios which have a low correlation with the index return should not be used high leverage factors for hedging, due to the higher volatility and target-shortfall-probability.

  14. Molecular beam epitaxy a short history

    CERN Document Server

    Orton, J W


    This volume describes the development of molecular beam epitaxy from its origins in the 1960s through to the present day. It begins with a short historical account of other methods of crystal growth, both bulk and epitaxial, to set the subject in context, emphasising the wide range of semiconductor materials employed. This is followed by an introduction to molecular beams and their use in the Stern-Gerlach experiment and the development of the microwave MASER.

  15. Automatic location of short circuit faults

    Energy Technology Data Exchange (ETDEWEB)

    Lehtonen, M. [VTT Energy, Espoo (Finland); Hakola, T.; Antila, E. [ABB Power Oy, Helsinki (Finland); Seppaenen, M. [North-Carelian Power Company (Finland)


    In this presentation, the automatic location of short circuit faults on medium voltage distribution lines, based on the integration of computer systems of medium voltage distribution network automation is discussed. First the distribution data management systems and their interface with the substation telecontrol, or SCADA systems, is studied. Then the integration of substation telecontrol system and computerised relay protection is discussed. Finally, the implementation of the fault location system is presented and the practical experience with the system is discussed

  16. Short-time Lyapunov exponent analysis (United States)

    Vastano, J. A.


    A new technique for analyzing complicated fluid flows in numerical simulations has been successfully tested. The analysis uses short time Lyapunov exponent contributions and the associated Lyapunov perturbation fields. A direct simulation of the Taylor-Couette flow just past the onset of chaos demonstrated that this new technique marks important times during the system evolution and identifies the important flow features at those times. This new technique will now be applied to a 'minimal' turbulent channel.

  17. Subthreshold pair production in short laser pulses


    Nousch, T.; Seipt, D.; Kampfer, B.; Titov, A. I.


    The $e^+e^-$ pair production by a probe photon traversing a linearly polarized laser pulse is treated as generalized nonlinear Breit-Wheeler process. For short laser pulses with very few oscillations of the electromagnetic field we find below the perturbative weak-field threshold $\\sqrt{s} = 2m$ a similar enhancement of the pair production rate as for circular polarization. The strong subthreshold enhancement is traced back to the finite bandwidth of the laser pulse. A folding model is develo...

  18. Subnanosecond Short Wavelength Generation Using Optical Fibers. (United States)


    was focussed by a *short focal length lens (4-5 cm) into quartz fibers of 200 - 300 um * diameter. These were solid core with plastic or glass ...October, 1983]. 3. "Gas in Glass - experiments The intention of these experiments was to provide Reman-active * media within a quartz fiber in order to...200 um hollow quartz capillary tubes surrounded by a larger glass tube for protection, and with two liquid cells at each end, as shown in Figure (5

  19. Automatic location of short circuit faults

    Energy Technology Data Exchange (ETDEWEB)

    Lehtonen, M. [VTT Energy, Espoo (Finland); Hakola, T.; Antila, E. [ABB Power Oy (Finland); Seppaenen, M. [North-Carelian Power Company (Finland)


    In this chapter, the automatic location of short circuit faults on medium voltage distribution lines, based on the integration of computer systems of medium voltage distribution network automation is discussed. First the distribution data management systems and their interface with the substation telecontrol, or SCADA systems, is studied. Then the integration of substation telecontrol system and computerized relay protection is discussed. Finally, the implementation of the fault location system is presented and the practical experience with the system is discussed

  20. The long and the short of it


    Denman, Peter


    The Morning train; November Wedding and Other Poems; The Wind Dog; While these three collections could hardly be said to constitute a representative sample, they do prompt the thought that perhaps we are seeing the emergence of a new convention in the arrangement of poetry collections. Each of these books offers a gathering of short lyric length pieces, together with at least one longer poem or sequence. Paul Muldoon has repeatedly followed this practice of concluding with a long poem, and in...