Science.gov

Sample records for extremely abundant sirnas

  1. The lithium abundance in extreme halo stars

    SciTech Connect

    Hobbs, L.M.; Thorburn, J.A. )

    1991-07-01

    New observations are reported of atmospheric Li abundances for six extremely metal-poor dwarfs with Fe-H ratios not higher than {minus}2.59 and T(e) not lower than 5950 K. The spectra were obtained in 1990 at Kitt Peak National Observatory, using the echelle spectrograph with the UV Fast camera. The resulting Li abundances for these stars range between N(Li) values of 1.99 and 2.24, where N(Li) = 12 + log (Li/H). These results agree with the abundances reported previously for five other metal-poor dwarfs with the Fe/H ratios not above {minus}2.60. The invariance of Li abundance in these 11 stars indicates a primordial origin for most of the Li observed in these Galactic stars. 23 refs.

  2. Extreme possible variations of the deuterium abundance within the Galaxy

    NASA Astrophysics Data System (ADS)

    Delbourgo-Salvador, P.; Audouze, J.; Vidal-Madjar, A.

    1987-03-01

    In order to reconcile the present baryonic densities deduced respectively from the primordial abundances of D and 4He, some recent chemical evolution models imply that D could have been destroyed more thoroughly during the Galaxy evolution than what was previously predicted. Under the conditions outlined by these models, the present abundance of D may vary by factors as large as 50 in different parts of the Galaxy. If such variations are not observed, this implies that the ratio X(D)prim/X(D)present is not large (2 - 3): the simplest Big Bang models may then be unable to reconcile the baryonic densities predicted by D and 4He respectively.

  3. Genome-wide identification of endogenous RNA-directed DNA methylation loci associated with abundant 21-nucleotide siRNAs in Arabidopsis

    PubMed Central

    Zhao, Jian-Hua; Fang, Yuan-Yuan; Duan, Cheng-Guo; Fang, Rong-Xiang; Ding, Shou-Wei; Guo, Hui-Shan

    2016-01-01

    In Arabidopsis, the 24-nucleotide (nt) small interfering RNAs (siRNAs) mediates RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of transposable elements (TEs). In the present study, we examined genome-wide changes in DNA methylation and siRNA accumulation in Arabidopsis induced by expression of the Cucumber mosaic virus silencing suppressor protein 2b known to directly bind to both the 21/24-nt siRNAs as well as their associated Argonaute proteins. We demonstrated a genome-wide reduction of CHH and CHG methylation in the 2b-transgenic plants. We found that 2b suppressed RdDM not only at the previously annotated loci directed by 24-nt siRNAs but also a new set of loci associated with 21/22-nt siRNAs. Further analysis showed that the reduced methylation of TEs and coding genes targeted by 21/22-nt siRNAs was associated with sequestration of the duplex siRNAs by the 2b protein but not with changes in either siRNA production or transcription. Notably, we detected both the deletion and/or the transposition of multicopy TEs associated with 2b-induced hypomethylation, suggesting potential TE reactivation. We propose that the silencing of many TEs in Arabidopsis is controlled by the 24- and 21-nt endogenous siRNAs analogous to Drosophila TE silencing by PIWI-interacting RNAs and siRNAs. PMID:27786269

  4. Lithium abundance in a turnoff halo star on an extreme orbit

    NASA Astrophysics Data System (ADS)

    Spite, M.; Spite, F.; Caffau, E.; Bonifacio, P.

    2015-10-01

    Context. The lithium abundance in turnoff stars of the old population of our Galaxy is remarkably constant in the metallicity interval -2.8 < [Fe/H] < -2.0, defining a plateau. The Li abundance of these turnoff stars is clearly lower than the abundance predicted by the primordial nucleosynthesis in the frame of the standard Big Bang nucleosynthesis. Different scenarios have been proposed for explaining this discrepancy, along with the very low scatter of the lithium abundance around the plateau. Aims: The recently identified very high velocity star, WISE J0725-2351 appears to belong to the old Galactic population, and appears to be an extreme halo star on a bound, retrograde Galactic orbit. In this paper, we study the abundance ratios and, in particular the lithium abundance, in this star. Methods: The available spectra (ESO-Very Large Telescope) are analyzed and the abundances of Li, C, Na, Mg, Al, Si, Ca, Sc, Ti, Cr, Mn, Fe, Co, Ni, Sr and Ba are determined. Results: The abundance ratios in WISE J0725-2351 are those typical of old turnoff stars. The lithium abundance in this star is in close agreement with the lithium abundance found in the metal-poor turnoff stars located at moderate distance from the Sun. This high velocity star confirms, in an extreme case, that the very small scatter of the lithium plateau persists independent of the dynamic and kinematic properties of the stars. Based on observations obtained at the ESO Paranal Observatory, Chile Programmes 093.D-0127, PI: S. Geier and 189.B-0925, PI: S. Trager.Table 2 (line by line abundances of the elements) is only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/582/A74

  5. Distribution, abundance and diversity of the extremely halophilic bacterium Salinibacter ruber

    PubMed Central

    Antón, Josefa; Peña, Arantxa; Santos, Fernando; Martínez-García, Manuel; Schmitt-Kopplin, Philippe; Rosselló-Mora, Ramon

    2008-01-01

    Since its discovery in 1998, representatives of the extremely halophilic bacterium Salinibacter ruber have been found in many hypersaline environments across the world, including coastal and solar salterns and solar lakes. Here, we review the available information about the distribution, abundance and diversity of this member of the Bacteroidetes. PMID:18957079

  6. First high-precision differential abundance analysis of extremely metal-poor stars

    NASA Astrophysics Data System (ADS)

    Reggiani, Henrique; Meléndez, Jorge; Yong, David; Ramírez, Ivan; Asplund, Martin

    2016-02-01

    Context. Studies of extremely metal-poor stars indicate that chemical abundance ratios [X/Fe] have a root mean square scatter as low as 0.05 dex (12%). It remains unclear whether this reflects observational uncertainties or intrinsic astrophysical scatter arising from physical conditions in the interstellar medium at early times. Aims: We measure differential chemical abundance ratios in extremely metal-poor stars to investigate the limits of precision and to understand whether cosmic scatter or observational errors are dominant. Methods: We used high-resolution (R ~ 95 000) and high signal-to-noise (S/N = 700 at 5000 Å) HIRES/Keck spectra to determine high-precision differential abundances between two extremely metal-poor stars through a line-by-line differential approach. We determined stellar parameters for the star G64-37 with respect to the standard star G64-12. We performed EW measurements for the two stars for the lines recognized in both stars and performed spectral synthesis to study the carbon abundances. Results: The differential approach allowed us to obtain errors of σ(Teff) = 27 K, σ(log g) = 0.06 dex, σ( [Fe/H] ) = 0.02 dex and σ(vt) = 0.06 km s-1. We estimated relative chemical abundances with a precision as low as σ([X/Fe]) ≈ 0.01 dex. The small uncertainties demonstrate that there are genuine abundance differences larger than the measurement errors. The observed Li difference cannot be explained by the difference in mass because the less massive star has more Li. Conclusions: It is possible to achieve an abundance precision around ≈ 0.01-0.05 dex for extremely metal-poor stars, which opens new windows on the study of the early chemical evolution of the Galaxy. Table A.1 is also available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (ftp://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/586/A67

  7. Lithium abundances in extremely metal-poor turn-off stars

    NASA Astrophysics Data System (ADS)

    Sbordone, L.; Bonifacio, P.; Caffau, E.

    We discuss the current status of the sample of Lithium abundances in extremely metal poor (EMP) turn-off (TO) stars collected by our group, and compare it with the available literature results. In the last years, evidences have accumulated of a progressive disruption of the Spite plateau in stars of extremely low metallicity. What appears to be a flat, thin plateau above [Fe/H]˜-2.8 turns, at lower metallicities, into a broader distribution for which the plateau level constitutes the upper limit, but more and more stars show lower Li abundances. The sample we have collected currently counts abundances or upper limits for 44 EMP TO stars between [Fe/H]=-2.5 and -3.5, plus the ultra-metal poor star SDSS J102915+172927 at [Fe/H]=-4.9. The ``meltdown'' of the Spite plateau is quite evident and, at the current status of the sample, does not appear to be restricted to the cool end of the effective temperature distribution. SDSS J102915+172927 displays an extreme Li depletion that contrasts with its otherwise quite ordinary set of [X/Fe] ratios.

  8. NEON AND CNO ABUNDANCES FOR EXTREME HELIUM STARS-A NON-LTE ANALYSIS

    SciTech Connect

    Pandey, Gajendra; Lambert, David L. E-mail: dll@astro.as.utexas.edu

    2011-02-01

    A non-LTE (NLTE) abundance analysis was carried out for three extreme helium stars (EHes): BD+10{sup 0} 2179, BD-9{sup 0} 4395, and LS IV+6{sup 0} 002, from their optical spectra with NLTE model atmospheres. NLTE TLUSTY model atmospheres were computed with H, He, C, N, O, and Ne treated in NLTE. Model atmosphere parameters were chosen from consideration of fits to observed He I line profiles and ionization equilibria of C and N ions. The program SYNSPEC was then used to determine the NLTE abundances for Ne as well as H, He, C, N, and O. LTE neon abundances from Ne I lines in the EHes: LSE 78, V1920 Cyg, HD 124448, and PV Tel, are derived from published models and an estimate of the NLTE correction applied to obtain the NLTE Ne abundance. We show that the derived abundances of these key elements, including Ne, are well matched with semi-quantitative predictions for the EHe resulting from a cold merger (i.e., no nucleosynthesis during the merger) of an He white dwarf with a C-O white dwarf.

  9. Detailed Abundances in Extremely Metal Poor Dwarf Stars Extracted from SDSS

    NASA Astrophysics Data System (ADS)

    Sbordone, L.; Bonifacio, P.; Caffau, E.; Ludwig, H.-G.

    2012-08-01

    We report on the result of an ongoing campaign to determine chemical abundances in extremely metal poor (EMP) turn-off (TO) stars selected from the Sloan Digital Sky Survey (SDSS) low resolution spectra. This contribution focuses principally on the largest part of the sample (18 stars out of 29), observed with UVES@VLT and analyzed by means of the automatic abundance analysis code MyGIsFOS to derive atmosphere parameters and detailed compositions. The most significant findings include i) the detection of a C-rich, strongly Mg-enhanced star ([Mg/Fe]=1.45); ii) a group of Mn-rich stars ([Mn/Fe]>-0.4); iii) a group of Ni-rich stars ([Ni/Fe]>0.2). Li is measured in twelve stars, while for three upper limits are derived.

  10. Oxygen abundance from strong-line methods at extremely low metallicities

    NASA Astrophysics Data System (ADS)

    Morales-Luis, A. B.; Sánchez Almeida, J.; Pérez Montero, E.; Muñoz-Tuñon, C.; Aguerri, J. A. L.; Vilchez, J. M.; Terlevich, E.; Terlevich, R.

    2013-05-01

    The determination of oxygen abundance in nebulae requires measuring a significant number of emission lines distributed along a wide spectral range. The required measurements are hard to obtain at high redshift, where sources are very faint, and where the accessible spectral range is limited. These difficulties are often overcome using empirical relationships between the oxygen abundance and the fluxes in a small number of strong lines. The so-called strong-line methods are often the only practical alternative for metallicity estimate at high redshift. In this sense, the low metallicities range is particularly important since high redshift objects are primitive and so of low metallic content. One of the most widely used relationships links the oxygen with the ratio between [NII]6583 and Hα. This relationship shows a large scatter at low metallicity. In an effort to bring down the errors, we re-calibrated the relationship using a large sample of extremely metal-poor galaxies. The SDSS spectra of the galaxies were all analyzed in the same way to minimize systematic errors. To our surprise, the decrease of scatter reveals that the ratio [N{II}]6583 to Hα seems to be independent of metallicity at low oxygen abundance (12+log[{O}/{H}] < 7.6). This result casts doubts on the metallicities of high-redshift objects based on the relationship. We explain how the re-calibration was carried (including the sample selection and the abundance determinations). In addition, we try explain what produces the lack of correlation.

  11. AGB star intershell abundances inferred from UV spectra of extremely hot post-AGB stars

    NASA Astrophysics Data System (ADS)

    Werner, K.; Rauch, T.; Reiff, E.; Kruk, J. W.

    2009-04-01

    The hydrogen-deficiency in extremely hot post-AGB stars of spectral class PG1159 is probably caused by a (very) late helium-shell flash or a AGB final thermal pulse that consumes the hydrogen envelope, exposing the usually-hidden intershell region. Thus, the photospheric element abundances of these stars allow us to draw conclusions about details of nuclear burning and mixing processes in the precursor AGB stars. We compare predicted element abundances to those determined by quantitative spectral analyses performed with advanced non-LTE model atmospheres. A good qualitative and quantitative agreement is found for many species (He, C, N, O, Ne, F, Si, Ar) but discrepancies for others (P, S, Fe) point at shortcomings in stellar evolution models for AGB stars. Almost all of the chemical trace elements in these hot stars can only be identified in the UV spectral range. The Far Ultraviolet Spectroscopic Explorer and the Hubble Space Telescope played a crucial role for this research.

  12. First Stars. III. A detailed elemental abundance study of four extremely metal-poor giant stars

    NASA Astrophysics Data System (ADS)

    François, P.; Depagne, E.; Hill, V.; Spite, M.; Spite, F.; Plez, B.; Beers, T. C.; Barbuy, B.; Cayrel, R.; Andersen, J.; Bonifacio, P.; Molaro, P.; Nordström, B.; Primas, F.

    2003-06-01

    This paper reports detailed abundance analyses for four extremely metal-poor (XMP) giant stars with [Fe/H]<-3.8, based on high-resolution, high-S/N spectra from the ESO VLT (Kueyen/UVES) and LTE model atmosphere calculations. The derived [alpha /Fe] ratios in our sample exhibit a small dispersion, confirming previous findings in the literature, i.e. a constant overabundance of the alpha -elements with a very small (if any) dependence on [Fe/H]. In particular, the very small scatter we determine for [Si/Fe] suggests that this element shows a constant overabundance at very low metallicity, a conclusion which could not have been derived from the widely scattered [Si/Fe] values reported in the literature for less metal-poor stars. For the iron-peak elements, our precise abundances for the four XMP stars in our sample confirm the decreasing trend of Cr and Mn with decreasing [Fe/H], as well as the increasing trend for Co and the absence of any trend for Sc and Ni. In contrast to the significant spread of the ratios [Sr/Fe] and [Ba/Fe], we find [Sr/Ba] in our sample to be roughly solar, with a much lower dispersion than previously found for stars in the range -3.5 < [Fe/H] < -2.5. Based on observations made with the ESO Very Large Telescope at Paranal Observatory, Chile (Large Programme ID 165.N-0276(A)). The complete version of Table 5 is only available in electronic form at the CDS via anonymous ftp to cdsarc.u-strasbg.fr (130.79.125.5) or via http:/ /cdsweb.u-strasbg.fr/cgi-bin/qcat?J /A+A/403/1105

  13. Extreme abundance ratios in the polluted atmosphere of the cool white dwarf NLTT 19868

    NASA Astrophysics Data System (ADS)

    Kawka, Adela; Vennes, Stéphane

    2016-05-01

    We present an analysis of intermediate-dispersion spectra and photometric data of the newly identified cool, polluted white dwarf NLTT 19868. The spectra obtained with X-shooter on the Very Large Telescope-Melipal show strong lines of calcium, and several lines of magnesium, aluminium and iron. We use these spectra and the optical-to-near-infrared spectral energy distribution to constrain the atmospheric parameters of NLTT 19868. Our analysis shows that NLTT 19868 is iron poor with respect to aluminium and calcium. A comparison with other cool, polluted white dwarfs shows that the Fe to Ca abundance ratio (Fe/Ca) varies by up to approximately two orders of magnitudes over a narrow temperature range with NLTT 19868 at one extremum in the Fe/Ca ratio and, in contrast, NLTT 888 at the other extremum. The sample shows evidence of extreme diversity in the composition of the accreted material: in the case of NLTT 888, the inferred composition of the accreted matter is akin to iron-rich planetary core composition, while in the case of NLTT 19868 it is close to mantle composition depleted by subsequent chemical separation at the bottom of the convection zone.

  14. Chemical Abundances in the Extremely Carbon-rich and Xenon-rich Halo Planetary Nebula H4-1

    NASA Astrophysics Data System (ADS)

    Otsuka, Masaaki; Tajitsu, Akito

    2013-12-01

    We performed detailed chemical abundance analysis of the extremely metal-poor ([Ar/H] ~ -2) halo planetary nebula (PN) H4-1 based on the multi-wavelength spectra from Subaru/HDS, GALEX, SDSS, and Spitzer/IRS and determined the abundances of 10 elements. The C and O abundances were derived from collisionally excited lines (CELs) and are almost consistent with abundances from recombination lines (RLs). We demonstrated that the large discrepancy in the C abundance between CEL and RL in H4-1 can be solved using the temperature fluctuation model. We reported the first detection of the [Xe III] λ5846 line in H4-1 and determination of its elemental abundance ([Xe/H] > +0.48). H4-1 is the most Xe-rich PN among the Xe-detected PNe. The observed abundances are close to the theoretical prediction by a 2.0 M ⊙ single star model with an initially element rich ([r/Fe] = +2.0 dex) rapid neutron-capture process (r-process). The observed Xe abundance would be a product of the r-process in primordial supernovae. The [C/O]-[Ba/(Eu or Xe)] diagram suggests that the progenitor of H4-1 shares the evolution with carbon-enhanced metal-poor (CEMP)-r/s and CEMP-no stars. The progenitor of H4-1 is presumably a binary formed in an r-process-rich environment.

  15. Chemical abundances in the extremely carbon-rich and xenon-rich halo planetary nebula H4-1

    SciTech Connect

    Otsuka, Masaaki; Tajitsu, Akito E-mail: tajitsu@subaru.naoj.org

    2013-12-01

    We performed detailed chemical abundance analysis of the extremely metal-poor ([Ar/H] ∼ –2) halo planetary nebula (PN) H4-1 based on the multi-wavelength spectra from Subaru/HDS, GALEX, SDSS, and Spitzer/IRS and determined the abundances of 10 elements. The C and O abundances were derived from collisionally excited lines (CELs) and are almost consistent with abundances from recombination lines (RLs). We demonstrated that the large discrepancy in the C abundance between CEL and RL in H4-1 can be solved using the temperature fluctuation model. We reported the first detection of the [Xe III] λ5846 line in H4-1 and determination of its elemental abundance ([Xe/H] > +0.48). H4-1 is the most Xe-rich PN among the Xe-detected PNe. The observed abundances are close to the theoretical prediction by a 2.0 M {sub ☉} single star model with an initially element rich ([r/Fe] = +2.0 dex) rapid neutron-capture process (r-process). The observed Xe abundance would be a product of the r-process in primordial supernovae. The [C/O]-[Ba/(Eu or Xe)] diagram suggests that the progenitor of H4-1 shares the evolution with carbon-enhanced metal-poor (CEMP)-r/s and CEMP-no stars. The progenitor of H4-1 is presumably a binary formed in an r-process-rich environment.

  16. Snow cover and extreme winter warming events control flower abundance of some, but not all species in high arctic Svalbard.

    PubMed

    Semenchuk, Philipp R; Elberling, Bo; Cooper, Elisabeth J

    2013-08-01

    The High Arctic winter is expected to be altered through ongoing and future climate change. Winter precipitation and snow depth are projected to increase and melt out dates change accordingly. Also, snow cover and depth will play an important role in protecting plant canopy from increasingly more frequent extreme winter warming events. Flower production of many Arctic plants is dependent on melt out timing, since season length determines resource availability for flower preformation. We erected snow fences to increase snow depth and shorten growing season, and counted flowers of six species over 5 years, during which we experienced two extreme winter warming events. Most species were resistant to snow cover increase, but two species reduced flower abundance due to shortened growing seasons. Cassiope tetragona responded strongly with fewer flowers in deep snow regimes during years without extreme events, while Stellaria crassipes responded partly. Snow pack thickness determined whether winter warming events had an effect on flower abundance of some species. Warming events clearly reduced flower abundance in shallow but not in deep snow regimes of Cassiope tetragona, but only marginally for Dryas octopetala. However, the affected species were resilient and individuals did not experience any long term effects. In the case of short or cold summers, a subset of species suffered reduced reproductive success, which may affect future plant composition through possible cascading competition effects. Extreme winter warming events were shown to expose the canopy to cold winter air. The following summer most of the overwintering flower buds could not produce flowers. Thus reproductive success is reduced if this occurs in subsequent years. We conclude that snow depth influences flower abundance by altering season length and by protecting or exposing flower buds to cold winter air, but most species studied are resistant to changes. Winter warming events, often occurring

  17. Snow cover and extreme winter warming events control flower abundance of some, but not all species in high arctic Svalbard

    PubMed Central

    Semenchuk, Philipp R; Elberling, Bo; Cooper, Elisabeth J

    2013-01-01

    Abstract The High Arctic winter is expected to be altered through ongoing and future climate change. Winter precipitation and snow depth are projected to increase and melt out dates change accordingly. Also, snow cover and depth will play an important role in protecting plant canopy from increasingly more frequent extreme winter warming events. Flower production of many Arctic plants is dependent on melt out timing, since season length determines resource availability for flower preformation. We erected snow fences to increase snow depth and shorten growing season, and counted flowers of six species over 5 years, during which we experienced two extreme winter warming events. Most species were resistant to snow cover increase, but two species reduced flower abundance due to shortened growing seasons. Cassiope tetragona responded strongly with fewer flowers in deep snow regimes during years without extreme events, while Stellaria crassipes responded partly. Snow pack thickness determined whether winter warming events had an effect on flower abundance of some species. Warming events clearly reduced flower abundance in shallow but not in deep snow regimes of Cassiope tetragona, but only marginally for Dryas octopetala. However, the affected species were resilient and individuals did not experience any long term effects. In the case of short or cold summers, a subset of species suffered reduced reproductive success, which may affect future plant composition through possible cascading competition effects. Extreme winter warming events were shown to expose the canopy to cold winter air. The following summer most of the overwintering flower buds could not produce flowers. Thus reproductive success is reduced if this occurs in subsequent years. We conclude that snow depth influences flower abundance by altering season length and by protecting or exposing flower buds to cold winter air, but most species studied are resistant to changes. Winter warming events, often

  18. Abundant carbon substrates drive extremely high sulfate reduction rates and methane fluxes in Prairie Pothole Wetlands.

    PubMed

    Dalcin Martins, Paula; Hoyt, David W; Bansal, Sheel; Mills, Christopher T; Tfaily, Malak; Tangen, Brian A; Finocchiaro, Raymond G; Johnston, Michael D; McAdams, Brandon C; Solensky, Matthew J; Smith, Garrett J; Chin, Yu-Ping; Wilkins, Michael J

    2017-01-24

    Inland waters are increasingly recognized as critical sites of methane emissions to the atmosphere, but the biogeochemical reactions driving such fluxes are less well understood. The Prairie Pothole Region (PPR) of North America is one of the largest wetland complexes in the world, containing millions of small, shallow wetlands. The sediment pore waters of PPR wetlands contain some of the highest concentrations of dissolved organic carbon (DOC) and sulfur species ever recorded in terrestrial aquatic environments. Using a suite of geochemical and microbiological analyses, we measured the impact of sedimentary carbon and sulfur transformations in these wetlands on methane fluxes to the atmosphere. This research represents the first study of coupled geochemistry and microbiology within the PPR and demonstrates how the conversion of abundant labile DOC pools into methane results in some of the highest fluxes of this greenhouse gas to the atmosphere ever reported. Abundant DOC and sulfate additionally supported some of the highest sulfate reduction rates ever measured in terrestrial aquatic environments, which we infer to account for a large fraction of carbon mineralization in this system. Methane accumulations in zones of active sulfate reduction may be due to either the transport of free methane gas from deeper locations or the co-occurrence of methanogenesis and sulfate reduction. If both respiratory processes are concurrent, any competitive inhibition of methanogenesis by sulfate-reducing bacteria may be lessened by the presence of large labile DOC pools that yield noncompetitive substrates such as methanol. Our results reveal some of the underlying mechanisms that make PPR wetlands biogeochemical hotspots, which ultimately leads to their critical, but poorly recognized role in regional greenhouse gas emissions.

  19. Extremely abundant antimicrobial peptides existed in the skins of nine kinds of Chinese odorous frogs.

    PubMed

    Yang, Xinwang; Lee, Wen-Hui; Zhang, Yun

    2012-01-01

    Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structure-functional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics.

  20. Probing the Site for r-Process Nucleosynthesis with Abundances of Barium and Magnesium in Extremely Metal-poor Stars.

    PubMed

    Tsujimoto; Shigeyama; Yoshii

    2000-03-01

    We suggest that if the astrophysical site for r-process nucleosynthesis in the early Galaxy is confined to a narrow mass range of Type II supernova (SN II) progenitors, with a lower mass limit of Mms=20 M middle dot in circle, a unique feature in the observed distribution of [Ba/Mg] versus [Mg/H] for extremely metal-poor stars can be adequately reproduced. We associate this feature, a bifurcation of the observed elemental ratios into two branches in the Mg abundance interval -3.7abundance ratios of stars which were formed in the dense shells of the interstellar medium swept up by SNe II with Mms<20 M middle dot in circle that do not synthesize r-process elements, and it applies to stars with observed Mg abundances in the range &sqbl0;Mg&solm0;H&sqbr0;<-2.7. The Ba abundances in these stars reflect those of the interstellar gas at the (later) time of their formation. The existence of a [Ba/Mg] i-branch strongly suggests that SNe II that are associated with stars of progenitor mass Mms

  1. The Chemical Abundances of Stars in the Halo (CASH) Project. II. New Extremely Metal-poor Stars

    NASA Astrophysics Data System (ADS)

    Krugler, Julie A.; Frebel, A.; Roederer, I. U.; Sneden, C.; Shetrone, M.; Beers, T.; Christlieb, N.

    2011-01-01

    We present new abundance results from the Chemical Abundances of Stars in the Halo (CASH) project. The 500 CASH spectra were observed using the Hobby-Eberly Telescope in "snapshot" mode and are analyzed using an automated stellar parameter and abundance pipeline called CASHCODE. For the 20 most metal-poor stars of the CASH sample we have obtained high resolution spectra using the Magellan Telescope in order to test the uncertainties and systematic errors associated with the snapshot quality (i.e., R 15,000 and S/N 65) HET spectra and to calibrate the newly developed CASHCODE by making a detailed comparison between the stellar parameters and abundances determined from the high resolution and snapshot spectra. We find that the CASHCODE stellar parameters (effective temperature, surface gravity, metallicity, and microturbulence) agree well with the results of the manual analysis of the high resolution spectra. We present the abundances of three newly discovered stars with [Fe/H] < -3.5. For the entire pilot sample, we find typical halo abundance ratios with alpha-enhancement and Fe-peak depletion and a range of n-capture elements. The full CASH sample will be used to derive statistically robust abundance trends and frequencies (e.g. carbon and n-capture), as well as placing constraints on nucleosynthetic processes that occurred in the early universe.

  2. Solar abundances of rock-forming elements, extreme oxygen and hydrogen in a young polluted white dwarf

    NASA Astrophysics Data System (ADS)

    Farihi, J.; Koester, D.; Zuckerman, B.; Vican, L.; Gänsicke, B. T.; Smith, N.; Walth, G.; Breedt, E.

    2016-12-01

    The Teff = 20 800 K white dwarf WD 1536+520 is shown to have broadly solar abundances of the major rock-forming elements O, Mg, Al, Si, Ca, and Fe, together with a strong relative depletion in the volatile elements C and S. In addition to the highest metal abundances observed to date, including log (O/He) = -3.4, the helium-dominated atmosphere has an exceptional hydrogen abundance at log (H/He) = -1.7. Within the uncertainties, the metal-to-metal ratios are consistent with the accretion of an H2O-rich and rocky parent body, an interpretation supported by the anomalously high trace hydrogen. The mixed atmosphere yields unusually short diffusion time-scales for a helium atmosphere white dwarf, of no more than a few hundred years, and equivalent to those in a much cooler, hydrogen-rich star. The overall heavy element abundances of the disrupted parent body deviate modestly from a bulk Earth pattern, and suggest the deposition of some core-like material. The total inferred accretion rate is 4.2 × 109 g s-1, and at least four times higher than for any white dwarf with a comparable diffusion time-scale. Notably, when accretion is exhausted in this system, both metals and hydrogen will become undetectable within roughly 300 Myr, thus supporting a scenario where the trace hydrogen is related to the ongoing accretion of planetary debris.

  3. The Chemical Abundances of Stars in the Halo (CASH) Project. II. A Sample of 14 Extremely Metal-poor Stars

    NASA Astrophysics Data System (ADS)

    Hollek, Julie K.; Frebel, Anna; Roederer, Ian U.; Sneden, Christopher; Shetrone, Matthew; Beers, Timothy C.; Kang, Sung-ju; Thom, Christopher

    2011-11-01

    We present a comprehensive abundance analysis of 20 elements for 16 new low-metallicity stars from the Chemical Abundances of Stars in the Halo (CASH) project. The abundances have been derived from both Hobby-Eberly Telescope High Resolution Spectrograph snapshot spectra (R ~15, 000) and corresponding high-resolution (R ~35, 000) Magellan Inamori Kyocera Echelle spectra. The stars span a metallicity range from [Fe/H] from -2.9 to -3.9, including four new stars with [Fe/H] < -3.7. We find four stars to be carbon-enhanced metal-poor (CEMP) stars, confirming the trend of increasing [C/Fe] abundance ratios with decreasing metallicity. Two of these objects can be classified as CEMP-no stars, adding to the growing number of these objects at [Fe/H]< - 3. We also find four neutron-capture-enhanced stars in the sample, one of which has [Eu/Fe] of 0.8 with clear r-process signatures. These pilot sample stars are the most metal-poor ([Fe/H] <~ -3.0) of the brightest stars included in CASH and are used to calibrate a newly developed, automated stellar parameter and abundance determination pipeline. This code will be used for the entire ~500 star CASH snapshot sample. We find that the pipeline results are statistically identical for snapshot spectra when compared to a traditional, manual analysis from a high-resolution spectrum. Based on observations obtained with the Hobby-Eberly Telescope, which is a joint project of the University of Texas at Austin, the Pennsylvania State University, Stanford University, Ludwig-Maximilians-Universität München, and Georg-August-Universität Göttingen. Based on observations gathered with the 6.5 m Magellan Telescopes located at Las Campanas Observatory, Chile.

  4. Primary and Secondary siRNAs in Geminivirus-induced Gene Silencing

    PubMed Central

    Rajeswaran, Rajendran; Gubaeva, Ekaterina G.; Zvereva, Anna S.; Windels, David; Vazquez, Franck; Blevins, Todd; Farinelli, Laurent; Pooggin, Mikhail M.

    2012-01-01

    In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3′-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5′-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs. PMID:23028332

  5. Minimizing off-target effects by using diced siRNAs for RNA interference

    PubMed Central

    Myers, Jason W; Chi, Jen-Tsan; Gong, Delquin; Schaner, Marci E; Brown, Patrick O; Ferrell, James E

    2006-01-01

    Microarray studies have shown that individual synthetic small interfering RNAs (siRNAs) can have substantial off-target effects. Pools of siRNAs, produced by incubation of dsRNAs with recombinant Dicer or RNase III, can also be used to silence genes. Here we show that diced siRNA pools are highly complex, containing hundreds of different individual siRNAs. This high complexity could either compound the problem of off-target effects, since the number of potentially problematic siRNAs is high, or it could diminish the problem, since the concentration of any individual problematic siRNA is low. We therefore compared the off-target effects of diced siRNAs to chemically synthesized siRNAs. In agreement with previous reports, we found that two chemically synthesized siRNAs targeted against p38α MAPK (MAPK14) induced off-target changes in the abundance of hundreds of mRNAs. In contrast, three diced siRNA pools against p38α MAPK had almost no off-target effects. The off-target effects of a synthetic siRNA were reduced when the siRNA was diluted 3-fold in a diced pool and completely alleviated when it was diluted 30- or 300-fold, suggesting that when problematic siRNAs are present within a diced pool, their absolute concentration is too low to result in significant off-target effects. These data rationalize the observed high specificity of RNA interference in C. elegans and D. melanogaster, where gene suppression is mediated by endogenously-generated diced siRNA pools, and provide a strategy for improving the specificity of RNA interference experiments and screens in mammalian cells. PMID:19771225

  6. Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

    PubMed Central

    Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.

    2016-01-01

    We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

  7. Fundamental Physics with the Hubble Frontier Fields: Constraining Dark Matter Models with the Abundance of Extremely Faint and Distant Galaxies

    NASA Astrophysics Data System (ADS)

    Menci, N.; Merle, A.; Totzauer, M.; Schneider, A.; Grazian, A.; Castellano, M.; Sanchez, N. G.

    2017-02-01

    We show that the measured abundance of ultra-faint lensed galaxies at z≈ 6 in the Hubble Frontier Fields (HFF) provides stringent constraints on the parameter space of (i) dark matter models based on keV sterile neutrinos; (ii) “fuzzy” wavelike dark matter models, based on Bose–Einstein condensates of ultra-light particles. For the case of sterile neutrinos, we consider two production mechanisms: resonant production through mixing with active neutrinos and the decay of scalar particles. For the former model, we derive constraints for the combination of sterile neutrino mass {m}ν and mixing parameter {\\sin }2(2θ ) which provide the tightest lower bounds on the mixing angle (and hence on the lepton asymmetry) derived so far by methods independent of baryonic physics. For the latter we compute the allowed combinations of the scalar mass, its coupling to the Higgs field, and the Yukawa coupling of scalar to sterile neutrinos. We compare our results to independent existing astrophysical bounds on sterile neutrinos in the same mass range. For the case of “fuzzy” dark matter, we show that the observed number density ≈ 1/{{Mpc}}3 of high-redshift galaxies in the HFF sets a lower limit {m}\\psi ≥slant 8\\cdot {10}-22 eV (at the 3-σ confidence level) on the particle mass, a result that strongly disfavors wavelike bosonic dark matter as a viable model for structure formation. We discuss the impact on our results of uncertainties due to systematics in the selection of highly magnified, faint galaxies at high redshift.

  8. Low to Extremely Low Water Abundances Measured in Nominally Anhydrous Minerals in Mafic to Granitic Apollo Rock Clasts

    NASA Technical Reports Server (NTRS)

    Simon, J. I.; Christoffersen, R.; Wang, J.; Alexander, C. M. O'D.; Mills, R. D.; Hauri, E. H.

    2017-01-01

    Lunar sample-based volatile studies have focused on assessing the inventory and distribution of water in the Moon. Some have focused on the relatively young mare basalts and pyroclastic glasses, which result from partial melting of the relatively young lunar mantle. Less certain is the water inventory for the oldest materials available, which have the greater potential to record the earliest history of volatiles in the Moon (and thus provide evidence for the "wet" vs. "dry" accretion hypotheses of the Earth-Moon system. Studies of volatiles in ancient lunar rocks have largely focused on apatite. One recent FTIR (Fourier Transform Infrared Radiometer) study of plagioclase reported a relatively "wet" (approximately 320 parts per million) magma for primordial ferroan anorthosites (FANs). Another, a NanoSIMS study of alkali feldspar, reported a "wet" (approximately 1 weight percentage) felsic magma, but due to the differentiation processes required for silicic magmatism in the lunar crust, predicted an essentially "dry" (less than 100 parts per million) bulk Moon. Thus, despite evidence that appears to complicate the early "dry" Moon paradigm, there is no apparent unanimity among the measurements, even those on apatite. This disparity is clearly seen by the order of magnitude different water estimates for lunar "alkali-rich suite rocks" (Fig. 1). Some of the apparent differences may be explained by recent improvements in the apatite-based water estimates that better account for relative compatibilities of OH-, Cl, and F. In the present work, we seek to expand our understanding of the volatile abundances in early formed lunar magmas, their source reservoirs, and to address the potential role that felsic magmas play on the lunar hydrogen budget over time by employing NanoSIMS analysis of nominally anhydrous minerals.

  9. Strategies for in vivo delivery of siRNAs: recent progress.

    PubMed

    Higuchi, Yuriko; Kawakami, Shigeru; Hashida, Mitsuru

    2010-06-01

    RNA interference (RNAi) is a post-transcriptional gene-silencing mechanism that involves the degradation of messenger RNA in a highly sequence-specific manner. Double-stranded small interfering RNA (siRNA), consisting of 21-25 nucleotides, can induce RNAi and inhibit the expression of target proteins. Therefore, siRNA is considered a promising therapeutic for treatment of a variety of diseases, including genetic and viral diseases, and cancer. Clinical trials of siRNA are ongoing or have been planned, although some issues need to be addressed. For example, cellular uptake of naked siRNA is extremely low due to its polyanionic nature. Furthermore, siRNA is easily degraded by enzymes in blood, tissues, and cells. Several types of chemically modified siRNA have been produced and investigated to improve stability; these have involved modification of the siRNA backbone, the sugar moiety, and the nucleotide bases of antisense and/or sense strands. Because the accumulation at the target site after administration is extremely low, even if stability is improved, an effective delivery system is required to induce RNAi at the site of action. Delivery strategies can be categorized into physical methods, conjugation methods, and drug delivery system carrier-mediated methods. Physical techniques can enhance siRNA uptake at a specific tissue site using electroporation, pressure, mechanical massage, etc. Terminal modification of siRNAs can enhance their resistance to degradation by exonucleases in serum and tissue. Moreover, modification with a suitable ligand can achieve targeted delivery. Several types of carrier for drug delivery have been developed for siRNA in addition to traditional cationic liposome and cationic polymer systems. Ultrasound and microbubbles or liposomal bubbles have also been used in combination with a carrier for siRNA delivery. New materials with unique characteristics such as carbon nanotubes, gold nanoparticles, and gold nanorods have attracted attention

  10. Drawing siRNAs of viral origin out from plant siRNAs libraries.

    PubMed

    Miozzi, Laura; Pantaleo, Vitantonio

    2015-01-01

    Viruses are obligate intracellular entities that infect all forms of life. In plants, invading viral nucleic acids trigger RNA silencing machinery and it results in the accumulation of viral short interfering RNAs (v-siRNAs). The study of v-siRNAs population in biological samples has become a major part of many research projects aiming to identify viruses infecting them, including unknown viruses, even at extremely low titer. Currently, siRNA populations are investigated by high-throughput sequencing approaches, which generate very large data sets. The major difficulty in these studies is to properly analyze such huge amount of data. In this regard, easy-to-use bioinformatics tools to groom and decipher siRNA libraries and to draw out v-siRNAs are needed. Here we describe a workflow, which permit users with little experience in bioinformatics to draw out v-siRNAs from raw data sequences obtained by Illumina technology. Such pipeline has been released in the context of Galaxy, an open source Web-based platform for bioinformatics analyses.

  11. Engineering approaches in siRNA delivery.

    PubMed

    Barba, Anna Angela; Cascone, Sara; Caccavo, Diego; Lamberti, Gaetano; Chiarappa, Gianluca; Abrami, Michela; Grassi, Gabriele; Grassi, Mario; Tomaiuolo, Giovanna; Guido, Stefano; Brucato, Valerio; Carfì Pavia, Francesco; Ghersi, Giulio; La Carrubba, Vincenzo; Abbiati, Roberto Andrea; Manca, Davide

    2017-02-14

    siRNAs are very potent drug molecules, able to silence genes involved in pathologies development. siRNAs have virtually an unlimited therapeutic potential, particularly for the treatment of inflammatory diseases. However, their use in clinical practice is limited because of their unfavorable properties to interact and not to degrade in physiological environments. In particular they are large macromolecules, negatively charged, which undergo rapid degradation by plasmatic enzymes, are subject to fast renal clearance/hepatic sequestration, and can hardly cross cellular membranes. These aspects seriously impair siRNAs as therapeutics. As in all the other fields of science, siRNAs management can be advantaged by physical-mathematical descriptions (modeling) in order to clarify the involved phenomena from the preparative step of dosage systems to the description of drug-body interactions, which allows improving the design of delivery systems/processes/therapies. This review analyzes a few mathematical modeling approaches currently adopted to describe the siRNAs delivery, the main procedures in siRNAs vectors' production processes and siRNAs vectors' release from hydrogels, and the modeling of pharmacokinetics of siRNAs vectors. Furthermore, the use of physical models to study the siRNAs vectors' fate in blood stream and in the tissues is presented. The general view depicts a framework maybe not yet usable in therapeutics, but with promising possibilities for forthcoming applications.

  12. SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity

    SciTech Connect

    Kubo, Takanori; Yanagihara, Kazuyoshi; Takei, Yoshifumi; Mihara, Keichiro; Sato, Yuichiro; Seyama, Toshio

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.

  13. Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium Caldicellulosiruptor obsidiansis Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

    SciTech Connect

    Giannone, Richard J; Lochner, Adriane; Keller, Martin; Antranikian, Garabed; Graham, David E; Hettich, Robert {Bob} L

    2011-01-01

    Mass spectrometric analysis of Caldicellulosiruptor obsidiansis cultures grown on four different carbon sources identified 65% of the cells predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47-0549 together with the COB47-1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47-0096 and COB47-0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 {beta}-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When C. obsidiansis was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation.

  14. Heavy metals in a light white dwarf: abundances of the metal-rich, extremely low-mass GALEX J1717+6757

    NASA Astrophysics Data System (ADS)

    Hermes, J. J.; Gänsicke, B. T.; Koester, D.; Bours, M. C. P.; Townsley, D. M.; Farihi, J.; Marsh, T. R.; Littlefair, Stuart; Dhillon, V. S.; Gianninas, A.; Breedt, E.; Raddi, R.

    2014-10-01

    Using the Hubble Space Telescope, we detail the first abundance analysis enabled by far-ultraviolet spectroscopy of a low-mass (≃0.19 M⊙) white dwarf (WD), GALEX J1717+6757, which is in a 5.9-h binary with a fainter, more-massive companion. We see absorption from nine metals, including roughly solar abundances of Ca, Fe, Ti, and P. We detect a significantly sub-solar abundance of C, and put upper limits on N and O that are also markedly sub-solar. Updated diffusion calculations indicate that all metals should settle out of the atmosphere of this 14 900 K, log g = 5.67 WD in the absence of radiative forces in less than 20 yr, orders of magnitude faster than the cooling age of hundreds of Myr. We demonstrate that ongoing accretion of rocky material that is often the cause of atmospheric metals in isolated, more massive WDs is unlikely to explain the observed abundances in GALEX J1717+6757. Using new radiative levitation calculations, we determine that radiative forces can counteract diffusion and support many but not all of the elements present in the atmosphere of this WD; radiative levitation cannot, on its own, explain all of the observed abundance patterns, and additional mechanisms such as rotational mixing may be required. Finally, we detect both primary and secondary eclipses using ULTRACAM high-speed photometry, which we use to constrain the low-mass WD radius and rotation rate as well as update the ephemeris from the discovery observations of this WD+WD binary.

  15. Distinct and concurrent pathways of Pol II- and Pol IV-dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.

    PubMed

    Sasaki, Taku; Lee, Tzuu-fen; Liao, Wen-Wei; Naumann, Ulf; Liao, Jo-Ling; Eun, Changho; Huang, Ya-Yi; Fu, Jason L; Chen, Pao-Yang; Meyers, Blake C; Matzke, Antonius J M; Matzke, Marjori

    2014-07-01

    Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II (Pol II)- and Pol IV-dependent pathways of siRNA biogenesis. At the original silencer locus, Pol-II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant siRNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template.

  16. VARIABLE AND EXTREME IRRADIATION CONDITIONS IN THE EARLY SOLAR SYSTEM INFERRED FROM THE INITIAL ABUNDANCE OF {sup 10}Be IN ISHEYEVO CAIs

    SciTech Connect

    Gounelle, Matthieu; Chaussidon, Marc; Rollion-Bard, Claire

    2013-02-01

    A search for short-lived {sup 10}Be in 21 calcium-aluminum-rich inclusions (CAIs) from Isheyevo, a rare CB/CH chondrite, showed that only 5 CAIs had {sup 10}B/{sup 11}B ratios higher than chondritic correlating with the elemental ratio {sup 9}Be/{sup 11}B, suggestive of in situ decay of this key short-lived radionuclide. The initial ({sup 10}Be/{sup 9}Be){sub 0} ratios vary between {approx}10{sup -3} and {approx}10{sup -2} for CAI 411. The initial ratio of CAI 411 is one order of magnitude higher than the highest ratio found in CV3 CAIs, suggesting that the more likely origin of CAI 411 {sup 10}Be is early solar system irradiation. The low ({sup 26}Al/{sup 27}Al){sub 0} [{<=} 8.9 Multiplication-Sign 10{sup -7}] with which CAI 411 formed indicates that it was exposed to gradual flares with a proton fluence of a few 10{sup 19} protons cm{sup -2}, during the earliest phases of the solar system, possibly the infrared class 0. The irradiation conditions for other CAIs are less well constrained, with calculated fluences ranging between a few 10{sup 19} and 10{sup 20} protons cm{sup -2}. The variable and extreme value of the initial {sup 10}Be/{sup 9}Be ratios in carbonaceous chondrite CAIs is the reflection of the variable and extreme magnetic activity in young stars observed in the X-ray domain.

  17. Recent advances in siRNA delivery.

    PubMed

    Sarisozen, Can; Salzano, Giuseppina; Torchilin, Vladimir P

    2015-12-01

    In the 1990s an unexpected gene-silencing phenomena in plants, the later called RNA interference (RNAi), perplexed scientists. Following the proof of activity in mammalian cells, small interfering RNAs (siRNAs) have quickly crept into biomedical research as a new powerful tool for the potential treatment of different human diseases based on altered gene expression. In the past decades, several promising data from ongoing clinical trials have been reported. However, despite surprising successes in many pre-clinical studies, concrete obstacles still need to be overcome to translate therapeutic siRNAs into clinical reality. Here, we provide an update on the recent advances of RNAi-based therapeutics and highlight novel synthetic platforms for the intracellular delivery of siRNAs.

  18. DNA repair investigations using siRNA.

    PubMed

    Miller, Holly; Grollman, Arthur P

    2003-06-11

    Small interfering RNA (siRNA) is a revolutionary tool for the experimental modulation of gene expression, in many cases making redundant the need for specific gene mutations and allowing examination of the effect of modulating essential genes. It has now been shown that siRNA phenotypes resulting from stable transfection with short hairpin RNA (shRNA) can be transmitted through the mouse germ line and Rosenquist and his colleagues have used shRNA, which is processed in vivo to siRNA, to create germline transgenic mice in which a target DNA repair gene has been silenced. Here, Holly Miller and Arthur P. Grollman give the background of these discoveries, provide an overview of current uses, and look at future applications of this research.

  19. Abundances in Przybylski's star

    NASA Astrophysics Data System (ADS)

    Cowley, C. R.; Ryabchikova, T.; Kupka, F.; Bord, D. J.; Mathys, G.; Bidelman, W. P.

    2000-09-01

    We have derived abundances for 54 elements in the extreme roAp star HD101065. ESO spectra with a resolution of about 80000, and S/N of 200 or more were employed. The adopted model has Teff=6600K, and log(g)=4.2. Because of the increased line opacity and consequent low gas pressure, convection plays no significant role in the temperature structure. Lighter elemental abundances through the iron group scatter about standard abundance distribution (SAD) (solar) values. Iron and nickel are about one order of magnitude deficient while cobalt is enhanced by 1.5dex. Heavier elements, including the lanthanides, generally follow the solar pattern but enhanced by 3 to 4dex. Odd-Z elements are generally less abundant than their even-Z neighbours. With a few exceptions (e.g. Yb), the abundance pattern among the heavy elements is remarkably coherent, and resembles a displaced solar distribution.

  20. Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system.

    PubMed

    Sardo, C; Farra, R; Licciardi, M; Dapas, B; Scialabba, C; Giammona, G; Grassi, M; Grassi, G; Cavallaro, G

    2015-07-30

    Small interfering RNAs (siRNAs) have the potential to be of therapeutic value for many human diseases. So far, however, a serious obstacle to their therapeutic use is represented by the absence of appropriate delivery systems able to protect them from degradation and to allow an efficient cellular uptake. In this work we developed a siRNA delivery system based on inulin (Inu), an abundant and natural polysaccharide. Inu was functionalized via the conjugation with diethylenetriamine (DETA) residues to form the complex Inu-DETA. We studied the size, surface charge and the shape of the Inu-DETA/siRNA complexes; additionally, the cytotoxicity, the silencing efficacy and the cell uptake-mechanisms were studied in the human bronchial epithelial cells (16HBE) and in the hepatocellular carcinoma derived cells (JHH6). The results presented here indicate that Inu-DETA copolymers can effectively bind siRNAs, are highly cytocompatible and, in JHH6, can effectively deliver functional siRNAs. Optimal delivery is observed using a weight ratio Inu-DETA/siRNA of 4 that corresponds to polyplexes with an average size of 600nm and a slightly negative surface charge. Moreover, the uptake and trafficking mechanisms, mainly based on micropinocytosis and clatrin mediated endocytosis, allow the homogeneous diffusion of siRNA within the cytoplasm of JHH6. Notably, in 16 HBE where the trafficking mechanism (caveolae mediated endocytosis) does not allow an even distribution of siRNA within the cell cytoplasm, no significant siRNA activity is observed. In conclusion, we developed a novel inulin-based siRNA delivery system able to efficiently release siRNA in JHH6 with negligible cytotoxicity thus opening the way for further testing in more complex in vivo models.

  1. siRNA for Influenza Therapy

    PubMed Central

    Barik, Sailen

    2010-01-01

    Influenza virus is one of the most prevalent and ancient infections in humans. About a fifth of world’s population is infected by influenza virus annually, leading to high morbidity and mortality, particularly in infants, the elderly and the immunocompromised. In the US alone, influenza outbreaks lead to roughly 30,000 deaths each year. Current vaccines and anti-influenza drugs are of limited use due to high mutation rate of the virus and side effects. In recent years, RNA interference, triggered by synthetic short interfering RNA (siRNA), has rapidly evolved as a potent antiviral regimen. Properly designed siRNAs have been shown to function as potent inhibitors of influenza virus replication. The siRNAs outperform traditional small molecule antivirals in a number of areas, such as ease of design, modest cost, and fast turnaround. Although specificity and tissue delivery remain major bottlenecks in the clinical applications of RNAi in general, intranasal application of siRNA against respiratory viruses including, but not limited to influenza virus, has experienced significant success and optimism, which is reviewed here. PMID:21994689

  2. Bioengineered nanoparticles for siRNA delivery.

    PubMed

    Kozielski, Kristen L; Tzeng, Stephany Y; Green, Jordan J

    2013-01-01

    Short interfering RNA (siRNA) has been an important laboratory tool in the last two decades and has allowed researchers to better understand the functions of nonprotein-coding genes through RNA interference (RNAi). Although RNAi holds great promise for this purpose as well as for treatment of many diseases, efforts at using siRNA have been hampered by the difficulty of safely and effectively introducing it into cells of interest, both in vitro and in vivo. To overcome this challenge, many biomaterials and nanoparticles (NPs) have been developed and optimized for siRNA delivery, often taking cues from the DNA delivery field, although different barriers exist for these two types of molecules. In this review, we discuss general properties of biomaterials and nanoparticles that are necessary for effective nucleic acid delivery. We also discuss specific examples of bioengineered materials, including lipid-based NPs, polymeric NPs, inorganic NPs, and RNA-based NPs, which clearly illustrate the problems and successes in siRNA delivery.

  3. Bioengineered Nanoparticles for siRNA delivery

    PubMed Central

    Kozielski, Kristen L.; Tzeng, Stephany Y.; Green, Jordan J.

    2014-01-01

    Short interfering RNA (siRNA) has been an important laboratory tool in the last two decades and has allowed researchers to better understand the functions of non-protein-coding genes through RNA interference (RNAi). Although RNAi holds great promise for this purpose as well as for treatment of many diseases, efforts at using siRNA have been hampered by the difficulty of safely and effectively introducing it into cells of interest, both in vitro and in vivo. To overcome this challenge, many biomaterials and nanoparticles (NPs) have been developed and optimized for siRNA delivery, often taking cues from the DNA delivery field, although different barriers exist for these two types of molecules. In this review, we discuss general properties of biomaterials and nanoparticles that are necessary for effective nucleic acid delivery. We also discuss specific examples of bioengineered materials, including lipid-based NPs, polymeric NPs, inorganic NPs, and RNA-based NPs, which clearly illustrate the problems and successes in siRNA delivery. PMID:23821336

  4. Dendrimers for siRNA Delivery

    PubMed Central

    Biswas, Swati; Torchilin, Vladimir P.

    2013-01-01

    Since the discovery of the “starburst polymer”, later renamed as dendrimer, this class of polymers has gained considerable attention for numerous biomedical applications, due mainly to the unique characteristics of this macromolecule, including its monodispersity, uniformity, and the presence of numerous functionalizable terminal groups. In recent years, dendrimers have been studied extensively for their potential application as carriers for nucleic acid therapeutics, which utilize the cationic charge of the dendrimers for effective dendrimer-nucleic acid condensation. siRNA is considered a promising, versatile tool among various RNAi-based therapeutics, which can effectively regulate gene expression if delivered successfully inside the cells. This review reports on the advancements in the development of dendrimers as siRNA carriers. PMID:24275946

  5. De Novo Reconstruction of Consensus Master Genomes of Plant RNA and DNA Viruses from siRNAs

    PubMed Central

    Seguin, Jonathan; Rajeswaran, Rajendran; Malpica-López, Nachelli; Martin, Robert R.; Kasschau, Kristin; Dolja, Valerian V.; Otten, Patricia; Farinelli, Laurent; Pooggin, Mikhail M.

    2014-01-01

    Virus-infected plants accumulate abundant, 21–24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this ‘siRNA omics’ approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense. PMID:24523907

  6. Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis.

    PubMed

    Zhong, Si-Hui; Liu, Jun-Zhong; Jin, Hua; Lin, Lin; Li, Qun; Chen, Ying; Yuan, Yue-Xing; Wang, Zhi-Yong; Huang, Hai; Qi, Yi-Jun; Chen, Xiao-Ya; Vaucheret, Hervé; Chory, Joanne; Li, Jianming; He, Zu-Hua

    2013-05-28

    Owing to their sessile nature, plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly and reversibly to daily and seasonal temperature changes. However, our knowledge of how plants sense and respond to warming ambient temperatures is rather limited. Here we show that an increase in growth temperature from 22 °C to 30 °C effectively inhibited transgene-induced posttranscriptional gene silencing (PTGS) in Arabidopsis. Interestingly, warmth-induced PTGS release exhibited transgenerational epigenetic inheritance. We discovered that the warmth-induced PTGS release occurred during a critical step that leads to the formation of double-stranded RNA (dsRNA) for producing small interfering RNAs (siRNAs). Deep sequencing of small RNAs and RNA blot analysis indicated that the 22-30 °C increase resulted in a significant reduction in the abundance of many trans-acting siRNAs that require dsRNA for biogenesis. We discovered that the temperature increase reduced the protein abundance of SUPPRESSOR OF GENE SILENCING 3, as a consequence, attenuating the formation of stable dsRNAs required for siRNA biogenesis. Importantly, SUPPRESSOR OF GENE SILENCING 3 overexpression released the warmth-triggered inhibition of siRNA biogenesis and reduced the transgenerational epigenetic memory. Thus, our study reveals a previously undescribed association between warming temperatures, an epigenetic system, and siRNA biogenesis.

  7. Alkane-modified short polyethyleneimine for siRNA delivery.

    PubMed

    Schroeder, Avi; Dahlman, James E; Sahay, Gaurav; Love, Kevin T; Jiang, Shan; Eltoukhy, Ahmed A; Levins, Christopher G; Wang, Yingxia; Anderson, Daniel G

    2012-06-10

    RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer-lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.

  8. Predicting siRNA efficacy based on multiple selective siRNA representations and their combination at score level

    NASA Astrophysics Data System (ADS)

    He, Fei; Han, Ye; Gong, Jianting; Song, Jiazhi; Wang, Han; Li, Yanwen

    2017-03-01

    Small interfering RNAs (siRNAs) may induce to targeted gene knockdown, and the gene silencing effectiveness relies on the efficacy of the siRNA. Therefore, the task of this paper is to construct an effective siRNA prediction method. In our work, we try to describe siRNA from both quantitative and qualitative aspects. For quantitative analyses, we form four groups of effective features, including nucleotide frequencies, thermodynamic stability profile, thermodynamic of siRNA-mRNA interaction, and mRNA related features, as a new mixed representation, in which thermodynamic of siRNA-mRNA interaction is introduced to siRNA efficacy prediction for the first time to our best knowledge. And then an F-score based feature selection is employed to investigate the contribution of each feature and remove the weak relevant features. Meanwhile, we encode the siRNA sequence and existed empirical design rules as a qualitative siRNA representation. These two kinds of siRNA representations are combined to predict siRNA efficacy by supported Vector Regression (SVR) at score level. The experimental results indicate that our method may select the features with powerful discriminative ability and make the two kinds of siRNA representations work at full capacity. The prediction results also demonstrate that our method can outperform other popular siRNA efficacy prediction algorithms.

  9. Predicting siRNA efficacy based on multiple selective siRNA representations and their combination at score level

    PubMed Central

    He, Fei; Han, Ye; Gong, Jianting; Song, Jiazhi; Wang, Han; Li, Yanwen

    2017-01-01

    Small interfering RNAs (siRNAs) may induce to targeted gene knockdown, and the gene silencing effectiveness relies on the efficacy of the siRNA. Therefore, the task of this paper is to construct an effective siRNA prediction method. In our work, we try to describe siRNA from both quantitative and qualitative aspects. For quantitative analyses, we form four groups of effective features, including nucleotide frequencies, thermodynamic stability profile, thermodynamic of siRNA-mRNA interaction, and mRNA related features, as a new mixed representation, in which thermodynamic of siRNA-mRNA interaction is introduced to siRNA efficacy prediction for the first time to our best knowledge. And then an F-score based feature selection is employed to investigate the contribution of each feature and remove the weak relevant features. Meanwhile, we encode the siRNA sequence and existed empirical design rules as a qualitative siRNA representation. These two kinds of siRNA representations are combined to predict siRNA efficacy by supported Vector Regression (SVR) at score level. The experimental results indicate that our method may select the features with powerful discriminative ability and make the two kinds of siRNA representations work at full capacity. The prediction results also demonstrate that our method can outperform other popular siRNA efficacy prediction algorithms. PMID:28317874

  10. Simple gene silencing using the trans-acting siRNA pathway.

    PubMed

    Jacobs, Thomas B; Lawler, Noah J; LaFayette, Peter R; Vodkin, Lila O; Parrott, Wayne A

    2016-01-01

    In plants, particular micro-RNAs (miRNAs) induce the production of a class of small interfering RNAs (siRNA) called trans-acting siRNA (ta-siRNA) that lead to gene silencing. A single miRNA target is sufficient for the production of ta-siRNAs, which target can be incorporated into a vector to induce the production of siRNAs, and ultimately gene silencing. The term miRNA-induced gene silencing (MIGS) has been used to describe such vector systems in Arabidopsis. Several ta-siRNA loci have been identified in soybean, but, prior to this work, few of the inducing miRNAs have been experimentally validated, much less used to silence genes. Nine ta-siRNA loci and their respective miRNA targets were identified, and the abundance of the inducing miRNAs varies dramatically in different tissues. The miRNA targets were experimentally verified by silencing a transgenic GFP gene and two endogenous genes in hairy roots and transgenic plants. Small RNAs were produced in patterns consistent with the utilization of the ta-siRNA pathway. A side-by-side experiment demonstrated that MIGS is as effective at inducing gene silencing as traditional hairpin vectors in soybean hairy roots. Soybean plants transformed with MIGS vectors produced siRNAs and silencing was observed in the T1 generation. These results complement previous reports in Arabidopsis by demonstrating that MIGS is an efficient way to produce siRNAs and induce gene silencing in other species, as shown with soybean. The miRNA targets identified here are simple to incorporate into silencing vectors and offer an effective and efficient alternative to other gene silencing strategies.

  11. In-depth sequencing of the siRNAs associated with peach latent mosaic viroid infection

    PubMed Central

    2010-01-01

    Background It has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd), a model RNA genome, the regions that are most susceptible to RNA interference machinery. Results The RNA isolated from an infected tree have been used to sequence in parallel viroid species and small non-coding RNA species. Specifically, PLMVd RNAs were amplified, cloned and sequenced according to a conventional approach, while small non-coding RNAs were determined by high-throughput sequencing. The first led to the typing of 18 novel PLMVd variants. The second provided a library of small RNAs including 880 000 sequences corresponding to PLMVd-derived siRNAs, which makes up 11.2% of the sequences of the infected library. These siRNAs contain mainly 21-22 nucleotide RNAs and are equivalently distributed between the plus and the minus polarities of the viroid. They cover the complete viroid genome, although the amount varies depending on the regions. These regions do not necessarily correlate with the double-stranded requirement to be a substrate for Dicer-like enzymes. We noted that some sequences encompass the hammerhead self-cleavage site, indicating that the circular conformers could be processed by the RNA-silencing machinery. Finally, a bias in the relative abundance of the nature of the 5' nucleotides was observed (A, U >> G, C). Conclusions The approach used provided us a quantitative representation of the PLMVd-derived siRNAs retrieved from infected peach trees. These siRNAs account for a relatively large proportion of the small non-coding RNAs. Surprisingly, the siRNAs from some regions of the PLMVd genome appear over-represented, although these

  12. siRNA and cancer immunotherapy.

    PubMed

    Ghafouri-Fard, Soudeh; Ghafouri-Fard, Somayyeh

    2012-09-01

    Immunotherapeutic approaches have been gaining attention in the field of cancer treatment because of their possible ability to eradicate cancer cells as well as metastases by recruiting the host immune system. On the other hand, RNA-based therapeutics with the ability to silence expression of specific targets are currently under clinical investigation for various disorders including cancer. As the mechanisms of tumor evasion from the host immune system are versatile, different molecules have the capacity to be targeted by RNAi technology in order to enhance the immune response against tumors. This technology has been used to silence specific targets in tumor cells, as well as immune cells in cancer cell lines, animal models and clinical trials. siRNAs can also stimulate innate immune responses through activation of Toll-like receptors. Although currently clinical trials of the application of siRNA in cancer immunotherapy are few, it is predicted that in future this technology will be used broadly in cancer treatment.

  13. Dendrimeric siRNA for Efficient Gene Silencing.

    PubMed

    Hong, Cheol Am; Eltoukhy, Ahmed A; Lee, Hyukjin; Langer, Robert; Anderson, Daniel G; Nam, Yoon Sung

    2015-06-01

    Programmable molecular self-assembly of siRNA molecules provides precisely controlled generation of dendrimeric siRNA nanostructures. The second-generation dendrimers of siRNA can be effectively complexed with a low-molecular-weight, cationic polymer (poly(β-amino ester), PBAE) to generate stable nanostructures about 160 nm in diameter via strong electrostatic interactions. Condensation and gene silencing efficiencies increase with the increased generation of siRNA dendrimers due to a high charge density and structural flexibility.

  14. How extreme are extremes?

    NASA Astrophysics Data System (ADS)

    Cucchi, Marco; Petitta, Marcello; Calmanti, Sandro

    2016-04-01

    High temperatures have an impact on the energy balance of any living organism and on the operational capabilities of critical infrastructures. Heat-wave indicators have been mainly developed with the aim of capturing the potential impacts on specific sectors (agriculture, health, wildfires, transport, power generation and distribution). However, the ability to capture the occurrence of extreme temperature events is an essential property of a multi-hazard extreme climate indicator. Aim of this study is to develop a standardized heat-wave indicator, that can be combined with other indices in order to describe multiple hazards in a single indicator. The proposed approach can be used in order to have a quantified indicator of the strenght of a certain extreme. As a matter of fact, extremes are usually distributed in exponential or exponential-exponential functions and it is difficult to quickly asses how strong was an extreme events considering only its magnitude. The proposed approach simplify the quantitative and qualitative communication of extreme magnitude

  15. Bone site-specific delivery of siRNA

    PubMed Central

    Liu, Xinli

    2016-01-01

    Abstract Small interfering RNAs (siRNA) have enormous potential as therapeutics to target and treat various bone disorders such as osteoporosis and cancer bone metastases. However, effective and specific delivery of siRNA therapeutics to bone and bone-specific cells in vivo is very challenging. To realize the full therapeutic potential of siRNA in treating bone disorders, a safe and efficient, tissue- and cell-specific delivery system must be developed. This review focuses on recent advances in bone site-specific delivery of siRNA at the tissue or cellular level. Bone-targeted nanoparticulate siRNA carriers and various bone-targeted moieties such as bisphosphonates, oligopeptides (Asp)8 and (AspSerSer)6, and aptamers are highlighted. Incorporation of these bone-seeking targeting moieties into siRNA carriers allows for recognition of different sub-tissue functional domains of bone and also specific cell types residing in bone tissue. It also provides a means for bone-formation surface-, bone-resorption surface-, or osteoblast-specific targeting and transportation of siRNA therapeutics. The discussion mainly focuses on systemic and local bone-specific delivery of siRNA in osteoporosis and bone metastasis preclinical models. PMID:26642236

  16. Nonviral delivery of synthetic siRNAs in vivo

    PubMed Central

    Akhtar, Saghir; Benter, Ibrahim F.

    2007-01-01

    Sequence-specific gene silencing using small interfering RNA (siRNA) is a Nobel prize–winning technology that is now being evaluated in clinical trials as a potentially novel therapeutic strategy. This article provides an overview of the major pharmaceutical challenges facing siRNA therapeutics, focusing on the delivery strategies for synthetic siRNA duplexes in vivo, as this remains one of the most important issues to be resolved. This article also highlights the importance of understanding the genocompatibility/toxicogenomics of siRNA delivery reagents in terms of their impact on gene-silencing activity and specificity. Collectively, this information is essential for the selection of optimally acting siRNA delivery system combinations for the many proposed applications of RNA interference. PMID:18060020

  17. Effect of Surface Properties on Liposomal siRNA Delivery

    PubMed Central

    Xia, Yuqiong; Tian, Jie; Chen, Xiaoyuan

    2015-01-01

    Liposomes are one of the most widely investigated carriers for siRNA delivery. The surface properties of liposomal carriers, including the surface charge, PEGylation, and ligand modification can significantly affect the gene silencing efficiency. Three barriers of systemic siRNA delivery (long blood circulation, efficient tumor penetration and efficient cellular uptake/endosomal escape) are analyzed on liposomal carriers with different surface charges, PEGylations and ligand modifications. Cationic formulations dominate siRNA delivery and neutral formulations also have good performance while anionic formulations are generally not proper for siRNA delivery. The PEG dilemma (prolonged blood circulation vs. reduced cellular uptake/endosomal escape) and the side effect of repeated PEGylated formulation (accelerated blood clearance) were discussed. Effects of ligand modification on cationic and neutral formulations were analyzed. Finally, we summarized the achievements in liposomal siRNA delivery, outlined existing problems and provided some future perspectives. PMID:26695117

  18. Experimental validation of the importance of seed complement frequency to siRNA specificity.

    PubMed

    Anderson, Emily M; Birmingham, Amanda; Baskerville, Scott; Reynolds, Angela; Maksimova, Elena; Leake, Devin; Fedorov, Yuriy; Karpilow, Jon; Khvorova, Anastasia

    2008-05-01

    Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2-7) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3' UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3' UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.

  19. Bio-inspired pulmonary surfactant-modified nanogels: A promising siRNA delivery system.

    PubMed

    De Backer, Lynn; Braeckmans, Kevin; Stuart, Marc C A; Demeester, Jo; De Smedt, Stefaan C; Raemdonck, Koen

    2015-05-28

    Inhalation therapy with small interfering RNA (siRNA) is a promising approach in the treatment of pulmonary disorders. However, clinical translation is severely limited by the lack of suitable delivery platforms. In this study, we aim to address this limitation by designing a novel bioinspired hybrid nanoparticle with a core-shell nanoarchitecture, consisting of a siRNA-loaded dextran nanogel (siNG) core and a pulmonary surfactant (Curosurf®) outer shell. The decoration of siNGs with a surfactant shell enhances the colloidal stability and prevents siRNA release in the presence of competing polyanions, which are abundantly present in biofluids. Additionally, the impact of the surfactant shell on the biological efficacy of the siNGs is determined in lung cancer cells. The presence of the surfactants substantially reduces the cellular uptake of siNGs. Remarkably, the lowered intracellular dose does not impede the gene silencing effect, suggesting a crucial role of the pulmonary surfactant in the intracellular processing of the nanoparticles. In order to surmount the observed reduction in cellular dose, folate is incorporated as a targeting ligand in the pulmonary surfactant shell to incite receptor-mediated endocytosis. The latter substantially enhances both cellular uptake and gene silencing potential, achieving efficient knockdown at siRNA concentrations in the low nanomolar range.

  20. Small-interfering RNAs (siRNAs) as a promising tool for ocular therapy

    PubMed Central

    Guzman-Aranguez, A; Loma, P; Pintor, J

    2013-01-01

    RNA interference (RNAi) can be used to inhibit the expression of specific genes in vitro and in vivo, thereby providing an extremely useful tool for investigating gene function. Progress in the understanding of RNAi-based mechanisms has opened up new perspectives in therapeutics for the treatment of several diseases including ocular disorders. The eye is currently considered a good target for RNAi therapy mainly because it is a confined compartment and, therefore, enables local delivery of small-interfering RNAs (siRNAs) by topical instillation or direct injection. However, delivery strategies that protect the siRNAs from degradation and are suitable for long-term treatment would be help to improve the efficacy of RNAi-based therapies for ocular pathologies. siRNAs targeting critical molecules involved in the pathogenesis of glaucoma, retinitis pigmentosa and neovascular eye diseases (age-related macular degeneration, diabetic retinopathy and corneal neovascularization) have been tested in experimental animal models, and clinical trials have been conducted with some of them. This review provides an update on the progress of RNAi in ocular therapeutics, discussing the advantages and drawbacks of RNAi-based therapeutics compared to previous treatments. PMID:23937539

  1. Repression of multiple CYP2D genes in mouse primary hepatocytes with a single siRNA construct.

    PubMed

    Elraghy, Omaima; Baldwin, William S

    2015-01-01

    The Cyp2d subfamily is the second most abun-dant subfamily of hepatic drug-metabolizing CYPs. In mice, there are nine Cyp2d members that are believed to have redundant catalytic activity. We are testing and optimizing the ability of one short interfering RNA (siRNA) construct to knockdown the expression of multiple mouse Cyp2ds in primary hepatocytes. Expression of Cyp2d10, Cyp2d11, Cyp2d22, and Cyp2d26 was observed in the primary male mouse hepatocytes. Cyp2d9, which is male-specific and growth hormone-dependent, was not expressed in male primary hepatocytes, potentially because of its dependence on pulsatile growth hormone release from the anterior pituitary. Several different siRNAs at different concentrations and with different reagents were used to knockdown Cyp2d expression. siRNA constructs designed to repress only one construct often mildly repressed several Cyp2d isoforms. A construct designed to knockdown every Cyp2d isoform provided the best results, especially when incubated with transfection reagents designed specifically for primary cell culture. Interestingly, a construct designed to knockdown all Cyp2d isoforms, except Cyp2d10, caused a 2.5× increase in Cyp2d10 expression, presumably because of a compensatory response. However, while RNA expression is repressed 24 h after siRNA treatment, associated changes in Cyp2d-mediated metabolism are tenuous. Overall, this study provides data on the expression of murine Cyp2ds in primary cell lines, valuable information on designing siRNAs for silencing multiple murine CYPs, and potential pros and cons of using siRNA as a tool for repressing Cyp2d and estimating Cyp2d's role in murine xenobiotic metabolism.

  2. Chemical and structural diversity of siRNA molecules.

    PubMed

    Nawrot, Barbara; Sipa, Katarzyna

    2006-01-01

    Short interfering RNAs (siRNAs) are 21-23 nt long double-stranded oligoribonucleotides which in mammalian cells exhibit a potency for sequence-specific gene silencing via an RNA interference (RNAi) pathway. It has been already proven that exogenous, chemically synthesized siRNA molecules are effective inhibitors of gene expression and are widely applied for analysis of protein function and proteomics-based target identification. Moreover, since their discovery siRNA molecules have been implemented as potential candidates for therapeutic applications. Variously modified siRNA molecules containing sugar modifications (2'-OMe, -F, -O-allyl, -amino, orthoesters and LNA analogues), internucleotide phospodiester bond modifications (phosphorothioates, boranophosphates), base modifications (s(2)U) as well as 3'-terminal cholesterol-conjugated constructs were investigated as potential candidates for effective inhibition of gene expression. This chapter reviews an impact of chemical and structural modifications of siRNA molecules on their serum and thermal stability, cellular and in vivo activity, cellular uptake, biodistribution and cytotoxicity. Functional analysis of chemically modified siRNA molecules allows for better understanding of the mechanism of the RNA interference process as well as demonstrates immense efforts in optimizing in vivo potency of siRNA molecules for RNAi-based drug design.

  3. Strategies for siRNA navigation to desired cells.

    PubMed

    Sioud, Mouldy

    2015-01-01

    Whilst small interfering (si) RNAs have emerged as a promising therapeutic modality for treating a diversity of human diseases, delivery constitutes the most serious obstacle to siRNA drug development. As the most used delivery agents can enter all cell types, specificity must be built into the delivery agents or directly attached to the siRNA molecules. The use of antibodies, peptides, Peptide-Fc fusions, aptamers, and other targeting ligands has now enabled efficient gene silencing in the desired cell populations/tissues in vitro and in vivo. The present review summarizes these current innovations, which are important for the design of safe therapeutic siRNAs.

  4. Non-viral Methods for siRNA Delivery

    PubMed Central

    Gao, Kun; Huang, Leaf

    2009-01-01

    RNA interference (RNAi) as a mechanism to selectively degrade messenger RNA (mRNA) expression has emerged as a potential novel approach for drug target validation and the study of functional genomics. Small interfering RNAs (siRNA) therapeutics has developed rapidly and already there are clinical trials ongoing or planned. Although other challenges remain, delivery strategies for siRNA become the main hurdle that must be resolved prior to the full-scale clinical development of siRNA therapeutics. This article provides an overview of the current delivery strategies for synthetic siRNA, focusing on the targeted, self-assembled nanoparticles which show potential to become a useful and efficient tool in cancer therapy. PMID:19115957

  5. Chaperonin–Dendrimer Conjugates for siRNA Delivery

    PubMed Central

    Nussbaumer, Martin G.; Duskey, Jason T.; Rother, Martin; Renggli, Kasper; Chami, Mohamed

    2016-01-01

    The group II chaperonin thermosome (THS) is a hollow protein nanoparticle that can encapsulate macromolecular guests. Two large pores grant access to the interior of the protein cage. Poly(amidoamine) (PAMAM) is conjugated into THS to act as an anchor for small interfering RNA (siRNA), allowing to load the THS with therapeutic payload. THS–PAMAM protects siRNA from degradation by RNase A and traffics KIF11 and GAPDH siRNA into U87 cancer cells. By modification of the protein cage with the cell‐penetrating peptide TAT, RNA interference is also induced in PC‐3 cells. THS–PAMAM protein–polymer conjugates are therefore promising siRNA transfection reagents and greatly expand the scope of protein cages in drug delivery applications. PMID:27840795

  6. Generation of siRNA Nanosheets for Efficient RNA Interference

    NASA Astrophysics Data System (ADS)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  7. Characterization of viral siRNA populations in honey bee colony collapse disorder.

    PubMed

    Chejanovsky, Nor; Ophir, Ron; Schwager, Michal Sharabi; Slabezki, Yossi; Grossman, Smadar; Cox-Foster, Diana

    2014-04-01

    Colony Collapse Disorder (CCD), a special case of collapse of honey bee colonies, has resulted in significant losses for beekeepers. CCD-colonies show abundance of pathogens which suggests that they have a weakened immune system. Since honey bee viruses are major players in colony collapse and given the important role of viral RNA interference (RNAi) in combating viral infections we investigated if CCD-colonies elicit an RNAi response. Deep-sequencing analysis of samples from CCD-colonies from US and Israel revealed abundant small interfering RNAs (siRNA) of 21-22 nucleotides perfectly matching the Israeli acute paralysis virus (IAPV), Kashmir virus and Deformed wing virus genomes. Israeli colonies showed high titers of IAPV and a conserved RNAi-pattern of matching the viral genome. That was also observed in sample analysis from colonies experimentally infected with IAPV. Our results suggest that CCD-colonies set out a siRNA response that is specific against predominant viruses associated with colony losses.

  8. MysiRNA: improving siRNA efficacy prediction using a machine-learning model combining multi-tools and whole stacking energy (ΔG).

    PubMed

    Mysara, Mohamed; Elhefnawi, Mahmoud; Garibaldi, Jonathan M

    2012-06-01

    The investigation of small interfering RNA (siRNA) and its posttranscriptional gene-regulation has become an extremely important research topic, both for fundamental reasons and for potential longer-term therapeutic benefits. Several factors affect the functionality of siRNA including positional preferences, target accessibility and other thermodynamic features. State of the art tools aim to optimize the selection of target siRNAs by identifying those that may have high experimental inhibition. Such tools implement artificial neural network models as Biopredsi and ThermoComposition21, and linear regression models as DSIR, i-Score and Scales, among others. However, all these models have limitations in performance. In this work, a neural-network trained new siRNA scoring/efficacy prediction model was developed based on combining two existing scoring algorithms (ThermoComposition21 and i-Score), together with the whole stacking energy (ΔG), in a multi-layer artificial neural network. These three parameters were chosen after a comparative combinatorial study between five well known tools. Our developed model, 'MysiRNA' was trained on 2431 siRNA records and tested using three further datasets. MysiRNA was compared with 11 alternative existing scoring tools in an evaluation study to assess the predicted and experimental siRNA efficiency where it achieved the highest performance both in terms of correlation coefficient (R(2)=0.600) and receiver operating characteristics analysis (AUC=0.808), improving the prediction accuracy by up to 18% with respect to sensitivity and specificity of the best available tools. MysiRNA is a novel, freely accessible model capable of predicting siRNA inhibition efficiency with improved specificity and sensitivity. This multiclassifier approach could help improve the performance of prediction in several bioinformatics areas. MysiRNA model, part of MysiRNA-Designer package [1], is expected to play a key role in siRNA selection and evaluation.

  9. Kinesin spindle protein SiRNA slows tumor progression.

    PubMed

    Marra, Emanuele; Palombo, Fabio; Ciliberto, Gennaro; Aurisicchio, Luigi

    2013-01-01

    The kinesin spindle protein (KSP), a member of the kinesin superfamily of microtubule-based motors, plays a critical role in mitosis as it mediates centrosome separation and bipolar spindle assembly and maintenance. Inhibition of KSP function leads to cell cycle arrest at mitosis with the formation of monoastral microtubule arrays, and ultimately, to cell death. Several KSP inhibitors are currently being studied in clinical trials and provide new opportunities for the development of novel anticancer therapeutics. RNA interference (RNAi) may represent a powerful strategy to interfere with key molecular pathways involved in cancer. In this study, we have established an efficient method for intratumoral delivery of siRNA. We evaluated short interfering RNA (siRNA) duplexes targeting luciferase as surrogate marker or KSP sequence. To examine the potential feasibility of RNAi therapy, the siRNA was transfected into pre-established lesions by means of intratumor electro-transfer of RNA therapeutics (IERT). This technology allowed cell permeation of the nucleic acids and to efficiently knock down gene expression, albeit transiently. The KSP-specific siRNA drastically reduced outgrowth of subcutaneous melanoma and ovarian cancer lesions. Our results show that intratumoral electro-transfer of siRNA is feasible and KSP-specific siRNA may provide a novel strategy for therapeutic intervention.

  10. Transdermal Delivery of siRNA through Microneedle Array

    NASA Astrophysics Data System (ADS)

    Deng, Yan; Chen, Jiao; Zhao, Yi; Yan, Xiaohui; Zhang, Li; Choy, Kwongwai; Hu, Jun; Sant, Himanshu J.; Gale, Bruce K.; Tang, Tao

    2016-02-01

    Successful development of siRNA therapies has significant potential for the treatment of skin conditions (alopecia, allergic skin diseases, hyperpigmentation, psoriasis, skin cancer, pachyonychia congenital) caused by aberrant gene expression. Although hypodermic needles can be used to effectively deliver siRNA through the stratum corneum, the major challenge is that this approach is painful and the effects are restricted to the injection site. Microneedle arrays may represent a better way to deliver siRNAs across the stratum corneum. In this study, we evaluated for the first time the ability of the solid silicon microneedle array for punching holes to deliver cholesterol-modified housekeeping gene (Gapdh) siRNA to the mouse ear skin. Treating the ear with microneedles showed permeation of siRNA in the skin and could reduce Gapdh gene expression up to 66% in the skin without accumulation in the major organs. The results showed that microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively.

  11. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    NASA Astrophysics Data System (ADS)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  12. Technologies for Controlled, Local Delivery of siRNA

    PubMed Central

    Sarett, Samantha M.; Nelson, Christopher E.; Duvall, Craig L.

    2015-01-01

    The discovery of RNAi in the late 1990s unlocked a new realm of therapeutic possibilities by enabling potent and specific silencing of theoretically any desired genetic target. Better elucidation of the mechanism of action, the impact of chemical modifications that stabilize and reduce nonspecific effects of siRNA molecules, and the key design considerations for effective delivery systems has spurred progress toward developing clinically-successful siRNA therapies. A logical aim for initial siRNA translation is local therapies, as delivering siRNA directly to its site of action helps to ensure that a sufficient dose reaches the target tissue, lessens the potential for off-target side effects, and circumvents the substantial systemic delivery barriers. While topical siRNA delivery has progressed into numerous clinical trials, an enormous opportunity also exists to develop sustained-release, local delivery systems that enable both spatial and temporal control of gene silencing. This review focuses on material platforms that establish both localized and controlled gene silencing, with emphasis on the systems that show most promise for clinical translation. PMID:26476177

  13. Lipid-based vectors for siRNA delivery

    PubMed Central

    Zhang, Shubiao; Zhi, Defu; Huang, Leaf

    2016-01-01

    siRNA therapeutics has developed rapidly and already there are clinical trials ongoing or planned; however, the delivery of siRNA into cells, tissues or organs remains to be a major obstacle. Lipid-based vectors hold the most promising position among non-viral vectors, as they have a similar structure to cell or organelle membranes. But when used in the form of liposomes, these vectors have shown some problems. Therefore, either the nature of lipids themselves or forms used should be improved. As a novel class of lipid like materials, lipidoids have the advantages of easy synthesis and the ability for delivering siRNA to obtain excellent silencing activity. However, the toxicities of lipidoids have not been thoroughly studied. pH responsive lipids have also gained great attention recently, though some of the amine-based lipids are not novel in terms of chemical structures. More complex self-assembly structures, such as LPD (LPH) and LCP, may provide a good solution to siRNA delivery. They have demonstrated controlled particle morphology and size and siRNA delivery activity for both in vitro and in vivo. PMID:22994300

  14. FRET-labeled siRNA probes for tracking assembly and disassembly of siRNA nanocomplexes.

    PubMed

    Alabi, Christopher A; Love, Kevin T; Sahay, Gaurav; Stutzman, Tina; Young, Whitney T; Langer, Robert; Anderson, Daniel G

    2012-07-24

    The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability, and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which may be difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (K(D)) and intracellular disassembly half-life for several in-house and proprietary commercial DVs. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing.

  15. Lentivirus-expressed siRNA vectors against Alzheimer disease.

    PubMed

    Peng, Kevin A; Masliah, Eliezer

    2010-01-01

    Amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease, and the accumulation of APP products ultimately leads to the familiar histopathological and clinical manifestations associated with this most common form of dementia. A protein that has been shown to promote APP accumulation is beta-secretase (beta-site APP cleaving enzyme 1, or BACE1), which is increased in the cerebrospinal fluid in those affected with Alzheimer disease. Through in vivo studies using APP transgenic mice, we demonstrated that decreasing the expression of BACE1 via lentiviral vector delivery of BACE1 siRNA has the potential for significantly reducing the cleavage of APP, accumulation of these products, and consequent neurodegeneration. As such, lentiviral-expressed siRNA against BACE1 is a therapeutic possibility in the treatment of Alzheimer disease. We detail the use of lentivirus-expressed siRNA as a method to ameliorate Alzheimer disease neuropathology in APP transgenic mice.

  16. Characterization of rice black-streaked dwarf virus- and rice stripe virus-derived siRNAs in singly and doubly infected insect vector Laodelphax striatellus.

    PubMed

    Li, Junmin; Andika, Ida Bagus; Shen, Jiangfeng; Lv, Yuanda; Ji, Yongqiang; Sun, Liying; Chen, Jianping

    2013-01-01

    Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5'- and 3'-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.

  17. Gene silencing by chemically modified siRNAs.

    PubMed

    Engels, Joachim W

    2013-03-25

    RNA interference (RNAi) has not only already risen as a gold standard for validating gene function in basic science studies, but also holds great promise as a new therapeutic paradigm. Advantages of RNAi-based therapeutics include relatively fast initial screening and the ability to target proteins not yet addressable by traditional drug design strategies. In this review we describe the development of chemically modified small inhibiting siRNAs and their application as potential therapeutics during the past decade. Focus is on proper siRNA design, choice of chemical modification and how to circumvent immunogenicity as well as off-target effects.

  18. Innovative delivery of siRNA to solid tumors by super carbonate apatite.

    PubMed

    Wu, Xin; Yamamoto, Hirofumi; Nakanishi, Hiroyuki; Yamamoto, Yuki; Inoue, Akira; Tei, Mitsuyoshi; Hirose, Hajime; Uemura, Mamoru; Nishimura, Junichi; Hata, Taishi; Takemasa, Ichiro; Mizushima, Tsunekazu; Hossain, Sharif; Akaike, Toshihiro; Matsuura, Nariaki; Doki, Yuichiro; Mori, Masaki

    2015-01-01

    RNA interference (RNAi) technology is currently being tested in clinical trials for a limited number of diseases. However, systemic delivery of small interfering RNA (siRNA) to solid tumors has not yet been achieved in clinics. Here, we introduce an in vivo pH-sensitive delivery system for siRNA using super carbonate apatite (sCA) nanoparticles, which is the smallest class of nanocarrier. These carriers consist simply of inorganic ions and accumulate specifically in tumors, yet they cause no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA abundantly accumulated in the cytoplasm of tumor cells at 4 h, indicating quick achievement of endosomal escape. sCA-survivin-siRNA induced apoptosis in HT29 tumors and significantly inhibited in vivo tumor growth of HCT116, to a greater extent than two other in vivo delivery reagents. With innovative in vivo delivery efficiency, sCA could be a useful nanoparticle for the therapy of solid tumors.

  19. Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing

    PubMed Central

    Zheng, Xianwu; Zhu, Jianhua; Kapoor, Avnish; Zhu, Jian-Kang

    2007-01-01

    Argonautes (AGOs) are conserved proteins that contain an RNA-binding PAZ domain and an RNase H-like PIWI domain. In Arabidopsis, except for AGO1, AGO4 and AGO7, the roles of seven other AGOs in gene silencing are not known. We found that a mutation in AGO6 partially suppresses transcriptional gene silencing in the DNA demethylase mutant ros1-1. In ago6-1ros1-1 plants, RD29A promoter short interfering RNAs (siRNAs) are less abundant, and cytosine methylation at both transgenic and endogenous RD29A promoters is reduced, compared to that in ros1-1. Interestingly, the ago4-1 mutation has a stronger suppression of the transcriptional silencing phenotype of ros1-1 mutant. Analysis of cytosine methylation at the endogenous MEA-ISR, AtREP2 and SIMPLEHAT2 loci revealed that the CpNpG and asymmetric methylation levels are lower in either of the ago6-1 and ago4-1 single mutants than those in the wild type, and the levels are the lowest in the ago6-1ago4-1 double mutant. These results suggest that AGO6 is important for the accumulation of specific heterochromatin-related siRNAs, and for DNA methylation and transcriptional gene silencing, this function is partly redundant with AGO4. PMID:17332757

  20. Improved nucleic acid descriptors for siRNA efficacy prediction

    PubMed Central

    Sciabola, Simone; Cao, Qing; Orozco, Modesto; Faustino, Ignacio; Stanton, Robert V.

    2013-01-01

    Although considerable progress has been made recently in understanding how gene silencing is mediated by the RNAi pathway, the rational design of effective sequences is still a challenging task. In this article, we demonstrate that including three-dimensional descriptors improved the discrimination between active and inactive small interfering RNAs (siRNAs) in a statistical model. Five descriptor types were used: (i) nucleotide position along the siRNA sequence, (ii) nucleotide composition in terms of presence/absence of specific combinations of di- and trinucleotides, (iii) nucleotide interactions by means of a modified auto- and cross-covariance function, (iv) nucleotide thermodynamic stability derived by the nearest neighbor model representation and (v) nucleic acid structure flexibility. The duplex flexibility descriptors are derived from extended molecular dynamics simulations, which are able to describe the sequence-dependent elastic properties of RNA duplexes, even for non-standard oligonucleotides. The matrix of descriptors was analysed using three statistical packages in R (partial least squares, random forest, and support vector machine), and the most predictive model was implemented in a modeling tool we have made publicly available through SourceForge. Our implementation of new RNA descriptors coupled with appropriate statistical algorithms resulted in improved model performance for the selection of siRNA candidates when compared with publicly available siRNA prediction tools and previously published test sets. Additional validation studies based on in-house RNA interference projects confirmed the robustness of the scoring procedure in prospective studies. PMID:23241392

  1. siRNA Delivery by Stimuli-Sensitive Nanocarriers

    PubMed Central

    Salzano, Giuseppina; Costa, Daniel F.; Torchilin, Vladimir P.

    2016-01-01

    Since its discovery in late 1990s, small interfering RNA (siRNA) has become a significant biopharmaceutical research tool and a powerful option for the treatment of different human diseases based on altered gene-expression. Despite promising data from many pre-clinical studies, concrete hurdles still need to be overcome to bring therapeutic siRNAs in clinic. The design of stimuli-sensitive nanopreparations for gene therapy is a lively area of the current research. Compared to conventional systems for siRNA delivery, this type of platform can respond to local stimuli that are characteristics of the pathological area of interest, allowing the release of nucleic acids at the desired site. Acidic pH, abnormal levels of enzymes, altered redox potential and magnetic field are examples of stimuli exploited in the design of stimuli-sensitive nanoparticles. In this review, we discuss on recent stimuli-sensitive strategies for siRNA delivery and we highlight on the potential of combining multiple stimuli-sensitive strategies in the same nano-platform for a better therapeutic outcome. PMID:26486143

  2. Beryllium and Boron abundances in population II stars

    NASA Technical Reports Server (NTRS)

    1995-01-01

    The scientific focus of this program was to undertake UV spectroscopic abundance analyses of extremely metal poor stars with attention to determining abundances of light elements such as beryllium and boron. The abundances are likely to reflect primordial abundances within the early galaxy and help to constrain models for early galactic nucleosynthesis. The general metal abundances of these stars are also important for understanding stellar evolution.

  3. Mucus barrier-triggered disassembly of siRNA nanocarriers

    NASA Astrophysics Data System (ADS)

    Thomsen, Troels B.; Li, Leon; Howard, Kenneth A.

    2014-10-01

    The mucus overlying mucosal epithelial surfaces presents not only a biological barrier to the penetration of potential pathogens, but also therapeutic modalities including RNAi-based nanocarriers. Movement of nanomedicines across the mucus barriers of the gastrointestinal mucosa is modulated by interactions of the nanomedicine carriers with mucin glycoproteins inside the mucus, potentiated by the large surface area of the nanocarrier. We have developed a fluorescence activation-based reporter system showing that the interaction between polyanionic mucins and the cationic chitosan/small interfering RNA (siRNA) nanocarriers (polyplexes) results in the disassembly and consequent triggered release of fluorescent siRNA. The quantity of release was found to be dependent on the molar ratio between chitosan amino groups and siRNA phosphate groups (NP ratio) of the polyplexes with a maximal estimated 48.6% release of siRNA over 30 min at NP 60. Furthermore, a microfluidic in vitro model of the gastrointestinal mucus barrier was used to visualize the dynamic interaction between chitosan/siRNA nanocarriers and native purified porcine stomach mucins. We observed strong interactions and aggregations at the mucin-liquid interface, followed by an NP ratio dependent release and consequent diffusion of siRNA across the mucin barrier. This work describes a new model of interaction at the nanocarrier-mucin interface and has important implications for the design and development of nucleic acid-based nanocarrier therapeutics for mucosal disease treatments and also provides insights into nanoscale pathogenic processes.The mucus overlying mucosal epithelial surfaces presents not only a biological barrier to the penetration of potential pathogens, but also therapeutic modalities including RNAi-based nanocarriers. Movement of nanomedicines across the mucus barriers of the gastrointestinal mucosa is modulated by interactions of the nanomedicine carriers with mucin glycoproteins inside the

  4. siRNA Machinery in Whitefly (Bemisia tabaci)

    PubMed Central

    Upadhyay, Santosh Kumar; Dixit, Sameer; Sharma, Shailesh; Singh, Harpal; Kumar, Jitesh; Verma, Praveen C.; Chandrashekar, K.

    2013-01-01

    Background RNA interference has been emerged as an utmost tool for the control of sap sucking insect pests. Systemic response is necessary to control them in field condition. Whitefly is observed to be more prone to siRNA in recent studies, however the siRNA machinery and mechanism is not well established. Methodology/Principal Findings To identify the core siRNA machinery, we curated transcriptome data of whitefly from NCBI database. Partial mRNA sequences encoding Dicer2, R2D2, Argonaute2 and Sid1 were identified by tblastn search of homologous sequences from Aphis glycines and Tribolium castaneum. Complete encoding sequences were obtained by RACE, protein sequences derived by Expasy translate tool and confirmed by blastp analysis. Conserved domain search and Prosite-Scan showed similar domain architecture as reported in homologs from related insects. We found helicase, PAZ, RNaseIIIa, RNaseIIIb and double-stranded RNA-binding fold (DSRBF) in Dicer2; DsRBD in R2D2; and PAZ and PIWI domains in Argonaute2. Eleven transmembrane domains were detected in Sid1. Sequence homology and phylogenetic analysis revealed that RNAi machinery of whitefly is close to Aphids. Real-time PCR analysis showed similar expression of these genes in different developmental stages as reported in A. glycines and T. castaneum. Further, the expression level of above genes was quite similar to the housekeeping gene actin. Conclusions/Significance Availability of core siRNA machinery including the Sid1 and their universal expression in reasonable quantity indicated significant response of whitefly towards siRNA. Present report opens the way for controlling whitefly, one of the most destructive crop insect pest. PMID:24391810

  5. Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots.

    PubMed

    Yang, Yingzhen; Jittayasothorn, Yingyos; Chronis, Demosthenis; Wang, Xiaohong; Cousins, Peter; Zhong, Gan-Yuan

    2013-01-01

    Root-knot nematodes (RKNs) infect many annual and perennial crops and are the most devastating soil-born pests in vineyards. To develop a biotech-based solution for controlling RKNs in grapes, we evaluated the efficacy of plant-derived RNA interference (RNAi) silencing of a conserved RKN effector gene, 16D10, for nematode resistance in transgenic grape hairy roots. Two hairpin-based silencing constructs, containing a stem sequence of 42 bp (pART27-42) or 271 bp (pART27-271) of the 16D10 gene, were transformed into grape hairy roots and compared for their small interfering RNA (siRNA) production and efficacy on suppression of nematode infection. Transgenic hairy root lines carrying either of the two RNAi constructs showed less susceptibility to nematode infection compared with control. Small RNA libraries from four pART27-42 and two pART27-271 hairy root lines were sequenced using an Illumina sequencing technology. The pART27-42 lines produced hundred times more 16D10-specific siRNAs than the pART27-271 lines. On average the 16D10 siRNA population had higher GC content than the 16D10 stem sequences in the RNAi constructs, supporting previous observation that plant dicer-like enzymes prefer GC-rich sequences as substrates for siRNA production. The stems of the 16D10 RNAi constructs were not equally processed into siRNAs. Several hot spots for siRNA production were found in similar positions of the hairpin stems in pART27-42 and pART27-271. Interestingly, stem sequences at the loop terminus produced more siRNAs than those at the stem base. Furthermore, the relative abundance of guide and passenger single-stranded RNAs from putative siRNA duplexes was largely correlated with their 5' end thermodynamic strength. This study demonstrated the feasibility of using a plant-derived RNAi approach for generation of novel nematode resistance in grapes and revealed several interesting molecular characteristics of transgene siRNAs important for optimizing plant RNAi constructs.

  6. Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain

    PubMed Central

    Nikan, Mehran; Osborn, Maire F; Coles, Andrew H; Godinho, Bruno MDC; Hall, Lauren M; Haraszti, Reka A; Hassler, Matthew R; Echeverria, Dimas; Aronin, Neil; Khvorova, Anastasia

    2016-01-01

    The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6–60 μg). Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73%) and cortex (up to 51%) after 1 week. Moreover, following a bilateral intrastriatal injection (60 μg), we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders. PMID:27504598

  7. siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster.

    PubMed

    Menon, Debashish U; Coarfa, Cristian; Xiao, Weimin; Gunaratne, Preethi H; Meller, Victoria H

    2014-11-18

    Highly differentiated sex chromosomes create a lethal imbalance in gene expression in one sex. To accommodate hemizygosity of the X chromosome in male fruit flies, expression of X-linked genes increases twofold. This is achieved by the male- specific lethal (MSL) complex, which modifies chromatin to increase expression. Mutations that disrupt the X localization of this complex decrease the expression of X-linked genes and reduce male survival. The mechanism that restricts the MSL complex to X chromatin is not understood. We recently reported that the siRNA pathway contributes to localization of the MSL complex, raising questions about the source of the siRNAs involved. The X-linked 1.688 g/cm(3) satellite related repeats (1.688(X) repeats) are restricted to the X chromosome and produce small RNA, making them an attractive candidate. We tested RNA from these repeats for a role in dosage compensation and found that ectopic expression of single-stranded RNAs from 1.688(X) repeats enhanced the male lethality of mutants with defective X recognition. In contrast, expression of double-stranded hairpin RNA from a 1.688(X) repeat generated abundant siRNA and dramatically increased male survival. Consistent with improved survival, X localization of the MSL complex was largely restored in these males. The striking distribution of 1.688(X) repeats, which are nearly exclusive to the X chromosome, suggests that these are cis-acting elements contributing to identification of X chromatin.

  8. Utilizing Selected Di- and Trinucleotides of siRNA to Predict RNAi Activity

    PubMed Central

    Han, Ye; Liu, Yuanning; Zhang, Hao; He, Fei; Shu, Chonghe

    2017-01-01

    Small interfering RNAs (siRNAs) induce posttranscriptional gene silencing in various organisms. siRNAs targeted to different positions of the same gene show different effectiveness; hence, predicting siRNA activity is a crucial step. In this paper, we developed and evaluated a powerful tool named “siRNApred” with a new mixed feature set to predict siRNA activity. To improve the prediction accuracy, we proposed 2-3NTs as our new features. A Random Forest siRNA activity prediction model was constructed using the feature set selected by our proposed Binary Search Feature Selection (BSFS) algorithm. Experimental data demonstrated that the binding site of the Argonaute protein correlates with siRNA activity. “siRNApred” is effective for selecting active siRNAs, and the prediction results demonstrate that our method can outperform other current siRNA activity prediction methods in terms of prediction accuracy. PMID:28243313

  9. Inhibition of liver fibrosis using vitamin A-coupled liposomes to deliver matrix metalloproteinase-2 siRNA in vitro

    PubMed Central

    LI, YIPING; LIU, FENG; DING, FENGAN; CHEN, PINGSHENG; TANG, MENG

    2015-01-01

    Hepatic fibrosis is a common form of wound healing in response to chronic liver injuries and can lead to more serious complications, including mortality. It is well-established that hepatic stellate cells (HSCs) are central mediators of hepatic fibrosis, and matrix metalloproteinase-2 (MMP-2) is important in the formation of liver fibrosis. In addition, HSCs are the primary cells secreting MMP-2 and extracellular matrix, therefore, there has been increasing interest in developing agents with high selectivity towards HSCs. However, no clinical drugs based on MMP-2, directed against HSCs, have been used to prevent fibrosis. Following consideration of the abundant vitamin A (VitA) receptors expressed on the cellular membrane of HSCs, the present study constructed VitA-coupled liposomes (VitA-lips) using dicyclohexylcarbodiimide-1, 3-diaminopentane condensation, rotatory film processing and ultrasonic oscillation. The results revealed that the liposomes exhibited low cytotoxicity and a suitable binding ability to MMP-2 small interference (si)RNA. Furthermore, the liposomes effectively delivered MMP-2 siRNA to the HSC-T6 cells. When HSCs were treated with the liposomes carrying MMP-2 siRNA (VitA-lip-MMP-2 siRNA), the mRNA expression and activity of MMP-2, and the protein expression levels of α-smooth muscle actin and type I collagen were significantly reduced. These results suggested that inhibition of the expression of MMP-2 in HSC-T6 cells may contribute to preventing hepatic fibrosis, and provided experimental support to the development of specific drugs against MMP-2 to prevent fibrogenesis in chronic liver disease. PMID:26017616

  10. Lipid flippases promote antiviral silencing and the biogenesis of viral and host siRNAs in Arabidopsis.

    PubMed

    Guo, Zhongxin; Lu, Jinfeng; Wang, Xianbing; Zhan, Binhui; Li, Wanxiang; Ding, Shou-Wei

    2017-02-07

    Dicer-mediated processing of virus-specific dsRNA into short interfering RNAs (siRNAs) in plants and animals initiates a specific antiviral defense by RNA interference (RNAi). In this study, we developed a forward genetic screen for the identification of host factors required for antiviral RNAi in Arabidopsis thaliana Using whole-genome sequencing and a computational pipeline, we identified aminophospholipid transporting ATPase 2 (ALA2) and the related ALA1 in the type IV subfamily of P-type ATPases as key components of antiviral RNAi. ALA1 and ALA2 are flippases, which are transmembrane lipid transporter proteins that transport phospholipids across cellular membranes. We found that the ala1/ala2 single- and double-mutant plants exhibited enhanced disease susceptibility to cucumber mosaic virus when the virus-encoded function to suppress RNAi was disrupted. Notably, the antiviral activity of both ALA1 and ALA2 was abolished by a single amino acid substitution known to inactivate the flippase activity. Genetic analysis revealed that ALA1 and ALA2 acted to enhance the amplification of the viral siRNAs by RNA-dependent RNA polymerase (RdRP) 1 (RDR1) and RDR6 and of the endogenous virus-activated siRNAs by RDR1. RNA virus replication by plant viral RdRPs occurs inside vesicle-like membrane invaginations induced by the recruitment of the viral RdRP and host factors to subcellular membrane microdomains enriched with specific phospholipids. Our results suggest that the phospholipid transporter activity of ALA1/ALA2 may be necessary for the formation of similar invaginations for the synthesis of dsRNA precursors of highly abundant viral and host siRNAs by the cellular RdRPs.

  11. Exosomes for targeted siRNA delivery across biological barriers.

    PubMed

    El Andaloussi, Samir; Lakhal, Samira; Mäger, Imre; Wood, Matthew J A

    2013-03-01

    Using oligonucleotide-based drugs to modulate gene expression has opened a new avenue for drug discovery. In particular small interfering RNAs (siRNAs) are being rapidly recognized as promising therapeutic tools, but their poor bioavailability limits the full realization of their clinical potential. In recent years, cumulating evidence has emerged for the role of membrane vesicles, secreted by most cells and found in all body fluids, as key mediators of information transmission between cells. Importantly, a sub-group of these termed exosomes, have recently been shown to contain various RNA species and to mediate their horizontal transfer to neighbouring- or distant recipient cells. Here, we provide a brief overview on membrane vesicles and their role in exchange of genetic information. We also describe how these natural carriers of genetic material can be harnessed to overcome the obstacle of poor delivery and allow efficient systemic delivery of exogenous siRNA across biological barriers such as the blood-brain barrier.

  12. Therapeutic siRNA: Principles, Challenges, and Strategies

    PubMed Central

    Gavrilov, Kseniya; Saltzman, W. Mark

    2012-01-01

    RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics. PMID:22737048

  13. Targeting siRNA Missiles to Her2+ Breast Cancer

    DTIC Science & Technology

    2009-06-01

    Nagai, H., Oniki, S., Oka, M., Horikawa, T., and Nishigori, C. (2006). Induction of cellular immunity against hair follicle melanocyte causes alopecia...Principal Investigator: Medina-Kauwe, Lali K. 4 INTRODUCTION: The purpose of this research is to test the hypothesis that recombinant Ad5 capsid...delivered the most potent cytotoxicity in order to incorporate it into the targeted complex and test it in vivo. We compared HER2- silencing siRNA

  14. Guide Strand 3'-End Modifications Regulate siRNA Specificity.

    PubMed

    Valenzuela, Rachel A P; Onizuka, Kazumitsu; Ball-Jones, Alexi A; Hu, Tiannan; Suter, Scott R; Beal, Peter A

    2016-12-14

    Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.

  15. Translational siRNA therapeutics using liposomal carriers: prospects & challenges.

    PubMed

    Bhavsar, Dhiraj; Subramanian, Krishnakumar; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2012-08-01

    Gene silencing has emerged as a promising strategy for molecular therapy of various malignant, viral, hereditary and inflammatory disorders. However, its translation from lab to clinic is yet to gain momentum due to the numerous problems that plague its development. A multi-functional siRNA delivery system with desired properties such as enhanced immune compatibility, target specificity, high cell uptake and excellent silencing efficiency is required to understand the challenges involved in the selection and modification of small interfering RNA (siRNA), factors influencing the complexation process and the response of the biological system to the formulation. Liposomes have been used as delivery systems due to its versatility in handling different types of drugs, tunable size, charge and surface functionalities that improve its effectiveness in vivo. This review highlights the challenges involved in gene silencing and describes the progression of liposomal systems used in gene silencing. The rationale in introducing chemical modifications in siRNA, synthesizing designer cationic lipids and evolution of hybrid liposomal systems has been elaborated, emphasizing their merits and short-comings. Finally, a description of the current state of clinical trials involving liposomal formulations has been included to provide an unbiased perspective of the future of liposomal systems and gene silencing tools as therapeutic tools.

  16. siRNA vs. shRNA: similarities and differences.

    PubMed

    Rao, Donald D; Vorhies, John S; Senzer, Neil; Nemunaitis, John

    2009-07-25

    RNA interference (RNAi) is a natural process through which expression of a targeted gene can be knocked down with high specificity and selectivity. Using available technology and bioinformatics investigators will soon be able to identify relevant bio molecular tumor network hubs as potential key targets for knockdown approaches. Methods of mediating the RNAi effect involve small interfering RNA (siRNA), short hairpin RNA (shRNA) and bi-functional shRNA. The simplicity of siRNA manufacturing and transient nature of the effect per dose are optimally suited for certain medical disorders (i.e. viral injections). However, using the endogenous processing machinery, optimized shRNA constructs allow for high potency and sustainable effects using low copy numbers resulting in less off-target effects, particularly if embedded in a miRNA scaffold. Bi-functional design may further enhance potency and safety of RNAi-based therapeutics. Remaining challenges include tumor selective delivery vehicles and more complete evaluation of the scope and scale of off-target effects. This review will compare siRNA, shRNA and bi-functional shRNA.

  17. Extreme Heat

    MedlinePlus

    ... Emergencies Biological Threats Chemical Threats Cyber Incident Drought Earthquakes Extreme Heat Explosions Floods Hazardous Materials Incidents Home ... Emergencies Biological Threats Chemical Threats Cyber ... Heat Explosions Floods Hazardous Materials Incidents Home ...

  18. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity

    PubMed Central

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-01-01

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects. PMID:27457182

  19. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity

    NASA Astrophysics Data System (ADS)

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-07-01

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects.

  20. Local administration of siRNA through Microneedle: Optimization, Bio-distribution, Tumor Suppression and Toxicity.

    PubMed

    Tang, Tao; Deng, Yan; Chen, Jiao; Zhao, Yi; Yue, Ruifeng; Choy, Kwong Wai; Wang, Chi Chiu; Du, Quan; Xu, Yan; Han, Linxiao; Chung, Tony Kwok Hung

    2016-07-26

    Although RNA interference may become a novel therapeutic approach for cancer treatment, target-site accumulation of siRNA to achieve therapeutic dosage will be a major problem. Microneedle represents a better way to deliver siRNAs and we have evaluated for the first time the capability of a silicon microneedle array for delivery of Gapdh siRNA to the skin in vivo and the results showed that the microneedle arrays could effectively deliver siRNA to relevant regions of the skin noninvasively. For the further study in this field, we evaluated the efficacy of the injectable microneedle device for local delivery of siRNA to the mouse xenograft. The results presented here indicate that local administration of siRNA through injectable microneedle could effectively deliver siRNA into the tumor region, and inhibit tumor progression without major adverse effects.

  1. miRNAs trigger widespread epigenetically activated siRNAs from transposons in Arabidopsis.

    PubMed

    Creasey, Kate M; Zhai, Jixian; Borges, Filipe; Van Ex, Frederic; Regulski, Michael; Meyers, Blake C; Martienssen, Robert A

    2014-04-17

    In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE 1 (DCL1)-dependent microRNAs (miRNAs), which also trigger 21-nucleotide secondary short interfering RNAs (siRNAs) via RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DCL4 and ARGONAUTE 1 (AGO1), whereas transcriptional gene silencing (TGS) of transposons is mediated by 24-nucleotide heterochromatic (het)siRNAs, RDR2, DCL3 and AGO4 (ref. 4). Transposons can also give rise to abundant 21-nucleotide 'epigenetically activated' small interfering RNAs (easiRNAs) in DECREASED DNA METHYLATION 1 (ddm1) and DNA METHYLTRANSFERASE 1 (met1) mutants, as well as in the vegetative nucleus of pollen grains and in dedifferentiated plant cell cultures. Here we show that easiRNAs in Arabidopsis thaliana resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than 50 miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nucleotide easiRNAs and severe infertility, but 24-nucleotide hetsiRNAs are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germ line. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing and by their hosts for genome defence.

  2. RIsearch2: suffix array-based large-scale prediction of RNA-RNA interactions and siRNA off-targets.

    PubMed

    Alkan, Ferhat; Wenzel, Anne; Palasca, Oana; Kerpedjiev, Peter; Rudebeck, Anders Frost; Stadler, Peter F; Hofacker, Ivo L; Gorodkin, Jan

    2017-01-20

    Intermolecular interactions of ncRNAs are at the core of gene regulation events, and identifying the full map of these interactions bears crucial importance for ncRNA functional studies. It is known that RNA-RNA interactions are built up by complementary base pairings between interacting RNAs and high level of complementarity between two RNA sequences is a powerful predictor of such interactions. Here, we present RIsearch2, a large-scale RNA-RNA interaction prediction tool that enables quick localization of potential near-complementary RNA-RNA interactions between given query and target sequences. In contrast to previous heuristics which either search for exact matches while including G-U wobble pairs or employ simplified energy models, we present a novel approach using a single integrated seed-and-extend framework based on suffix arrays. RIsearch2 enables fast discovery of candidate RNA-RNA interactions on genome/transcriptome-wide scale. We furthermore present an siRNA off-target discovery pipeline that not only predicts the off-target transcripts but also computes the off-targeting potential of a given siRNA. This is achieved by combining genome-wide RIsearch2 predictions with target site accessibilities and transcript abundance estimates. We show that this pipeline accurately predicts siRNA off-target interactions and enables off-targeting potential comparisons between different siRNA designs. RIsearch2 and the siRNA off-target discovery pipeline are available as stand-alone software packages from http://rth.dk/resources/risearch.

  3. Calcium phosphate nanoparticles-based systems for siRNA delivery

    PubMed Central

    Xu, Xiaochun; Li, Zehao; Zhao, Xueqin; Keen, Lawrence; Kong, Xiangdong

    2016-01-01

    Despite the enormous therapeutic potential of siRNA as a treatment strategy, the delivery is still a problem due to unfavorable biodistribution profiles and poor intracellular bioavailability. Calcium phosphate (CaP) co-precipitate has been used for nearly 40 years for in vitro transfection due to its non-toxic nature and simplicity of preparation. The surface charge of CaP will be tuned into positive by surface modification, which is important for siRNA loading and crossing cell membrane without enzymatic degradation. The new siRNA carrier system will also promote the siRNA escape from lysosome to achieve siRNA sustained delivery and high-efficiency silence. In this review, we focus on the current research activity in the development of CaP nanoparticles for siRNA delivery. These nanoparticles are mainly classified into lipid coated, polymer coated and various other types for discussion. PMID:27252888

  4. Indium-Labeling of siRNA for Small Animal SPECT Imaging.

    PubMed

    Jones, Steven; Merkel, Olivia

    2016-01-01

    Ever since the discovery of RNA interference (RNAi), therapeutic delivery of siRNA has attracted a lot of interest. However, due to the nature and structure of siRNA, a carrier is needed for any mode of systemic treatment. Furthermore, specific imaging techniques are required to trace where the deposition of the siRNA occurs throughout the body after treatment. Tracking in vivo siRNA biodistribution allows understanding and interpreting therapeutics effects and side effects. A great advantage of noninvasive imaging techniques such as SPECT imaging is that several time points can be assessed in the same subject. Thus, the time course of biodistribution or metabolic processes can be followed. Therefore, we have described an approach to modify siRNA with a DTPA (Diethylene Triamine Pentaacetic Acid) chelator in order to utilize an indium labeled siRNA for SPECT imaging. Here, we explain the details of the labeling and purification procedures.

  5. Hydroxychloroquine-conjugated gold nanoparticles for improved siRNA activity.

    PubMed

    Perche, F; Yi, Y; Hespel, L; Mi, P; Dirisala, A; Cabral, H; Miyata, K; Kataoka, K

    2016-06-01

    Current technology of siRNA delivery relies on pharmaceutical dosage forms to route maximal doses of siRNA to the tumor. However, this rationale does not address intracellular bottlenecks governing silencing activity. Here, we tested the impact of hydroxychloroquine conjugation on the intracellular fate and silencing activity of siRNA conjugated PEGylated gold nanoparticles. Addition of hydroxychloroquine improved endosomal escape and increased siRNA guide strand distribution to the RNA induced silencing complex (RISC), both crucial obstacles to the potency of siRNA. This modification significantly improved gene downregulation in cellulo. Altogether, our data suggest the benefit of this modification for the design of improved siRNA delivery systems.

  6. Addition of poly (propylene glycol) to multiblock copolymer to optimize siRNA delivery

    PubMed Central

    Dai, Zhi; Arévalo, Maria T; Li, Junwei; Zeng, Mingtao

    2014-01-01

    Previous studies have examined different strategies for siRNA delivery with varying degrees of success. These include use of viral vectors, cationic liposomes, and polymers. Several copolymers were designed and synthesized based on blocks of poly(ethylene glycol) PEG, poly(propylene glycol) PPG, and poly(l-lysine). These were designated as P1, P2, and P3. We studied the copolymer self-assembly, siRNA binding, particle size, surface potential, architecture of the complexes, and siRNA delivery. Silencing of GFP using copolymer P3 to deliver GFP-specific siRNA to Neuro-2a cells expressing GFP was almost as effective as using Lipofectamine 2000, with minimal cytotoxicity. Thus, we have provided a new copolymer platform for siRNA delivery that we can continue to modify for improved delivery of siRNA in vitro and eventually in vivo. PMID:24424156

  7. Addition of poly (propylene glycol) to multiblock copolymer to optimize siRNA delivery.

    PubMed

    Dai, Zhi; Arévalo, Maria T; Li, Junwei; Zeng, Mingtao

    2014-01-01

    Previous studies have examined different strategies for siRNA delivery with varying degrees of success. These include use of viral vectors, cationic liposomes, and polymers. Several copolymers were designed and synthesized based on blocks of poly(ethylene glycol) PEG, poly(propylene glycol) PPG, and poly(l-lysine). These were designated as P1, P2, and P3. We studied the copolymer self-assembly, siRNA binding, particle size, surface potential, architecture of the complexes, and siRNA delivery. Silencing of GFP using copolymer P3 to deliver GFP-specific siRNA to Neuro-2a cells expressing GFP was almost as effective as using Lipofectamine 2000, with minimal cytotoxicity. Thus, we have provided a new copolymer platform for siRNA delivery that we can continue to modify for improved delivery of siRNA in vitro and eventually in vivo.

  8. Construction of simple and efficient siRNA validation systems for screening and identification of effective RNAi-targeted sequences from mammalian genes.

    PubMed

    Tsai, Wen-Hui; Chang, Wen-Tsan

    2014-01-01

    RNA interference (RNAi) is an evolutionarily conserved mechanism of gene silencing induced by double-stranded RNAs (dsRNAs). Among the widely used dsRNAs, small interfering RNAs (siRNAs) and short hairpin RNAs have evolved as extremely powerful and the most popular gene silencing reagents. The key challenge to achieving efficient gene silencing especially for the purpose of therapeutics is mainly dependent on the effectiveness and specificity of the selected RNAi-targeted sequences. Practically, only a small number of dsRNAs are capable of inducing highly effective and sequence-specific gene silencing via RNAi mechanism. In addition, the efficiency of gene silencing induced by dsRNAs can only be experimentally examined based on inhibition of the target gene expression. Therefore, it is essential to develop a fully robust and comparative validation system for measuring the efficacy of designed dsRNAs. In this chapter, we focus our discussion on a reliable and quantitative reporter-based siRNA validation system that has been previously established in our laboratory. The system consists of a short synthetic DNA fragment containing an RNAi-targeted sequence of interest and two expression vectors for targeting reporter and triggering siRNA expressions. The efficiency of siRNAs is determined by their abilities to inhibit expression of the targeting reporters with easily quantified readouts including enhanced green fluorescence protein and firefly luciferase. Since only a readily available short synthetic DNA fragment is needed for constructing this reliable and efficient reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective RNAi-targeted sequences from mammalian genes but also implicates the use of RNAi-based dsRNA reagents for reverse functional genomics and molecular therapeutics.

  9. RNase non-sensitive and endocytosis independent siRNA delivery system: delivery of siRNA into tumor cells and high efficiency induction of apoptosis

    NASA Astrophysics Data System (ADS)

    Jiang, Xinglu; Wang, Guobao; Liu, Ru; Wang, Yaling; Wang, Yongkui; Qiu, Xiaozhong; Gao, Xueyun

    2013-07-01

    To date, RNase degradation and endosome/lysosome trapping are still serious problems for siRNA-based molecular therapy, although different kinds of delivery formulations have been tried. In this report, a cell penetrating peptide (CPP, including a positively charged segment, a linear segment, and a hydrophobic segment) and a single wall carbon nanotube (SWCNT) are applied together by a simple method to act as a siRNA delivery system. The siRNAs first form a complex with the positively charged segment of CPP via electrostatic forces, and the siRNA-CPP further coats the surface of the SWCNT via hydrophobic interactions. This siRNA delivery system is non-sensitive to RNase and can avoid endosome/lysosome trapping in vitro. When this siRNA delivery system is studied in Hela cells, siRNA uptake was observed in 98% Hela cells, and over 70% mRNA of mammalian target of rapamycin (mTOR) is knocked down, triggering cell apoptosis on a significant scale. Our siRNA delivery system is easy to handle and benign to cultured cells, providing a very efficient approach for the delivery of siRNA into the cell cytosol and cleaving the target mRNA therein.

  10. SiRNA delivery with functionalized carbon nanotubes.

    PubMed

    Varkouhi, Amir Khashayar; Foillard, Stéphanie; Lammers, Twan; Schiffelers, Raymond M; Doris, Eric; Hennink, Wim E; Storm, Gert

    2011-09-20

    Carbon nanotubes (CNTs) have been studied for drug, antigen and nucleic acid delivery both in vitro and in vivo. Due to their nano-needle structure, they are supposed to cross the plasma membrane and enter directly into the cytoplasm likely upon an endocytosis-independent mechanism without inducing cell death. In this study, two cationically functionalized CNTs (CNT-PEI and CNT-pyridinium) were investigated for siRNA delivery. Both functionalized CNTs complexed siRNA and showed 10-30% silencing activity and a cytotoxicity of 10-60%. However, in terms of reduced toxicity or increased silencing activity, CNT-PEI and CNT-pyridinium did not show an added value over PEI and other standard transfection systems. Probably, the type of functionalization of carbon nanotubes might be a key parameter to obtain an efficient and non-cytotoxic CNT-based delivery system. Nevertheless, in view of the present results and importantly also of the non-degradability of CNTs, preference should currently be given to designing biodegradable carriers which mimic the needle structure of CNTs.

  11. siRNAs induce efficient RNAi response in Bombyx mori embryos.

    PubMed

    Yamaguchi, Junichi; Mizoguchi, Takayuki; Fujiwara, Haruhiko

    2011-01-01

    Short interference RNA (siRNA) is widely used in mammalian cells. In insects, however, reports concerning the suitablility of siRNA in vivo is very limited compared with that of long dsRNA, which is thought to be more effective. There is insufficient information on the essential rules of siRNA design in insects, as very few siRNAs have been tested in this context. To establish an effective method of gene silencing using siRNA in vivo in insects, we determined the effects of siRNA on seven target genes. We designed siRNAs according to a new guideline and injected them into eggs of Bombyx mori. At the mRNA level, the expression of most of these genes was successfully silenced, down to less than half the constitutive level, which in some cases led to the development of distinctive phenotypes. In addition, we observed stronger effect of siRNA both on the mRNA level and the phenotype than that of long dsRNA under comparable conditions. These results indicate that direct injection of siRNA is an effective reverse-genetics tool for the analysis of embryogenesis in vivo in insects.

  12. Turning Squalene into Cationic Lipid Allows a Delivery of siRNA in Cultured Cells

    PubMed Central

    Bertrand, Jean-Rémi; Lucas, Claire; Pham, Ngoc Minh; Durieu, Catherine; Couvreur, Patrick; Desmaële, Didier

    2015-01-01

    Covalent binding of squalene to siRNA has already been shown to be an interesting way of delivering siRNA in vivo. Whether squalene derivatives could also be used to deliver siRNA in cells without covalent binding similar to usual transfection with cationic lipids is the question addressed in this article. Accordingly, we investigated the activity of two squalene derivatives bearing a quaternary ammonium head group and a guanidinium group, respectively. The second derivative displayed interesting properties for delivering siRNA into cells in vitro. PMID:25894614

  13. SiRNA Crosslinked Nanoparticles for the Treatment of Inflammation‐induced Liver Injury

    PubMed Central

    Tang, Yaqin; Zeng, Ziying; He, Xiao; Wang, Tingting

    2016-01-01

    RNA interference mediated by small interfering RNA (siRNA) provides a powerful tool for gene regulation, and has a broad potential as a promising therapeutic strategy. However, therapeutics based on siRNA have had limited clinical success due to their undesirable pharmacokinetic properties. This study presents pH‐sensitive nanoparticles‐based siRNA delivery systems (PNSDS), which are positive‐charge‐free nanocarriers, composed of siRNA chemically crosslinked with multi‐armed poly(ethylene glycol) carriers via acid‐labile acetal linkers. The unique siRNA crosslinked structure of PNSDS allows it to have minimal cytotoxicity, high siRNA loading efficiency, and a stimulus‐responsive property that enables the selective intracellular release of siRNA in response to pH conditions. This study demonstrates that PNSDS can deliver tumor necrosis factor alpha (TNF‐α) siRNA into macrophages and induce the efficient down regulation of the targeted gene in complete cell culture media. Moreover, PNSDS with mannose targeting moieties can selectively accumulate in mice liver, induce specific inhibition of macrophage TNF‐α expression in vivo, and consequently protect mice from inflammation‐induced liver damages. Therefore, this novel siRNA delivering platform would greatly improve the therapeutic potential of RNAi based therapies. PMID:28251047

  14. Development of streptavidin-based nanocomplex for siRNA delivery.

    PubMed

    Shukla, Ravi S; Tai, Wanyi; Mahato, Rubi; Jin, Wei; Cheng, Kun

    2013-12-02

    In our previous study, we have identified a PCBP2 siRNA that exhibits antifibrotic activity in rat hepatic stellate cells (HSCs) by inhibition of αCP2, a protein responsible for stabilization of the collagen α1 (I) mRNA in alcoholic liver fibrosis. This study aims to develop a streptavidin-based nanocomplex that can efficiently deliver the PCBP2 siRNA to HSCs. Biotin-siRNA and biotin-cholesterol were mixed with streptavidin to form the streptavidin-biotin complex, which was further condensed electrostatically with positively charged protamine to form the final multicomponent siRNA nanocomplex in the size range of 150-250 nm. The siRNA nanocomplex does not induce cytotoxicity in rat HSCs as compared to commercially available transfection agents. The cellular uptake efficiency of the siRNA nanocomplex is higher in rat HSCs than other cell lines, such as Caco-2 and PC-3, indicating that receptor-mediated endocytosis mainly contributes to the cellular uptake of the siRNA nanocomplex. The siRNA nanocomplex exhibits more than 85% silencing effect on the PCBP2 mRNA in HSCs. Stability study indicates that the nanocomplex can efficiently protect siRNA from degradation in the serum. The streptavidin-based multicomponent siRNA nanocomplex provides a promising strategy to deliver the PCBP2 siRNA to HSCs. Moreover, the nanocomplex can be used as a platform for other diseases by changing the siRNA sequence and targeting ligand.

  15. Integrated siRNA design based on surveying of features associated with high RNAi effectiveness

    PubMed Central

    Gong, Wuming; Ren, Yongliang; Xu, Qiqi; Wang, Yejun; Lin, Dong; Zhou, Haiyan; Li, Tongbin

    2006-01-01

    Background Short interfering RNAs have allowed the development of clean and easily regulated methods for disruption of gene expression. However, while these methods continue to grow in popularity, designing effective siRNA experiments can be challenging. The various existing siRNA design guidelines suffer from two problems: they differ considerably from each other, and they produce high levels of false-positive predictions when tested on data of independent origins. Results Using a distinctly large set of siRNA efficacy data assembled from a vast diversity of origins (the siRecords data, containing records of 3,277 siRNA experiments targeting 1,518 genes, derived from 1,417 independent studies), we conducted extensive analyses of all known features that have been implicated in increasing RNAi effectiveness. A number of features having positive impacts on siRNA efficacy were identified. By performing quantitative analyses on cooperative effects among these features, then applying a disjunctive rule merging (DRM) algorithm, we developed a bundle of siRNA design rule sets with the false positive problem well curbed. A comparison with 15 online siRNA design tools indicated that some of the rule sets we developed surpassed all of these design tools commonly used in siRNA design practice in positive predictive values (PPVs). Conclusion The availability of the large and diverse siRNA dataset from siRecords and the approach we describe in this report have allowed the development of highly effective and generally applicable siRNA design rule sets. Together with ever improving RNAi lab techniques, these design rule sets are expected to make siRNAs a more useful tool for molecular genetics, functional genomics, and drug discovery studies. PMID:17129386

  16. HIVsirDB: A Database of HIV Inhibiting siRNAs

    PubMed Central

    Thakur, Nishant; Sharma, Arun; Raghava, Gajendra P. S.; Kumar, Manoj

    2011-01-01

    Background Human immunodeficiency virus (HIV) is responsible for millions of deaths every year. The current treatment involves the use of multiple antiretroviral agents that may harm patients due to their toxic nature. RNA interference (RNAi) is a potent candidate for the future treatment of HIV, uses short interfering RNA (siRNA/shRNA) for silencing HIV genes. In this study, attempts have been made to create a database HIVsirDB of siRNAs responsible for silencing HIV genes. Descriptions HIVsirDB is a manually curated database of HIV inhibiting siRNAs that provides comprehensive information about each siRNA or shRNA. Information was collected and compiled from literature and public resources. This database contains around 750 siRNAs that includes 75 partially complementary siRNAs differing by one or more bases with the target sites and over 100 escape mutant sequences. HIVsirDB structure contains sixteen fields including siRNA sequence, HIV strain, targeted genome region, efficacy and conservation of target sequences. In order to facilitate user, many tools have been integrated in this database that includes; i) siRNAmap for mapping siRNAs on target sequence, ii) HIVsirblast for BLAST search against database, iii) siRNAalign for aligning siRNAs. Conclusion HIVsirDB is a freely accessible database of siRNAs which can silence or degrade HIV genes. It covers 26 types of HIV strains and 28 cell types. This database will be very useful for developing models for predicting efficacy of HIV inhibiting siRNAs. In summary this is a useful resource for researchers working in the field of siRNA based HIV therapy. HIVsirDB database is accessible at http://crdd.osdd.net/raghava/hivsir/. PMID:22022467

  17. Lipid-based systemic delivery of siRNA

    PubMed Central

    Tseng, Yu-Cheng; Mozumdar, Subho; Huang, Leaf

    2011-01-01

    RNAi technology has brought a new category of treatments for various diseases including genetic diseases, viral diseases, and cancer. Despite the great versatility of RNAi that can down regulate almost any protein in the cells, the delicate and precise machinery used for silencing is the same. The major challenge indeed for RNAi-based therapy is the delivery system. In this review, we start with the uniqueness and mechanism of RNAi machinery and the utility of RNAi in therapeutics. Then we discuss the challenges in systemic siRNA delivery by dividing them into two categories--kinetic and physical barriers. At the end, we discuss different strategies to overcome these barriers, especially focusing on the step of endosome escape. Toxicity issues and current successful examples for lipid-based delivery are also included in the review. PMID:19328215

  18. Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration.

    PubMed

    Ferrés-Coy, A; Galofré, M; Pilar-Cuéllar, F; Vidal, R; Paz, V; Ruiz-Bronchal, E; Campa, L; Pazos, Á; Caso, J R; Leza, J C; Alvarado, G; Montefeltro, A; Valdizán, E M; Artigas, F; Bortolozzi, A

    2016-03-01

    Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, ~80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive-like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant-like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies.

  19. Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes.

    PubMed

    Chandra, Partha K; Kundu, Anup K; Hazari, Sidhartha; Chandra, Sruti; Bao, Lili; Ooms, Tara; Morris, Gilbert F; Wu, Tong; Mandal, Tarun K; Dash, Srikanta

    2012-09-01

    Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5'-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection.

  20. Selecting effective siRNA target sequences by using Bayes' theorem.

    PubMed

    Takasaki, Shigeru

    2009-10-01

    Short interfering RNA (siRNA) has been widely used for studying gene functions in mammalian cells but varies markedly in its gene silencing efficacy. Although many design rules/guidelines for effective siRNAs based on various criteria have been reported recently, there are few consistencies among them. This makes it difficult to select effective siRNA sequences in mammalian genes. Another shortcoming of most previously reported methods is that they cannot estimate the probability that a candidate sequence will silence the target gene. The analytical prediction method proposed in the present study uses Bayes' theorem to select effective siRNA target sequences from many possible candidate sequences. It is quite different from the previous score-based siRNA design techniques and can predict the probability that a candidate siRNA sequence will be effective. The results of evaluating it by applying it to recently reported effective and ineffective siRNA sequences for various genes indicate that it would be useful for many other genes. It should therefore be useful for selecting siRNA sequences effective for mammalian genes.

  1. Lipid-based siRNA Delivery Systems: Challenges, Promises and Solutions Along the Long Journey.

    PubMed

    Sarisozen, Can; Salzano, Giuseppina; Torchilin, Vladimir P

    RNA interference (RNAi) is an evolutionary conserved highly specific gene-silencing mechanism initiated by small interfering RNA (siRNA) molecules. Fast-paced preclinical and clinical studies helped the siRNA technology become an efficient tool for undruggable targets in different diseases including genetic diseases, viral diseases and cancer. Despite great feature of siRNAs that can down-regulate any protein in the cells, the full potential and the success of the preclinical studies could not be translated into largely successful clinical outcomes. It has become clear that the possibility of overcoming the pitfalls for in vivo siRNA therapy fully depends on delivery systems. In this review, we start with the challenges and barriers for in vivo siRNA delivery. Then we briefly discuss the recent developments in siRNA modification technology. We specifically focused on siRNA lipidation and delivery approaches with special emphasis on the lipid based hybrid systems. Here we summarize the journey of lipid-based micelle-like nanoparticle systems that combine longevity in blood, effective cellular uptake and endosomal escape for successful siRNA delivery and discuss the multifunctional stimuli-sensitive systems based on lipids as the next generation smart systems.

  2. Silencing the roadblocks to effective triple-negative breast cancer treatments by siRNA nanoparticles.

    PubMed

    Parvani, Jenny G; Jackson, Mark W

    2017-04-01

    Over the past decade, RNA interference (RNAi) has been ubiquitously utilized to study biological function in vitro; however, limitations were associated with its utility in vivo More recently, small interfering RNA (siRNA) nanoparticles with improved biocompatibility have gained prevalence as a potential therapeutic option for the treatment of various diseases. The adaptability of siRNA nanoparticles enables the delivery of virtually any siRNA, which is especially advantageous for therapeutic applications in heterogeneous diseases that lack unifying molecular features, such as triple-negative breast cancer (TNBC). TNBC is an aggressive subtype of breast cancer that is stratified by the lack of estrogen receptor/progesterone receptor expression and HER2 amplification. There are currently no FDA-approved targeted therapies for the treatment of TNBCs, making cytotoxic chemotherapy the only treatment option available to these patients. In this review, we outline the current status of siRNA nanoparticles in clinical trials for cancer treatment and discuss the promising preclinical approaches that have utilized siRNA nanoparticles for TNBC treatment. Next, we address TNBC subtype-specific therapeutic interventions and highlight where and how siRNA nanoparticles fit into these strategies. Lastly, we point out ongoing challenges in the field of siRNA nanoparticle research that, if addressed, would significantly improve the efficacy of siRNA nanoparticles as a therapeutic option for cancer treatment.

  3. Efficient siRNA delivery system using carboxilated single-wall carbon nanotubes in cancer treatment.

    PubMed

    Neagoe, Ioana Berindan; Braicu, Cornelia; Matea, Cristian; Bele, Constantin; Florin, Graur; Gabriel, Katona; Veronica, Chedea; Irimie, Alexandru

    2012-08-01

    Several functionalized carbon nanotubes have been designed and tested for the purpose of nucleic acid delivery. In this study, the capacity of SWNTC-COOH for siRNA deliverey were investigated delivery in parallel with an efficient commercial system. Hep2G cells were reverse-transfected with 50 nM siRNA (p53 siRNA, TNF-alphasiRNA, VEGFsiRNA) using the siPORT NeoFX (Ambion) transfection agent in paralel with SWNTC-COOH, functionalised with siRNA. The highest level of gene inhibition was observed in the cases treated with p53 siRNA gene; in the case of transfection with siPort, the NeoFX value was 33.8%, while in the case of SWNTC-COOH as delivery system for p53 siRNA was 37.5%. The gene silencing capacity for VEGF was 53.7%, respectively for TNF-alpha 56.7% for siPORT NeoFX delivery systems versus 47.7% (VEGF) and 46.5% (TNF-alpha) for SWNTC-COOH delivery system. SWNTC-COOH we have been showed to have to be an efficient carrier system. The results from the inhibition of gene expresion for both transfection systems were confirmed at protein level. Overall, the lowest mRNA expression was confirmed at protein level, especially in the case of p53 siRNA and TNF-alpha siRNA transfection. Less efficient reduction protein expressions were observed in the case of VEGF siRNA, for both transfection systems at 24 h; only at 48 h, there was a statistically significant reduction of VEGF protein expression. SWCNT-COOH determined an efficient delivery of siRNA. SWNTC-COOH, combined with suitable tumor markers like p53 siRNA, TNFalpha siRNA or VEGF siRNA can be used for the efficient delivery of siRNA.

  4. Reconsideration of in silico siRNA design from a perspective of heterogeneous data integration: problems and solutions.

    PubMed

    Liu, Qi; Zhou, Han; Zhu, Ruixin; Xu, Ying; Cao, Zhiwei

    2014-03-01

    The success of RNA interference (RNAi) depends on the interaction between short interference RNAs (siRNAs) and mRNAs. Design of highly efficient and specific siRNAs has become a challenging issue in applications of RNAi. Here, we present a detailed survey on the state-of-the-art siRNAs design, focusing on several key issues with the current in silico RNAi studies, including: (i) inconsistencies among the proposed guidelines for siRNAs design and the incomplete list of siRNAs features, (ii) improper integration of the heterogeneous cross-platform siRNAs data, (iii) inadequate consideration of the binding specificity of the target mRNAs and (iv) reduction in the 'off-target' effect in siRNAs design. With these considerations, the popular in silico siRNAs design rules are reexamined and several inconsistent viewpoints toward siRNAs feature identifications are clarified. In addition, novel computational models for siRNAs design using state-of-art machine learning techniques are discussed, which focus on heterogeneous data integration, joint feature selection and customized siRNAs screening toward highly specific targets. We believe that addressing such issues in siRNA study will provide new clues for further improved design of more efficient and specific siRNAs in RNAi.

  5. siRNA and miRNA processing: new functions for Cajal bodies.

    PubMed

    Pontes, Olga; Pikaard, Craig S

    2008-04-01

    In diverse eukaryotes, micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) regulate important processes that include mRNA inactivation, viral defense, chromatin modification, and transposon silencing. Recently, nucleolus-associated Cajal bodies in plants have been implicated as sites of siRNA and miRNA biogenesis, whereas in animals siRNA and miRNA dicing occurs in the cytoplasm. The plant nucleolus also contains proteins of the nonsense-mediated mRNA decay pathway that in animals are found associated with cytoplasmic processing bodies (P-bodies). P-bodies also function in the degradation of mRNAs subjected to miRNA and siRNA targeting. Collectively, these observations suggest interesting variations in the way siRNAs and miRNAs can accomplish their similar functions in plants and animals.

  6. Non-Catalytic RISCs and Kinetics Determine Mammalian siRNA Sub-Cellular Localization.

    PubMed

    Ji, Fengmin; Liu, Lianyun; Tien, Ya-Hsin; Peng, Yi-Hsien; Lee, Hoong-Chien

    2015-01-01

    Small interfering RNAs (siRNAs) are fundamental to the regulation of cell function. Much is known about its gene interfering mechanism, but a kinetic description of it is still lacking. Here, we derived a set of reaction-diffusion equations for multiple RNA-induced silencing complex (RISC) pathways that give quantitative temporal and spatial descriptions of the siRNA process in mammalian cell, and are able to correctly describe all salient experimentally observed patterns of sub-cellular siRNA localization, including those that, at first glance, appear irreconcilable. These results suggest siRNA sub-cellular localization mainly concerns the non-catalytic RISC-target complex, and is caused by the selectiveness of RISC-target interaction and the permeability of the nuclear membrane to siRNA strands but not to RISC-target complexes. Our method is expected to be useful in devising RNAi based cell regulation strategies.

  7. ICS-283: a system for targeted intravenous delivery of siRNA.

    PubMed

    Schiffelers, Raymond M; Storm, Gert

    2006-05-01

    ICS-283 was developed within Intradigm Corporation as a system that is designed for the systemic delivery of therapeutic small interfering (siRNA) to sites of pathological angiogenesis. The non-viral siRNA delivery system is based on synthetic nanoparticles, known as Targe (Intradigm Corporation), which functions as a broad-platform technology to deliver siRNA to specific target cells in diseased tissues. The system is constructed to incorporate different functionalities that address critical needs for successful nucleic acid delivery. The TargeTran synthetic vector is a self-assembling, layered nanoparticle that protects and targets siRNA to specific cell types in pathological tissues. At present, ICS-283 is the only antiangiogenic siRNA delivery system that is designed for intravenous administration to treat angiogenesis-driven diseases.

  8. Multi-target siRNA: Therapeutic Strategy for Hepatocellular Carcinoma

    PubMed Central

    Li, Tiejun; Xue, Yuwen; Wang, Guilan; Gu, Tingting; Li, Yunlong; Zhu, York Yuanyuan; Chen, Li

    2016-01-01

    Multiple targets RNAi strategy is a preferred way to treat multigenic diseases, especially cancers. In the study, multi-target siRNAs were designed to inhibit NET-1, EMS1 and VEGF genes in hepatocellular carcinoma (HCC) cells. And multi-target siRNAs showed better silencing effects on NET-1, EMS1 and VEGF, compared with single target siRNA. Moreover, multi-target siRNA showed greater suppression effects on proliferation, migration, invasion, angiogenesis and induced apoptosis in HCC cells. The results suggested that multi-target siRNA might be a preferred strategy for cancer therapy and NET-1, EMS1 and VEGF could be effective targets for HCC treatments. PMID:27390607

  9. HSP47 siRNA conjugated with cationized gelatin microspheres suppresses peritoneal fibrosis in mice.

    PubMed

    Obata, Yoko; Nishino, Tomoya; Kushibiki, Toshihiro; Tomoshige, Ryuji; Xia, Zhiyin; Miyazaki, Masanobu; Abe, Katsushige; Koji, Takehiko; Tabata, Yasuhiko; Kohno, Shigeru

    2012-07-01

    Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for the biosynthesis and secretion of collagen and is expressed in the fibrotic peritoneum. In the present study, we evaluated the efficacy of HSP47 small interfering RNA (siRNA) to suppress the development of peritoneal fibrosis induced by chlorhexidine gluconate in mice. We initially confirmed that biodegradable cationized gelatin microspheres (CGMs) containing HSP47 siRNA could continuously release siRNA over 21 days as a result of microsphere degradation. We then determined that a single injection of CGMs incorporating HSP47 siRNA suppressed collagen expression and macrophage infiltration, thereby preventing peritoneal fibrosis. Therefore, we suggest that this controlled-release technology using HSP47 siRNA is a potential treatment for peritoneal fibrosis. Additionally, RNA interference combined with CGMs as a drug-delivery system may lead to new strategies for knocking down specific genes in vivo.

  10. Abundance estimation and conservation biology

    USGS Publications Warehouse

    Nichols, J.D.; MacKenzie, D.I.

    2004-01-01

    inference that increased recruitment was largely responsible for the improvements in population status and growth. However, various data sources also indicated that this increase in recruitment was likely a result of increased immigration rather than improved reproduction on the area. This latter inference is important from a conservation perspective in indicating the importance of birds in other locations to growth and health of the study population. Lukacs and Burnham presented material to be published elsewhere that dealt with the use of genetic markers in capture–recapture studies. The data sources for such studies are samples of hair or feces, which are then analyzed using molecular genetic techniques in order to determine individual genotypes with respect to a usually small number of loci. Two types of classification error can arise in such analyses. First, if only a small number of loci is examined, then there may be nonnegligible probabilities that multiple individual animals will have the same genotypes. The second type of error arises during the polymerase chain reaction (PCR) process and can result from failure of alleles to amplify (allelic dropout) or from PCR inhibitors in hair and feces that produce the appearance of false alleles or misprinting (Creel et al., 2003). Lukacs and Burnham developed models that formally incorporate possible misclassification of samples resulting from these errors. These models permit estimation of parameters such as abundance and survival in a manner that properly incorporates this uncertainty of individual identity. We anticipate that noninvasive sampling based on molecular genetic analyses of hair or feces will become extremely important for some species, and that the models of Lukacs and Burnham will become very popular for such analyses. MacKenzie & Nichols (2004) discuss the use of occupancy (proportion of patches or habitat area that is occupied) as a surrogate for abundance. In cases of territorial species and where

  11. Influence of Coronal Abundance Variations

    NASA Technical Reports Server (NTRS)

    Gurman, Joseph (Technical Monitor); DeLuca, Edward

    2005-01-01

    During the final year of this program we concentrated on understanding the how to constrain the models with the best available observations. Work on developing accurate temperature and density diagnostics fkom TRACE and CDS together with constrained fits of non-potential force free fields will be extremely useful in the guiding the next generation of coronal models. The program has produced three fully operation numerical codes that model multi-species of ions in coronal loops: Static models and constant flow models. The time dependent numerical models have not been completed. We have extended the steady flow investigations to study the effect these flows have on coronal structure as observed with TRACE. Coronal observations derive from heavy-ion emission; thus, we focus on the extent to which flow may modify coronal abundances by examining the heavy-ion abundance stratification within long-lived loops. We discuss the magnitudes of the physical effects modeled and compare simulated results with TRACE observations. These results can have a profound effect on the interpretation of TRACE observations.

  12. Abundances in Hot Evolved Stars

    NASA Astrophysics Data System (ADS)

    Werner, Klaus; Rauch, Thomas; Kruk, Jeffrey W.

    2009-05-01

    The hydrogen-deficiency in extremely hot post-AGB stars of spectral class PG1159 is probably caused by a (very) late helium-shell flash or a AGB final thermal pulse that consumes the hydrogen envelope, exposing the usually-hidden intershell region. Thus, the photospheric element abundances of these stars allow us to draw conclusions about details of nuclear burning and mixing processes in the precursor AGB stars. We compare predicted element abundances to those determined by quantitative spectral analyses performed with advanced non-LTE model atmospheres. A good qualitative and quantitative agreement is found for many species (He, C, N, O, Ne, F, Si, Ar) but discrepancies for others (P, S, Fe) point at shortcomings in stellar evolution models for AGB stars. Almost all of the chemical trace elements in these hot stars can only be identified in the UV spectral range. The Far Ultraviolet Spectroscopic Explorer and the Hubble Space Telescope played a crucial role for this research.

  13. OXYGEN ABUNDANCES IN CEPHEIDS

    SciTech Connect

    Luck, R. E.; Andrievsky, S. M.; Korotin, S. N.; Kovtyukh, V. V. E-mail: serkor@skyline.od.ua E-mail: scan@deneb1.odessa.ua

    2013-07-01

    Oxygen abundances in later-type stars, and intermediate-mass stars in particular, are usually determined from the [O I] line at 630.0 nm, and to a lesser extent, from the O I triplet at 615.7 nm. The near-IR triplets at 777.4 nm and 844.6 nm are strong in these stars and generally do not suffer from severe blending with other species. However, these latter two triplets suffer from strong non-local thermodynamic equilibrium (NLTE) effects and thus see limited use in abundance analyses. In this paper, we derive oxygen abundances in a large sample of Cepheids using the near-IR triplets from an NLTE analysis, and compare those abundances to values derived from a local thermodynamic equilibrium (LTE) analysis of the [O I] 630.0 nm line and the O I 615.7 nm triplet as well as LTE abundances for the 777.4 nm triplet. All of these lines suffer from line strength problems making them sensitive to either measurement complications (weak lines) or to line saturation difficulties (strong lines). Upon this realization, the LTE results for the [O I] lines and the O I 615.7 nm triplet are in adequate agreement with the abundance from the NLTE analysis of the near-IR triplets.

  14. [In vivo imaging of liposomal small interfering RNA (siRNA) trafficking by positron emission tomography].

    PubMed

    Ando, Hidenori; Yonenaga, Norihito; Asai, Tomohiro; Hatanaka, Kentaro; Koide, Hiroyuki; Tsuzuku, Takuma; Harada, Norihiro; Tsukada, Hideo; Oku, Naoto

    2012-01-01

    In the development of nucleic acid medicines such as small interfering RNA (siRNA) drugs, one problem is how to study the pharmacokinetics and pharmacodynamics, since the precise in vivo behavior of siRNA is hard to detect. In this research, to establish a highly sensitive detection system of siRNA biodistribution in the whole body, the technology of positron imaging was applied. First, a one-step synthetic method in which double-stranded siRNA was directly labeled by a positron emitter, (18)F, was developed. By using [(18)F]-labeled siRNA ([(18)F]-siRNA), the complex of siRNA and polycation liposomes (PCL) containing dicetylphosphate tetraethylenepentamine (TEPA-PCL) was prepared. Then, the biodistribution of the siRNA after intravenous administration to mice was analyzed by planar positron imaging system (PPIS). As a result, whereas naked [(18)F]-siRNA was immediately excreted in mouse bladder after administration, the complex with cationic liposome (CL) was trapped in the lungs. Furthermore, [(18)F]-siRNA carried with PEGylated CL (PL) was distributed throughout the body, suggesting that it circulated in the bloodstream for an extended period of time. Additionally, PET imaging revealed more detailed biodistribution of the siRNA than in vivo imaging system (IVIS) because PET imaging is not affected by the depth variation of target tissues. On the other hand, to induce high accumulation of siRNAs against c-myc, MDM2, and VEGF in tumor tissue, a tumor-targeting probe, RGD peptide, was grafted at the top of PEG chain in PEGylated TEPA-PCL and the effect of the complex on experimental lung metastasis of B16 melanoma was examined. The complex suppressed the progression of tumor. We believe that the positron imaging data would support the development of siRNA agent for clinical use.

  15. Dicetyl phosphate-tetraethylenepentamine-based liposomes for systemic siRNA delivery.

    PubMed

    Asai, Tomohiro; Matsushita, Saori; Kenjo, Eriya; Tsuzuku, Takuma; Yonenaga, Norihito; Koide, Hiroyuki; Hatanaka, Kentaro; Dewa, Takehisa; Nango, Mamoru; Maeda, Noriyuki; Kikuchi, Hiroshi; Oku, Naoto

    2011-03-16

    Dicetyl phosphate-tetraethylenepentamine (DCP-TEPA) conjugate was newly synthesized and formed into liposomes for efficient siRNA delivery. Formulation of DCP-TEPA-based polycation liposomes (TEPA-PCL) complexed with siRNA was examined by performing knockdown experiments using stable EGFP-transfected HT1080 human fibrosarcoma cells and siRNA for GFP. An adequate amount of DCP-TEPA in TEPA-PCL and N/P ratio of TEPA-PCL/siRNA complexes were determined based on the knockdown efficiency. Then, the biodistribution of TEPA-PCL modified with poly(ethylene glycol) (PEG) was examined in BALB/c mice. As a result, TEPA-PCL modified with PEG6000 avoided reticuloendothelial system uptake and showed long circulation in the bloodstream. On the other hand, PEGylation of TEPA-PCL/siRNA complexes caused dissociation of a portion of the siRNA from the liposomes. However, we found that the use of cholesterol-conjugated siRNA improved the interaction between TEPA-PCL and siRNA, which allowed PEGylation of TEPA-PCL/siRNA complexes without siRNA dissociation. In addition, TEPA-PCL complexed with cholesterol-conjugated siRNA showed potent knockdown efficiency in stable luciferase-transfected B16-F10 murine melanoma cells. Finally, the biodistribution of cholesterol-conjugated siRNA formulated in PEGylated TEPA-PCL was examined by performing near-infrared fluorescence imaging in Colon26 NL-17 murine carcinoma-bearing mice. Our results showed that tumor targeting with siRNA via systemic administration was achieved by using PEGylated TEPA-PCL combined with active targeting with Ala-Pro-Arg-Pro-Gly, a peptide used for targeting angiogenic endothelium.

  16. siRNA liposome-gold nanorod vectors for multispectral optoacoustic tomography theranostics

    NASA Astrophysics Data System (ADS)

    Taruttis, Adrian; Lozano, Neus; Nunes, Antonio; Jasim, Dhifaf A.; Beziere, Nicolas; Herzog, Eva; Kostarelos, Kostas; Ntziachristos, Vasilis

    2014-10-01

    Therapeutic applications of gene silencing using siRNA have seen increasing interest over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained.Therapeutic applications of gene silencing using siRNA have seen increasing interest over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained. Electronic supplementary information (ESI) available: Experimental section and dark-field microscopy in both tumors 24 h after injection of the complex have been included. See DOI: 10.1039/c4nr04164j

  17. Identification of miniature inverted-repeat transposable elements (MITEs) and biogenesis of their siRNAs in the Solanaceae: new functional implications for MITEs.

    PubMed

    Kuang, Hanhui; Padmanabhan, Chellappan; Li, Feng; Kamei, Ayako; Bhaskar, Pudota B; Ouyang, Shu; Jiang, Jiming; Buell, C Robin; Baker, Barbara

    2009-01-01

    Small RNAs regulate the genome by guiding transcriptional and post-transcriptional silencing machinery to specific target sequences, including genes and transposable elements (TEs). Although miniature inverted-repeat transposable elements (MITEs) are closely associated with euchromatic genes, the broader functional impact of these short TE insertions in genes is largely unknown. We identified 22 families of MITEs in the Solanaceae (MiS1-MiS22) and found abundant MiS insertions in Solanaceae genomic DNA and expressed sequence tags (EST). Several Solanaceae MITEs generate genome changes that potentially affect gene function and regulation, most notably, a MiS insertion that provides a functionally indispensable alternative exon in the tobacco mosaic virus N resistance gene. We show that MITEs generate small RNAs that are primarily 24 nt in length, as detected by Northern blot hybridization and by sequencing small RNAs of Solanum demissum, Nicotiana glutinosa, and Nicotiana benthamiana. Additionally, we show that stable RNAi lines silencing DICER-LIKE3 (DCL3) in tobacco and RNA-dependent RNA polymerase 2 (RDR2) in potato cause a reduction in 24-nt MITE siRNAs, suggesting that, as in Arabidopsis, TE-derived siRNA biogenesis is DCL3 and RDR2 dependent. We provide evidence that DICER-LIKE4 (DCL4) may also play a role in MITE siRNA generation in the Solanaceae.

  18. Insertion sequences enrichment in extreme Red sea brine pool vent.

    PubMed

    Elbehery, Ali H A; Aziz, Ramy K; Siam, Rania

    2017-03-01

    Mobile genetic elements are major agents of genome diversification and evolution. Limited studies addressed their characteristics, including abundance, and role in extreme habitats. One of the rare natural habitats exposed to multiple-extreme conditions, including high temperature, salinity and concentration of heavy metals, are the Red Sea brine pools. We assessed the abundance and distribution of different mobile genetic elements in four Red Sea brine pools including the world's largest known multiple-extreme deep-sea environment, the Red Sea Atlantis II Deep. We report a gradient in the abundance of mobile genetic elements, dramatically increasing in the harshest environment of the pool. Additionally, we identified a strong association between the abundance of insertion sequences and extreme conditions, being highest in the harshest and deepest layer of the Red Sea Atlantis II Deep. Our comparative analyses of mobile genetic elements in secluded, extreme and relatively non-extreme environments, suggest that insertion sequences predominantly contribute to polyextremophiles genome plasticity.

  19. The Abundance of Interstellar Fluorine

    NASA Technical Reports Server (NTRS)

    Lauroesch, James T.

    2005-01-01

    The primary objective of this program was to obtain FUSE observations of the interstellar absorption lines of F I at 951 and 954 Angstroms to derive the abundance of fluorine toward the star HD 164816. The nucleosynthetic source(s) of fluorine are still a matter of debate - the present day abundance of fluorine can potentially constrain models for pulsationally driven dredge-up in asymptotic giant branch stars. An accurate measure for the depletion behavior of fluorine will determine whether it may be detectable in QSO absorption line systems - an unambiguous detection of fluorine at suitably high redshifts would provide the best evidence to date for the neutrino process in massive stars. Furthermore, due to its extreme reactivity, measurement of the gas-phase interstellar fluorine abundance is important for models of grain chemistry. Despite the importance of measuring the interstellar fluorine abundance, at the time of our proposal only one previous detection has been made due to the low relative abundance of fluorine, the lack of lines outside the far-UV, and the blending of the available F I transitions with lines of Hz. The star HD 164816 is associated with the Lagoon nebula (M8), and at a distance of approximately 1.5 kpc probes both distant and local gas. Beginning April 8th, 2004 FUSE FP-Split observations of the star HD 164816 were obtained for this program. This data became available in the FUSE data archive May 21, 2004, and these observations were then downloaded and we began our analysis. Our analysis procedure has involved (1) fitting stellar models to the FUSE spectra, (2) using the multiple lines of Hz and N I at other wavelengths in the FUSE bandpass to derive column densities for the lines of H2 and N I which are blended with the F I features at 951 and 954 angstroms (3) the measurement of the column densities of F I and the species O I and C1 I which are important species for the dis-entangling of dust and nucleosynthetic effects. As discussed in

  20. Development and optimization of nanosomal formulations for siRNA delivery to the liver.

    PubMed

    Kundu, Anup K; Chandra, Partha K; Hazari, Sidhartha; Pramar, Yashoda V; Dash, Srikanta; Mandal, Tarun K

    2012-02-01

    The objective of this study is to develop an effective siRNA delivery system for successful delivery to the liver for the treatment of HCV. Nanosize liposomes (nanosomes) have been prepared using a mixture of cholesterol and DOTAP. A functional siRNA was encapsulated into nanosomes following condensation with protamine sulfate. The delivery of siRNA was optimized in an in vitro cell culture system. The efficacy of the formulations was evaluated by measuring functional gene silencing and cytotoxicity. Encapsulation of siRNA ≥ 7.4 nM resulted in successful delivery of siRNA to nearly 100% of cells. The formulations containing lipid-to-siRNA ratio ≥ 10.56:1 instantly cleared approximately 85% of HCV while maintaining cell viability at about 90%. The formulations were sonicated to further reduce the particle size. The size of these formulations was decreased up to 100 nm. However, there were no significant changes observed in zeta potential, or in siRNA encapsulation and integrity following sonication. The sonicated formulations also showed higher liver hepatocytes deposition and gene silencing properties. This study therefore provides a novel approach of siRNA delivery to liver hepatocytes, which can also be applied to treat HCV in chronic liver diseases.

  1. Therapeutic Effects of Myeloid Cell Leukemia-1 siRNA on Human Acute Myeloid Leukemia Cells

    PubMed Central

    Karami, Hadi; Baradaran, Behzad; Esfahani, Ali; Sakhinia, Masoud; Sakhinia, Ebrahim

    2014-01-01

    Purpose: Up-regulation of Mcl-1, a known anti-apoptotic protein, is associated with the survival and progression of various malignancies including leukemia. The aim of this study was to explore the effect of Mcl-1 small interference RNA (siRNA) on the proliferation and apoptosis of HL-60 acute myeloid leukemia (AML) cells. Methods: siRNA transfection was performed using Lipofectamine™2000 reagent. Relative mRNA and protein expressions were quantified by quantitative real-time PCR and Western blotting, respectively. Trypan blue assay was performed to assess tumor cell proliferation after siRNA transfection. The cytotoxic effect of Mcl-1 siRNA on leukemic cells was measured using MTT assay. Apoptosis was detected using ELISA cell death assay. Results: Mcl-1 siRNA clearly lowered both Mcl-1 mRNA and protein levels in a time-dependent manner, leading to marked inhibition of cell survival and proliferation. Furthermore, Mcl-1 down-regulation significantly enhanced the extent of HL-60 apoptotic cells. Conclusion: Our results suggest that the down-regulation of Mcl-1 by siRNA can effectively trigger apoptosis and inhibit the proliferation of leukemic cells. Therefore, Mcl-1 siRNA may be a potent adjuvant in AML therapy. PMID:24754007

  2. Coupling of double-stranded RNA synthesis and siRNA generation in fission yeast RNAi.

    PubMed

    Colmenares, Serafin U; Buker, Shane M; Buhler, Marc; Dlakić, Mensur; Moazed, Danesh

    2007-08-03

    The fission yeast centromeric repeats are transcribed and ultimately processed into small interfering RNAs (siRNAs) that are required for heterochromatin formation. siRNA generation requires dsRNA synthesis by the RNA-directed RNA polymerase complex (RDRC) and processing by the Dicer ribonuclease. Here we show that Dcr1, the fission yeast Dicer, is physically associated with RDRC. Dcr1 generates siRNAs in an ATP-dependent manner that requires its conserved N-terminal helicase domain. Furthermore, C-terminal truncations of Dcr1 that abolish its interaction with RDRC, but can generate siRNA in vitro, abolish siRNA generation and heterochromatic gene silencing in vivo. Finally, reconstitution experiments show that the association of Dcr1 with RDRC strongly stimulates the dsRNA synthesis activity of RDRC. Our results suggest that heterochromatic dsRNA synthesis and siRNA generation are physically coupled processes. This coupling has implications for cis-restriction of siRNA-mediated heterochromatin assembly and for mechanisms that give rise to siRNA strand polarity.

  3. Antineoplastic Effects of siRNA against TMPRSS2-ERG Junction Oncogene in Prostate Cancer

    PubMed Central

    Urbinati, Giorgia; Ali, Hafiz Muhammad; Rousseau, Quentin; Chapuis, Hubert; Desmaële, Didier; Couvreur, Patrick; Massaad-Massade, Liliane

    2015-01-01

    TMPRSS2-ERG junction oncogene is present in more than 50% of patients with prostate cancer and its expression is frequently associated with poor prognosis. Our aim is to achieve gene knockdown by siRNA TMPRSS2-ERG and then to assess the biological consequences of this inhibition. First, we designed siRNAs against the two TMPRSS2-ERG fusion variants (III and IV), most frequently identified in patients’ biopsies. Two of the five siRNAs tested were found to efficiently inhibit mRNA of both TMPRSS2-ERG variants and to decrease ERG protein expression. Microarray analysis further confirmed ERG inhibition by both siRNAs TMPRSS2-ERG and revealed one common down-regulated gene, ADRA2A, involved in cell proliferation and migration. The siRNA against TMPRSS2-ERG fusion variant IV showed the highest anti-proliferative effects: Significantly decreased cell viability, increased cleaved caspase-3 and inhibited a cluster of anti-apoptotic proteins. To propose a concrete therapeutic approach, siRNA TMPRSS2-ERG IV was conjugated to squalene, which can self-organize as nanoparticles in water. The nanoparticles of siRNA TMPRSS2-ERG-squalene injected intravenously in SCID mice reduced growth of VCaP xenografted tumours, inhibited oncoprotein expression and partially restored differentiation (decrease in Ki67). In conclusion, this study offers a new prospect of treatment for prostate cancer based on siRNA-squalene nanoparticles targeting TMPRSS2-ERG junction oncogene. PMID:25933120

  4. Bolaamphiphiles as carriers for siRNA delivery: From chemical syntheses to practical applications.

    PubMed

    Gupta, Kshitij; Afonin, Kirill A; Viard, Mathias; Herrero, Virginia; Kasprzak, Wojciech; Kagiampakis, Ioannis; Kim, Taejin; Koyfman, Alexey Y; Puri, Anu; Stepler, Marissa; Sappe, Alison; KewalRamani, Vineet N; Grinberg, Sarina; Linder, Charles; Heldman, Eliahu; Blumenthal, Robert; Shapiro, Bruce A

    2015-09-10

    In this study we have investigated a new class of cationic lipids--"bolaamphiphiles" or "bolas"--for their ability to efficiently deliver small interfering RNAs (siRNAs) to cancer cells. The bolas of this study consist of a hydrophobic chain with one or more positively charged head groups at each end. Recently, we reported that micelles of the bolas GLH-19 and GLH-20 (derived from vernonia oil) efficiently deliver siRNAs, while having relatively low toxicities in vitro and in vivo. Our previous studies validated that; bolaamphiphiles can be designed to vary the magnitude of siRNA shielding, its delivery, and its subsequent release. To further understand the structural features of bolas critical for siRNAs delivery, new structurally related bolas (GLH-58 and GLH-60) were designed and synthesized from jojoba oil. Both bolas have similar hydrophobic domains and contain either one, in GLH-58, or two, in GLH-60 positively charged head groups at each end of the hydrophobic core. We have computationally predicted and experimentally validated that GLH-58 formed more stable nano sized micelles than GLH-60 and performed significantly better in comparison to GLH-60 for siRNA delivery. GLH-58/siRNA complexes demonstrated better efficiency in silencing the expression of the GFP gene in human breast cancer cells at concentrations of 5μg/mL, well below the toxic dose. Moreover, delivery of multiple different siRNAs targeting the HIV genome demonstrated further inhibition of virus production.

  5. Structure-Guided Control of siRNA Off-Target Effects.

    PubMed

    Suter, Scott R; Sheu-Gruttadauria, Jessica; Schirle, Nicole T; Valenzuela, Rachel; Ball-Jones, Alexi A; Onizuka, Kazumitsu; MacRae, Ian J; Beal, Peter A

    2016-07-20

    Short interfering RNAs (siRNAs) are promising therapeutics that make use of the RNA interference (RNAi) pathway, but liabilities arising from the native RNA structure necessitate chemical modification for drug development. Advances in the structural characterization of components of the human RNAi pathway have enabled structure-guided optimization of siRNA properties. Here we report the 2.3 Å resolution crystal structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi pathway, bound to an siRNA guide strand bearing an unnatural triazolyl nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue inserts deeply into hAgo2's central RNA binding cleft and thus is able to modulate pairing between guide and target RNAs. The affinity of the hAgo2-siRNA complex for a seed-only matched target was significantly reduced by the triazolyl modification, while the affinity for a fully matched target was unchanged. In addition, siRNA potency for off-target repression was reduced (4-fold increase in IC50) by the modification, while on-target knockdown was improved (2-fold reduction in IC50). Controlling siRNA on-target versus microRNA (miRNA)-like off-target potency by projection of substituent groups into the hAgo2 central cleft from g1 is a new approach to enhance siRNA selectivity with a strong structural rationale.

  6. SiRNA sequence model: redesign algorithm based on available genome-wide libraries.

    PubMed

    Kozak, Karol

    2013-12-01

    The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

  7. Cationic derivatives of biocompatible hyaluronic acids for delivery of siRNA and antisense oligonucleotides.

    PubMed

    Han, Su-Eun; Kang, Hyungu; Shim, Ga Yong; Kim, Sun Jae; Choi, Han-Gon; Kim, Jiseok; Hahn, Sei Kwang; Oh, Yu-Kyoung

    2009-02-01

    In this study, we tested the use of cationic polymer derivatives of biocompatible hyaluronic acid (HA) as a delivery system of siRNA and antisense oligonucleotides. HA was modified with cationic polymer polyethylenimine (PEI). When compared with PEI alone, cationic PEI derivatives of HA (HA-PEI) provided increased cellular delivery of Small interfering RNA (siRNA) in B16F1, A549, HeLa, and Hep3B tumor cells. Indeed, more than 95% of the cells were positive for siRNA following its delivery with HA-PEI. A survivin-specific siRNA that was delivered using HA-PEI potently reduced the mRNA expression levels of the target gene in all of the cell lines. By contrast, survivin-specific siRNA delivered by PEI alone did not induce a significant reduction in mRNA levels. In green fluorescent protein (GFP)-expressing 293 T cells, a loss of GFP expression was evident in the cells that had been treated with GFP-specific siRNA and HA-PEI complex. The inhibition of target gene expression by antisense oligonucleotide G3139 was also enhanced after delivery with HA-PEI. Moreover, HA-PEI displayed lower cytotoxicity than PEI alone. These results suggest that HA-PEI could be further developed as biocompatible delivery systems of siRNA and antisense oligonucleotides for enhanced cellular uptake and inhibition of target gene expression.

  8. Assessment of siRNA pharmacokinetics using ELISA-based quantification.

    PubMed

    Kim, Eun-Joong; Park, Tae Gwan; Oh, Yu-Kyoung; Shim, Chang-Koo

    2010-04-02

    Here, we developed a novel ELISA-based assay for quantifying double-stranded intact siRNAs for in vivo pharmacokinetic analysis. The assay makes use of dual-labeled unmethylated or methylated siRNA, 5'-end-labeled on one strand with biotin (capture marker), and with dinitrophenol (detection marker), on the other end. This ELISA-based assay was linear over the range of 10-100 fmol/ml, with a sensitivity (5.4 fmol/ml) 629-fold higher than fluorometric quantification methods. The coefficient of variation (CV) of the ELISA quantification was 9.4% for intra-assay and 12.1% for inter-assay. The assay was specific for double-stranded siRNAs. The intensity of the detected signal was reduced to background levels in the presence of single-stranded RNA. The ELISA-based assay revealed that the levels of methylated forms of siRNAs after transfection into A549 and HeLa cells were significantly higher than those of unmethylated siRNA forms. Applying this assay to a study of the pharmacokinetic profiles of intravenously administered siRNAs, we found that the higher blood concentrations were achieved using the methylated form of siRNAs than unmethylated form. Moreover, methylated siRNAs complexed to DOTAP-based cationic liposomes showed significantly higher and prolonged blood concentration-time profile, with 2.2-fold lower clearance rate (0.11+/-0.02 ml/min) as compared to the uncomplexed form. These results demonstrate the utility of an ELISA-based assay for evaluating chemically modified siRNAs and cationic delivery systems, particularly from a pharmacokinetic perspective.

  9. Consensus siRNA for inhibition of HCV genotype-4 replication

    PubMed Central

    Zekri, Abdel Rahman N; Bahnassy, Abeer A; El-Din, Hanaa M Alam; Salama, Hosny M

    2009-01-01

    Background HCV is circulating as a heterogeneous group of quasispecies. It has been addressed that siRNA can inhibit HCV replication in-vitro using HCV clone and/or replicon which have only one genotype. The current study was conducted to assess whether siRNA can inhibit different HCV genotypes with many quasispecies and to assess whether consensus siRNA have the same effect as regular siRNA. Methods We generated two chemically synthesized consensus siRNAs (Z3 and Z5) which cover most known HCV genotype sequences and quasispecies using Ambium system. Highly positive HCV patient's serum with nine quasispecies was transfected in-vitro to Huh-7 cell line which supports HCV genotype-4 replication. siRNA (Z3&Z5) were transfected according to Qiagen Porta-lipid technique and subsequently cultured for eight days. HCV replication was monitored by RT-PCR for detection of plus and minus strands. Real-time PCR was used for quantification of HCV, whereas detection of the viral core protein was performed by western blot. Results HCV RNA levels decreased 18-fold (P = 0.001) and 25-fold (P = 0.0005) in cells transfected with Z3 and Z5, respectively, on Day 2 post transfection and continued for Day 3 by Z3 and Day 7 by Z5. Reduction of core protein expression was reported at Day 2 post Z3 siRNA transfection and at Day 1 post Z5 siRNA, which was persistent for Day 4 for the former and for Day 6 for the latter. Conclusion Consensus siRNA could be used as a new molecular target therapy to effectively inhibit HCV replication in the presence of more than one HCV quasispecies. PMID:19173711

  10. DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization

    PubMed Central

    Heissig, Philipp; Klein, Philipp M.; Hadwiger, Philipp; Wagner, Ernst

    2016-01-01

    siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4–10) of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3′ end of the siRNA passenger strand was beneficial over the 5′ end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol–shielded polyplex to downregulate a luciferase gene in folate receptor–positive cells. PMID:26928236

  11. Serum-stabilized naked caspase-3 siRNA protects autotransplant kidneys in a porcine model.

    PubMed

    Yang, Cheng; Zhao, Tian; Zhao, Zitong; Jia, Yichen; Li, Long; Zhang, Yufang; Song, Mangen; Rong, Ruiming; Xu, Ming; Nicholson, Michael L; Zhu, Tongyu; Yang, Bin

    2014-10-01

    The naked small interfering RNA (siRNA) of caspase-3, a key player in ischemia reperfusion injury, was effective in cold preserved and hemoreperfused kidneys, but not autotransplanted kidneys in our porcine models. Here, chemically modified serum stabilized caspase-3 siRNAs were further evaluated. The left kidney was retrieved and infused by University of Wisconsin solution with/without 0.3 mg caspase-3 or negative siRNA into the renal artery for 24-hour cold storage (CS). After an intravenous injection of 0.9 mg siRNA and right-uninephrectomy, the left kidney was autotransplanted for 2 weeks. The effectiveness of caspase-3 siRNA was confirmed by caspase-3 knockdown in the post-CS and/or post-transplant kidneys with reduced apoptosis and inflammation, while the functional caspase-3 siRNA in vivo was proved by detected caspase-3 mRNA degradation intermediates. HMGB1 protein was also decreased in the post-transplanted kidneys; correlated positively with renal IL-1β mRNA, but negatively with serum IL-10 or IL-4. The minimal off-target effects of caspase-3 siRNA were seen with favorable systemic responses. More importantly, renal function, associated with active caspase-3, HMGB1, apoptosis, inflammation, and tubulointerstitial damage, was improved by caspase-3 siRNA. Taken together, the 2-week autotransplanted kidneys were protected when caspase-3 siRNA administrated locally and systemically, which provides important evidence for future clinical trials.

  12. FGFR3 silencing by siRNA inhibits invasion of A549 cells

    PubMed Central

    Li, Yuhua; Liu, Xiguang; Zhang, Hongjun; Jiang, Tao; Xiao, Wenjing; Zhao, Shufen; Yu, Xiaoyun; Han, Fanjie

    2016-01-01

    The present study identified that fibroblast growth factor receptor 3 (FGFR3) was significantly upregulated in bone metastasis of lung adenocarcinoma. RNA interference (RNAi) is a powerful approach for treating a wide range of human diseases, including cancer, through downregulating the expression of selected genes. In the present study, the invasiveness of A549 cells cultured in vitro was altered by small interfering (si)RNA targeting FGFR3, and the regulatory effect of silencing FGFR3 on the expression levels of E-cadherin and matrix metalloproteinase (MMP)9 was investigated. Human lung adenocarcinoma A549 cells were transfected with synthetic specific siRNAs targeting a fragment of the FGFR3 gene (namely, siRNA-855, siRNA-1447 and siRNA-2076) or with negative control (NC) siRNA. Cells were divided into five groups (A, siRNA-855 group; B, siRNA-1447 group; C, siRNA-2076 group; D, NC-siRNA group; and E, blank control group). The effect of the above siRNAs targeting FGFR3 on the invasion capacity of A549 cells was detected by Transwell assay. siRNAs against FGFR3 were transfected into A549 cells with by Lipofectamine® 2000, and the expression levels of FGFR3, E-cadherin and MMP9 were measured by reverse transcription-quantitative polymerase chain reaction and western blot assay. The experimental findings indicated that the expression levels of FGFR3 and MMP9 were significantly reduced in the siRNA-FGFR3-transfected groups (A-C groups), compared with those in the D and E groups (P<0.01). In addition, the expression levels of E-cadherin were markedly elevated in the A-C groups, compared with those in the D and E groups (P<0.01). There was no significant difference in E-cadherin expression between the A-C groups, or between the D and E groups (P>0.05). These results indicated that siRNA-FGFR3 was able to decrease the invasiveness of A549 cells, inhibit the expression of MMP9 and increase the expression of E-cadherin by downregulating the expression of FGFR3. Taken

  13. Simultaneous cytosolic delivery of a chemotherapeutic and siRNA using nanoparticle-stabilized nanocapsules

    NASA Astrophysics Data System (ADS)

    Hardie, Joseph; Jiang, Ying; Tetrault, Emily R.; Ghazi, Phaedra C.; Yesilbag Tonga, Gulen; Farkas, Michelle E.; Rotello, Vincent M.

    2016-09-01

    We report on nanoparticle-stabilized capsules (NPSCs) as a platform for the co-delivery of survivin-targeted siRNA and tamoxifen. These capsules feature an inner oil core that provides a carrier for tamoxifen, and is coated on the surface with positively charged nanoparticles self-assembled with siRNA. The multifaceted chemical nature of the NPSC system enables the simultaneous delivery of both payloads directly into the cytosol in vitro. The NPSC co-delivery of tamoxifen and survivin-targeted siRNA into breast cancer cells disables the pathways that inhibit apoptosis, resulting in enhanced breast cell death.

  14. Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance.

    PubMed

    Zhao, Mingmin; San León, David; Mesel, Frida; García, Juan Antonio; Simón-Mateo, Carmen

    2015-01-01

    The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.

  15. Surface abundances of ON stars

    NASA Astrophysics Data System (ADS)

    Martins, F.; Simón-Díaz, S.; Palacios, A.; Howarth, I.; Georgy, C.; Walborn, N. R.; Bouret, J.-C.; Barbá, R.

    2015-06-01

    Context. Massive stars burn hydrogen through the CNO cycle during most of their evolution. When mixing is efficient or when mass transfer in binary systems occurs, chemically processed material is observed at the surface of O and B stars. Aims: ON stars show stronger lines of nitrogen than morphologically normal counterparts. Whether this corresponds to the presence of material processed through the CNO cycle is not known. Our goal is to answer this question. Methods: We performed a spectroscopic analysis of a sample of ON stars with atmosphere models. We determined the fundamental parameters as well as the He, C, N, and O surface abundances. We also measured the projected rotational velocities. We compared the properties of the ON stars to those of normal O stars. Results: We show that ON stars are usually rich in helium. Their CNO surface abundances are fully consistent with predictions of nucleosynthesis. ON stars are more chemically evolved and rotate - on average - faster than normal O stars. Evolutionary models including rotation cannot account for the extreme enrichment observed among ON main sequence stars. Some ON stars are members of binary systems, but others are single stars as indicated by stable radial velocities. Mass transfer is therefore not a simple explanation for the observed chemical properties. Conclusions: We conclude that ON stars show extreme chemical enrichment at their surface, consistent with nucleosynthesis through the CNO cycle. Its origin is not clear at present. Based on observations obtained 1) at the Anglo-Australian Telescope; 2) at the Canada-France-Hawaii Telescope (CFHT), which is operated by the National Research Council (NRC) of Canada, the Institut National des Science de l'Univers of the Centre National de la Recherche Scientifique (CNRS) of France, and the University of Hawaii; 3) at the ESO/La Silla Observatory under programs 081.D-2008, 083.D-0589, 086.D-0997; 4) the Nordic Optical Telescope, operated on the island of La

  16. Functional MnO nanoclusters for efficient siRNA delivery†

    PubMed Central

    Xing, Ruijun; Liu, Gang; Quan, Qimeng; Bhirde, Ashwinkumar; Zhang, Guofeng; Jin, Albert; Bryant, L. Henry; Zhang, Angela; Liang, Amy; Eden, Henry S.; Hou, Yanglong; Chen, Xiaoyuan

    2015-01-01

    A non-viral gene delivery nanovehicle based on Alkyl-PEI2k capped MnO nanoclusters was synthesized via a simple, facile method and used for efficient siRNA delivery and magnetic resonance imaging. PMID:21991584

  17. Low cytotoxicity fluorescent PAMAM dendrimer as gene carriers for monitoring the delivery of siRNA

    NASA Astrophysics Data System (ADS)

    Guan, Lingmei; Huang, Saipeng; Chen, Zhao; Li, Yanchao; Liu, Ke; Liu, Yang; Du, Libo

    2015-09-01

    Visual detection of gene vectors has attracted a great deal of attention due to the application of these vectors in monitoring and evaluating the effect of gene carriers in living cells. A non-viral vector, the fluorescent PAMAM dendrimer (F-PAMAM), was synthesized through conjugation of PAMAM dendrimers and fluorescein. In vitro and ex vivo experiments show that F-PAMAM exhibits superphotostability, low cytotoxicity and facilitates endocytosis by A549 cells. The vector has a high siRNA binding affinity and it increases the efficiency of cy5-siRNA delivery in A549 cells, in comparison with a cy5-siRNA monomer. Our results provide a new method for simultaneously monitoring the delivery of siRNA and its non-viral carriers in living cells.

  18. Cytoplasmic delivery of siRNAs to monocytes and dendritic cells via electroporation.

    PubMed

    Sioud, Mouldy

    2015-01-01

    RNA interference has been of great interest not only as a research tool to suppress gene expression but also as an emerging therapeutic strategy to silence disease genes. However, the therapeutic use of siRNA faces the in vivo delivery challenge. An alternative method that could potentially be used for siRNA delivery into primary immune cells for therapeutic purposes is an ex vivo route, whereby immune cells could be isolated from a patient, reprogrammed with siRNAs, and infused back into the same patient. This chapter describes siRNA delivery into human primary monocytes and dendritic cells using a standard electroporation technique. Dendritic cells occupy a central role in the immune system, orchestrating a wide repertoire of responses that span from the development of self-tolerance to the generation of protective CD8+ T cell immunity.

  19. Nanoengineered strategies for siRNA delivery: from target assessment to cancer therapeutic efficacy.

    PubMed

    Mishra, Dinesh Kumar; Balekar, Neelam; Mishra, Pradyumna Kumar

    2017-04-01

    The promise of RNA interference (RNAi) technology in cancer therapeutics aims to deliver small interfering RNA (siRNA) for silencing of gene expression in cell type-specific pathway. However, the challenge for the delivery of stable siRNA is hindered by an immune-hostile tumor microenvironment and physiological barriers of the circulatory system. Therefore, the development and validation of safe, stable, and efficient nanoengineered delivery systems are highly essential for effective delivery of siRNA into cancer cells. This review focuses on gene-silencing mechanisms, challenges to siRNA delivery, design and delivery of nanocarrier systems, ongoing clinical trials, and translational prospects for siRNA-mediated cancer therapeutics.

  20. Epigenome Editing of Potato by Grafting Using Transgenic Tobacco as siRNA Donor

    PubMed Central

    Hojo, Hatsune; Harada, Takeo

    2016-01-01

    In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops. PMID:27564864

  1. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight.

    PubMed

    Dahlman, James E; Barnes, Carmen; Khan, Omar F; Thiriot, Aude; Jhunjunwala, Siddharth; Shaw, Taylor E; Xing, Yiping; Sager, Hendrik B; Sahay, Gaurav; Speciner, Lauren; Bader, Andrew; Bogorad, Roman L; Yin, Hao; Racie, Tim; Dong, Yizhou; Jiang, Shan; Seedorf, Danielle; Dave, Apeksha; Singh Sandhu, Kamaljeet; Webber, Matthew J; Novobrantseva, Tatiana; Ruda, Vera M; Lytton-Jean, Abigail K R; Levins, Christopher G; Kalish, Brian; Mudge, Dayna K; Perez, Mario; Abezgauz, Ludmila; Dutta, Partha; Smith, Lynelle; Charisse, Klaus; Kieran, Mark W; Fitzgerald, Kevin; Nahrendorf, Matthias; Danino, Dganit; Tuder, Rubin M; von Andrian, Ulrich H; Akinc, Akin; Panigrahy, Dipak; Schroeder, Avi; Koteliansky, Victor; Langer, Robert; Anderson, Daniel G

    2014-08-01

    Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

  2. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight

    NASA Astrophysics Data System (ADS)

    Dahlman, James E.; Barnes, Carmen; Khan, Omar F.; Thiriot, Aude; Jhunjunwala, Siddharth; Shaw, Taylor E.; Xing, Yiping; Sager, Hendrik B.; Sahay, Gaurav; Speciner, Lauren; Bader, Andrew; Bogorad, Roman L.; Yin, Hao; Racie, Tim; Dong, Yizhou; Jiang, Shan; Seedorf, Danielle; Dave, Apeksha; Singh Sandhu, Kamaljeet; Webber, Matthew J.; Novobrantseva, Tatiana; Ruda, Vera M.; Lytton-Jean, Abigail K. R.; Levins, Christopher G.; Kalish, Brian; Mudge, Dayna K.; Perez, Mario; Abezgauz, Ludmila; Dutta, Partha; Smith, Lynelle; Charisse, Klaus; Kieran, Mark W.; Fitzgerald, Kevin; Nahrendorf, Matthias; Danino, Dganit; Tuder, Rubin M.; von Andrian, Ulrich H.; Akinc, Akin; Panigrahy, Dipak; Schroeder, Avi; Koteliansky, Victor; Langer, Robert; Anderson, Daniel G.

    2014-08-01

    Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

  3. Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells

    PubMed Central

    Ghildiyal, Megha; Seitz, Hervé; Horwich, Michael D.; Li, Chengjian; Du, Tingting; Lee, Soohyun; Xu, Jia; Kittler, Ellen L.W.; Zapp, Maria L.; Weng, Zhiping; Zamore, Phillip D.

    2009-01-01

    Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line. PMID:18403677

  4. RELATIVE ABUNDANCE MEASUREMENTS IN PLUMES AND INTERPLUMES

    SciTech Connect

    Guennou, C.; Hahn, M.; Savin, D. W.

    2015-07-10

    We present measurements of relative elemental abundances in plumes and interplumes. Plumes are bright, narrow structures in coronal holes that extend along open magnetic field lines far out into the corona. Previous work has found that in some coronal structures the abundances of elements with a low first ionization potential (FIP) <10 eV are enhanced relative to their photospheric abundances. This coronal-to-photospheric abundance ratio, commonly called the FIP bias, is typically 1 for elements with a high-FIP (>10 eV). We have used Extreme Ultraviolet Imaging Spectrometer observations made on 2007 March 13 and 14 over a ≈24 hr period to characterize abundance variations in plumes and interplumes. To assess their elemental composition, we used a differential emission measure analysis, which accounts for the thermal structure of the observed plasma. We used lines from ions of iron, silicon, and sulfur. From these we estimated the ratio of the iron and silicon FIP bias relative to that for sulfur. From the results, we have created FIP-bias-ratio maps. We find that the FIP-bias ratio is sometimes higher in plumes than in interplumes and that this enhancement can be time dependent. These results may help to identify whether plumes or interplumes contribute to the fast solar wind observed in situ and may also provide constraints on the formation and heating mechanisms of plumes.

  5. Systemic Administration of siRNA via cRGD-containing Peptide

    PubMed Central

    Huang, Yuanyu; Wang, Xiaoxia; Huang, Weiyan; Cheng, Qiang; Zheng, Shuquan; Guo, Shutao; Cao, Huiqing; Liang, Xing-Jie; Du, Quan; Liang, Zicai

    2015-01-01

    Although small interfering RNAs (siRNAs) have been demonstrated to specifically silence their target genes in disease models and clinical trials, in vivo siRNA delivery is still the technical bottleneck that limits their use in therapeutic applications. In this study, a bifunctional peptide named RGD10-10R was designed and tested for its ability to deliver siRNA in vitro and in vivo. Because of their electrostatic interactions with polyarginine (10R), negatively charged siRNAs were readily complexed with RGD10-10R peptides, forming spherical RGD10-10R/siRNA nanoparticles. In addition to enhancing their serum stability by preventing RNase from attacking siRNA through steric hindrance, peptide binding facilitated siRNA transfection into MDA-MB-231 cells, as demonstrated by FACS and confocal microscopy assays and by the repressed expression of target genes. When RGD10 peptide, a receptor competitor of RGD10-10R, was added to the transfection system, the cellular internalization of RGD10-10R/siRNA was significantly compromised, suggesting a mechanism of ligand/receptor interaction. Tissue distribution assays indicated that the peptide/siRNA complex preferentially accumulated in the liver and in several exocrine/endocrine glands. Furthermore, tumor-targeted delivery of siRNA was also demonstrated by in vivo imaging and cryosection assays. In summary, RGD10-10R might constitute a novel siRNA delivery tool that could potentially be applied in tumor treatment. PMID:26300278

  6. Safety of striatal infusion of siRNA in a transgenic Huntington's disease mouse model

    PubMed Central

    McGowan, Sarah; Mondo, Erica; Pfister, Edith; Mick, Eric; Friedline, Randall H.; Kim, Jason K.; Sapp, Ellen; DiFiglia, Marian; Aronin, Neil

    2016-01-01

    Background The immune system In Huntington's disease (HD) is activated and may overreact to some therapies. RNA interference using siRNA lowers mutant huntingtin (mHTT) protein but could increase immune responses. Objective To examine the innate immune response following siRNA infusion into the striatum of wild-type (WT) and HD transgenic (YAC128) mice. Methods siRNAs (2′-O-methyl phosphorothioated) were infused unilaterally into striatum of four month-old WT and YAC128 mice for 28 days. Microglia number and morphology (resting (normal), activated, dystrophic), cytokine levels, and DARPP32-positive neurons were measured in striatum immediately or 14 days post-infusion. Controls included contralateral untreated striatum, and PBS and sham treated striata. Results The striata of untreated YAC128 mice had significantly fewer resting microglia and more dystrophic microglia than WT mice, but no difference from WT in the proportion of activated microglia or total number of microglia. siRNA infusion increased the total number of microglia in YAC128 mice compared to PBS treated and untreated striata and increased the proportion of activated microglia in WT and YAC128 mice compared to untreated striata and sham treated groups. Cytokine levels were low and siRNA infusion resulted in only modest changes in those levels. siRNA infusion did not change the number of DARPP32-positive neurons. Conclusion Findings suggest that siRNA infusion may be a safe method for lowering mHTT levels in the striatum in young animals, since treatment does not produce a robust cytokine response or cause neurotoxicity. The potential long-term effects of a sustained increase in total and activated microglia after siRNA infusion in HD mice need to be explored. PMID:26444021

  7. pH-responsive hybrid quantum dots for targeting hypoxic tumor siRNA delivery.

    PubMed

    Zhu, HongYan; Zhang, ShengYu; Ling, Yong; Meng, GuoLiang; Yang, Yu; Zhang, Wei

    2015-12-28

    Hypoxia is a characteristic of cancer and plays a key role in tumorigenesis, angiogenesis and resistance to cancer therapies. SiRNA treatment is effective against hypoxic tumors by gene silencing. However, siRNA delivery to the hypoxic regions of solid tumors still presents a challenge due to the distance from blood vessels and the increased presence of efflux transporters. Therefore, tumor therapies would be improved through the immediate development of an effective siRNA delivery system to hypoxic regions. To this end, we synthesized a system to deliver HIF-1α siRNA into hypoxic tumor cells. The system consists of a functional shell composed of 2-deoxyglucose (DG)-polyethylene glycol (PEG) connected with the compound of lipoic acid, lysine and 9-poly-d-arginine (LA-Lys-9R) by a hydrazone bond and a core of CdTe quantum dots (QDs). The molecular structure of DG-PEG-LA-Lys-9R was confirmed by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The multifunctional CdTe QDs measured approximately 200 nm and showed excellent biocompatibility, perfect siRNA binding capability and enhanced hypoxic tumor targeting. Importantly, the system described here is pH-responsive with a hydrazone bond; therefore, it avoids GLUT1 receptor-mediated endocytic recycling, resulting in irreversible delivery of the siRNA. We used Western blots to confirm the superior gene silencing efficiency induced by the DG-PEG-LA-Lys-9R with hydrazone modified CdTe QDs. Here, we demonstrate high efficacy of the siRNA tumor delivery system using in vitro and in vivo experiments. In addition, these studies demonstrate that pH-responsive hybrid quantum dots show improved antitumor efficacy with decreased organ toxicity, indicating a promising siRNA delivery system for hypoxic cancer therapy.

  8. [Efficacy of siRNA on feline leukemia virus replication in vitro].

    PubMed

    Lehmann, Melanie; Weber, Karin; Rauch, Gisep; Hofmann-Lehmann, Regina; Hosie, Margaret J; Meli, Marina L; Hartmann, Katrin

    2015-01-01

    Feline leukemia virus (FeLV) can lead to severe clinical signs in cats. Until now, there is no effective therapy for FeLV-infected cats. RNA interference-based antiviral therapy is a new concept. Specific small interfering RNA (siRNA) are designed complementary to the mRNA of a target region, and thus inhibit replication. Several studies have proven efficacy of siRNAs in inhibiting virus replication. The aim of this study was to evaluate the inhibitory potential of siRNAs against FeLV replication in vitro. siRNAs against the FeLV env gene and the host cell surface receptor (feTHTR1) which is used by FeLV-A for entry as well as siRNA that were not complementary to the FeLV or cat genome, were tested. Crandell feline kidney cells (CrFK cells) were transfected with FeLV-A/Glasgow-1. On day 13, infected cells were transfected with siRNAs. As control, cells were mock-transfected or treated with azidothymidine (AZT) (5 μg/ml). Culture supernatants were analyzed for FeLV RNA using quantitative real-time RT-PCR and for FeLV p27 by ELISA every 24 hours for five days. All siRNAs significantly reduced viral RNA and p27 production, starting after 48 hours. The fact that non-complementary siRNAs also inhibited virus replication may lead to the conclusion that unspecific mechanisms rather than specific binding lead to inhibition.

  9. siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

    PubMed Central

    Kong, Zhenzhen; Shao, Qixiang; Su, Zhaoliang; Wang, Shengjun; Chen, Jianguo

    2016-01-01

    Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2Apro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID50) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2Apro region of the EV71 genome exerted antiviral effects in vitro. PMID:26886455

  10. Peptide- and Amine-Modified Glucan Particles for the Delivery of Therapeutic siRNA.

    PubMed

    Cohen, Jessica L; Shen, Yuefei; Aouadi, Myriam; Vangala, Pranitha; Tencerova, Michaela; Amano, Shinya U; Nicoloro, Sarah M; Yawe, Joseph C; Czech, Michael P

    2016-03-07

    Translation of siRNA technology into the clinic is limited by the need for improved delivery systems that target specific cell types. Macrophages are particularly attractive targets for RNAi therapy because they promote pathogenic inflammatory responses in a number of important human diseases. We previously demonstrated that a multicomponent formulation of β-1,3-d-glucan-encapsulated siRNA particles (GeRPs) can specifically and potently silence genes in mouse macrophages. A major advance would be to simplify the GeRP system by reducing the number of delivery components, thus enabling more facile manufacturing and future commercialization. Here we report the synthesis and evaluation of a simplified glucan-based particle (GP) capable of delivering siRNA in vivo to selectively silence macrophage genes. Covalent attachment of small-molecule amines and short peptides containing weak bases to GPs facilitated electrostatic interaction of the particles with siRNA and aided in the endosomal release of siRNA by the proton-sponge effect. Modified GPs were nontoxic and were efficiently internalized by macrophages in vitro. When injected intraperitoneally (i.p.), several of the new peptide-modified GPs were found to efficiently deliver siRNA to peritoneal macrophages in lean, healthy mice. In an animal model of obesity-induced inflammation, i.p. administration of one of the peptide-modified GPs (GP-EP14) bound to siRNA selectively reduced the expression of target inflammatory cytokines in the visceral adipose tissue macrophages. Decreasing adipose tissue inflammation resulted in an improvement of glucose metabolism in these metabolically challenged animals. Thus, modified GPs represent a promising new simplified system for the efficient delivery of therapeutic siRNAs specifically to phagocytic cells in vivo for modulation of inflammation responses.

  11. Topical Delivery of siRNA into Skin using SPACE-peptide Carriers

    PubMed Central

    Chen, Ming; Zakrewsky, Michael; Gupta, Vivek; Anselmo, Aaron C.; Slee, Deborah H.; Muraski, John A.; Mitragotri, Samir

    2014-01-01

    Short-interfering RNAs (siRNAs) offer a potential tool for the treatment of skin disorders. However, applications of siRNA for dermatological conditions are limited by their poor permeation across the stratum corneum of the skin and low penetration into skin’s viable cells. In this study, we report the use of SPACE-peptide in combination with a DOTAP-based ethosomal carrier system to enhance skin delivery of siRNA. A DOTAP-based SPACE Ethosomal System significantly enhanced siRNA penetration into porcine skin in vitro by 6.3±1.7-fold (p<0.01) with an approximately 10-fold (p<0.01) increase in epidermis accumulation of siRNA compared to that from an aqueous solution. Penetration of siRNA was also enhanced at the cellular level. Internalization of SPACE-peptide occurred in a concentration dependent manner marked by a shift in intracellular distribution from punctate spots to diffused cytoplasmic staining at a peptide concentration of 10 mg/mL. In vitro delivery of GAPDH siRNA by SPACE peptide led to 83.3±3.0% knockdown relative to the control. In vivo experiments performed using female BALB/C mice also confirmed the efficacy of DOTAP-SES in delivering GAPDH-siRNA into skin. Topical application of DOTAP-SES on mice skin resulted in 63.2%±7.7% of GAPDH knockdown, which was significantly higher than that from GAPDH-siRNA PBS (p<0.05). DOTAP-SES formulation reported here may open new opportunities for cutaneous siRNA delivery. PMID:24434423

  12. Polycationic nanoparticles for siRNA delivery: comparing ARGET ATRP and UV-initiated formulations.

    PubMed

    Forbes, Diane C; Peppas, Nicholas A

    2014-03-25

    In this work, we develop and evaluate polycationic nanoparticles for the delivery of small interfering RNA (siRNA). Delivery remains a major challenge for translating siRNA to the clinic, and overcoming the delivery challenge requires effective siRNA delivery vehicles that meet the demands of the specific delivery strategy. Cross-linked polycationic nanoparticle formulations were synthesized using ARGET ATRP or UV-initiated polymerization. The one-step, one-pot, surfactant-stabilized monomer-in-water synthesis technique may provide a simpler and faster alternative to complicated, multistep techniques and an alternative to methods that rely on toxic organic solvents. The polymer nanoparticles were synthesized using the cationic monomer 2-(diethylamino)ethyl methacrylate, the hydrophobic monomer tert-butyl methacrylate to tune pH responsiveness, the hydrophilic monomer poly(ethylene glycol) methyl ether methacrylate to improve biocompatibility, and cross-linking agent tetraethylene glycol dimethacrylate to enhance colloidal stability. Four formulations were evaluated for their suitability as siRNA delivery vehicles in vitro with the human embryonic kidney cell line HEK293T or the murine macrophage cell line RAW264.7. The polycationic nanoparticles demonstrated efficient and rapid loading of the anionic siRNA following complexation. Confocal microscopy as well as flow cytometry analysis of cells treated with polycationic nanoparticles loaded with fluorescently labeled siRNA demonstrated that the polycationic nanoparticles promoted cellular uptake of fluorescently labeled siRNA. Knockdown experiments using polycationic nanoparticles to deliver siRNA demonstrated evidence of knockdown, thus demonstrating potential as an alternative route to creating polycationic nanoparticles.

  13. Enhancing potency of siRNA targeting fusion genes by optimization outside of target sequence

    PubMed Central

    Gavrilov, Kseniya; Seo, Young-Eun; Tietjen, Gregory T.; Cui, Jiajia; Cheng, Christopher J.; Saltzman, W. Mark

    2015-01-01

    Canonical siRNA design algorithms have become remarkably effective at predicting favorable binding regions within a target mRNA, but in some cases (e.g., a fusion junction site) region choice is restricted. In these instances, alternative approaches are necessary to obtain a highly potent silencing molecule. Here we focus on strategies for rational optimization of two siRNAs that target the junction sites of fusion oncogenes BCR-ABL and TMPRSS2-ERG. We demonstrate that modifying the termini of these siRNAs with a terminal G-U wobble pair or a carefully selected pair of terminal asymmetry-enhancing mismatches can result in an increase in potency at low doses. Importantly, we observed that improvements in silencing at the mRNA level do not necessarily translate to reductions in protein level and/or cell death. Decline in protein level is also heavily influenced by targeted protein half-life, and delivery vehicle toxicity can confound measures of cell death due to silencing. Therefore, for BCR-ABL, which has a long protein half-life that is difficult to overcome using siRNA, we also developed a nontoxic transfection vector: poly(lactic-coglycolic acid) nanoparticles that release siRNA over many days. We show that this system can achieve effective killing of leukemic cells. These findings provide insights into the implications of siRNA sequence for potency and suggest strategies for the design of more effective therapeutic siRNA molecules. Furthermore, this work points to the importance of integrating studies of siRNA design and delivery, while heeding and addressing potential limitations such as restricted targetable mRNA regions, long protein half-lives, and nonspecific toxicities. PMID:26627251

  14. N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient siRNA delivery.

    PubMed

    Liu, Gang; Xie, Jin; Zhang, Fan; Wang, Zhiyong; Luo, Kui; Zhu, Lei; Quan, Qimeng; Niu, Gang; Lee, Seulki; Ai, Hua; Chen, Xiaoyuan

    2011-10-04

    Small-interfering RNA (siRNA) is an emerging class of therapeutics, which works by regulating the expression of a specific gene involved in disease progression. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. In this study, a nonviral nanoparticle gene carrier is developed and its efficiency for siRNA delivery and transfection is validated at both in vitro and in vivo levels. Such a nanocarrier, abbreviated as Alkyl-PEI2k-IO, was constructed with a core of iron oxide nanoparticles (IOs) and a shell of alkylated polyethyleneimine of 2000 Da [corrected] molecualr weight (Alkyl-PEI2k). It is found to be able to bind with siRNA, resulting in well-dispersed nanoparticles with a controlled clustering structure and narrow size distribution. Electrophoresis studies show that the Alkyl-PEI2k-IOs could retard siRNA completely at N:P ratios (i.e., PEI nitrogen to nucleic acid phosphate) above 10, protect siRNA from enzymatic degradation in serum, and release complexed siRNA efficiently in the presence of polyanionic heparin. The knockdown efficiency of the siRNA-loaded nanocarriers is assessed with 4T1 cells stably expressing luciferase (fluc-4T1) and further, with a fluc-4T1 xenograft model. Significant down-regulation of luciferase is observed, and unlike high-molecular-weight analogues, the Alkyl-PEI2k-coated IOs show good biocompatibility. In conclusion, Alkyl-PEI2k-IOs demonstrate highly efficient delivery of siRNA and an innocuous toxic profile, making it a potential carrier for gene therapy.

  15. Ammonia abundances in comets

    NASA Astrophysics Data System (ADS)

    Wyckoff, S.; Tegler, S.; Engel, L.

    The emission band strengths of the NH2 bands of Comets Halley, Hartley-Good, Thiele, and Borrelly were measured to determine the NH2 column densities for the comets. Production rates obtained using the Haser and vectorial models are in agreement within the observational errors, suggesting that a simple two-step decay model may be used to approximate the NH2 distribution in a comet's coma. Ammonia-to-water abundance ratios from 0.01 to 0.4 percent were found for the four comets. The ratio in Comet Halley is found to be Q(NH3)/Q(H2O) = 0.002 + or - 0.001. No significant difference in the ammonia abundance was found before or after perihelion in Comet Halley.

  16. Rigid nanoparticle-baseddelivery of anti-cancer siRNA: challenges and opportunities

    PubMed Central

    Wang, Zhiyong; Liu, Gang; Zheng, Hairong; Chen, Xiaoyuan

    2013-01-01

    Gene therapy is a promising strategy to treat various genetic and acquired diseases. Small interfering RNA (siRNA) is a revolutionary tool for gene therapy and the analysis of gene function. However, the development of a safe, efficient, and targetable non-viral siRNA delivery system remains a major challenge in gene therapy. An ideal delivery system should be able to encapsulate and protect the siRNA cargo from serum proteins, exhibit target tissue and cell specificity, penetrate the cell membrane, and release its cargo in the desired intracellular compartment. Nanomedicine has the potential to deal with these challenges faced by siRNA delivery. The unique characteristics of rigid nanoparticles mostly inorganic nanoparticles and allotropes of carbon nanomaterials, including high surface area, facile surface modification, controllable size, and excellent magnetic/optical/electrical properties, make them promising candidates for targeted siRNA delivery. In this review, recent progresses on rigid nanoparticle-based siRNA delivery systems will be summarized. PMID:24013011

  17. Nanotechnology As Potential Tool for siRNA Delivery in Parkinson's Disease.

    PubMed

    Cortés, Hernán; Alcalá-Alcalá, Sergio; Ávalos-Fuentes, Arturo; Mendoza-Muñoz, Nestor; Quintanar-Guerrero, David; Leyva-Gómez, Gerardo; Florán, Benjamín

    2017-03-21

    The lack of an outright treatment for Parkinson's disease (PD) is a pivotal concern in medicine and has driven the search for novel alternatives for treating the disease. Among the proposed approaches, small interfering RNA (siRNA)-based therapy is attracting significant attention as a potential method for the treatment of PD; however, siRNAs delivery possesses potential drawbacks, such as reduced stability in blood circulation and low capacity for reaching the target site. Therefore, siRNA delivery to the brain is a key issue that remains unsolved to date. In this regard, nanoparticles are being developed as carriers for siRNAs and represent an alternative to common vectors; the formulation of siRNAs in nanoparticles would possess several advantages over other vectors, such as controlled delivery and low toxicity profiles. Therefore, in this article we focus on siRNA-based approaches to PD and the latest advances for designing nanoparticles that effectively target siRNAs to the action site and that protect these against degradation in blood circulation. Additionally, we discuss the technological aspects for tackling the challenge that siRNAs targeting to the brain represents.

  18. In vitro validation of self designed "universal human Influenza A siRNA".

    PubMed

    Jain, Bhawana; Jain, Amita; Prakash, Om; Singh, Ajay Kr; Dangi, Tanushree; Singh, Mastan; Singh, K P

    2015-08-01

    The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected siRNA, which showed 73.6% inhibition in replication of Influenza A/pdmH1N1 and 62.1% inhibition in replication of Influenza A/H3N2 at 48 h post infection on MDCK cell line. This study provides a basis for the development of siRNA which can be used as universal anti-IAV therapeutic agent.

  19. Design of a multicomponent peptide-woven nanocomplex for delivery of siRNA.

    PubMed

    Jun, Eunsung; Kim, Soyoun; Kim, Jong-Ho; Cha, Kiweon; So, In-Seop; Son, Hye-Nam; Lee, Byung-Heon; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, Sang Yoon; Kim, In-San

    2015-01-01

    We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery.

  20. Specific subcellular localization of siRNAs delivered by lipoplex in MCF-7 breast cancer cells.

    PubMed

    Lavigne, Carole; Thierry, Alain R

    2007-10-01

    In order to better understand the mechanism of delivery of siRNAs by lipid-based vectors, we investigated the subcellular distribution of siRNAs directed against cyclin D1 delivered by the DLS system in the breast cancer cell line MCF-7. Cells were treated with cyclopentenone or 17beta-estradiol to modulate the level of expression of cyclin D1 mRNA. We qualitatively observed that siRNA localized to specific cytoplasmic compartments in the periphery of the nucleus in granular-like structures that do not correspond to early endosomal vesicles. In cells treated with either cyclopentenone or 17beta-estradiol cellular distribution of siRNAs was not affected but variations in the amount of siRNAs present in cells were found. We suggest these variations might be associated with the effects of cyclopentenone and 17beta-estradiol in cyclin D1 gene expression. Low cytotoxicity and highly cellular uptake of lipoplexes was observed in the presence of serum indicating that the DLS system could be a useful tool for siRNA vectorization in vitro and in vivo.

  1. Buffering Capacity and Size of siRNA Polyplexes Influence Cytokine Levels

    PubMed Central

    Leng, Qixin; Chou, Szu-Ting; Scaria, Puthupparampil V; Woodle, Martin C; Mixson, A James

    2012-01-01

    Induction of cytokines by small interfering RNA (siRNA) polyplexes has been a significant concern of researchers attempting to minimize the toxicity of this promising therapy. Although cationic carriers of siRNA are known to increase cytokine levels, few systematic studies have been done to determine what properties of the carrier are important to modulate cytokines. Because branched histidine-lysine (HK) peptides are effective carriers of siRNA and their sequence can be readily modified, we selected this class of carrier to determine which sequences of the peptide were important for cytokine induction. With the use of peripheral blood mononuclear cells (PBMCs), the HK peptide with a higher number of histidines (H3K(+H)4b) in complex with siRNA induced lower levels of cytokines compared with other HK (e.g., H2K4b, H3K4b, H3K(+N)4b) siRNA nanoplexes. Notably, these peptides' siRNA polyplexes showed a similar pattern of cytokine induction when injected intravenously in a mouse model, i.e., the HK with higher content of histidines induced cytokines the least. As indicated by the pH-sensitive dye within acidic endosomes, the greater pH-buffering capacity of H3K(+H)4b compared with other HK peptides may explain why cytokine levels were reduced. In addition to buffering capacity, the size of HK polyplexes markedly influenced cytokine production. PMID:23032972

  2. Endosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications

    PubMed Central

    Khormaee, Sariah; Choi, Yong; Shen, Michael J.; Xu, Biying; Wu, Haitao; Griffiths, Gary L.; Chen, Rongjun; Slater, Nigel K. H.; Park, John K.

    2013-01-01

    The use of small interfering RNAs (siRNAs) to down-regulate the expression of disease-associated proteins carries significant promise for the treatment of a variety of clinical disorders. One of the main barriers to the widespread clinical use of siRNAs, however, is their entrapment and degradation within the endolysosomal pathway of target cells. Here we report the trafficking and function of PP75, a non-toxic, biodegradable, lipid membrane disruptive anionic polymer composed of phenylalanine derivatized poly(L-lysine iso-phthalamide). PP75 is readily endocytosed by cells, safely permeabilizes endolysosomes in a pH dependent manner and facilitates the transfer of co-endocytosed materials directly into the cytoplasm. The covalent attachment of siRNAs to PP75 using disulfide linkages generates conjugates that effectively traffic siRNAs to the cytoplasm of target cells both in vitro and in vivo. In a subcutaneous malignant glioma tumor model, a locally delivered PP75-stathmin siRNA conjugate decreases stathmin expression in tumor cells and, in combination with the nitrosourea chemotherapy carmustine, is highly effective at inhibiting tumor growth. PP75 may be clinically useful for the local delivery of siRNAs, in particular for the treatment of solid tumors. PMID:24273480

  3. Low-Molecular-Weight Polyethyleneimine Grafted Polythiophene for Efficient siRNA Delivery.

    PubMed

    He, Pan; Hagiwara, Kyoji; Chong, Hui; Yu, Hsiao-hua; Ito, Yoshihiro

    2015-01-01

    Owing to its hydrophilicity, negative charge, small size, and labile degradation by endogenous nucleases, small interfering RNA (siRNA) delivery must be achieved by a carrier system. In this study, cationic copolymers composed of low-molecular-weight polyethylenimine and polythiophenes were synthesized and evaluated as novel self-tracking siRNA delivery vectors. The concept underlying the design of these copolymers is that hydrophobicity and rigidity of polythiophenes should enhance the transport of siRNA across the cell membrane and endosomal membrane. A gel retardation assay showed that the nanosized complexes formed between the copolymers and siRNA were stable even at a molar ratio of 1 : 2. The high cellular uptake (>80%) and localization of the copolymer vectors inside the cells were easily analyzed by tracking the fluorescence of polythiophene using fluorescent microscopy and cytometry. An in vitro luciferase knockdown (KD) assay in A549-luc cells demonstrated that the siRNA complexes with more hydrophobic copolymers achieved a higher KD efficiency of 52.8% without notable cytotoxicity, indicating protein-specific KD activity rather than solely the cytotoxicity of the materials. Our polythiophene copolymers should serve as novel, efficient, low cell toxicity, and label-free siRNA delivery systems.

  4. Pol IV-Dependent siRNA Production is Reduced in Brassica rapa

    PubMed Central

    Huang, Yi; Kendall, Timmy; Mosher, Rebecca A.

    2013-01-01

    Plants produce a diverse array of small RNA molecules capable of gene regulation, including Pol IV-dependent short interfering (p4-si)RNAs that trigger transcriptional gene silencing. Small RNA transcriptomes are available for many plant species, but mutations affecting the synthesis of Pol IV-dependent siRNAs are characterized only in Arabidopsis and maize, leading to assumptions regarding nature of p4-siRNAs in all other species. We have identified a mutation in the largest subunit of Pol IV, NRPD1, that impacts Pol IV activity in Brassica rapa, an agriculturally important relative of the reference plant Arabidopsis. Using this mutation we characterized the Pol IV-dependent and Pol IV-independent small RNA populations in B. rapa. In addition, our analysis demonstrates reduced production of p4-siRNAs in B. rapa relative to Arabidopsis. B. rapa genomic regions are less likely to generate p4-siRNAs than Arabidopsis but more likely to generate Pol IV-independent siRNAs, including 24 nt RNAs mapping to transposable elements. These observations underscore the diversity of small RNAs produced by plants and highlight the importance of genetic studies during small RNA analysis. PMID:24833221

  5. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  6. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes.

  7. Trends in the Binding of Cell Penetrating Peptides to siRNA: A Molecular Docking Study

    PubMed Central

    Gunathunge, B. G. C. M.; Wimalasiri, P. N.; Karunaratne, D. N.

    2017-01-01

    The use of gene therapeutics, including short interfering RNA (siRNA), is limited by the lack of efficient delivery systems. An appealing approach to deliver gene therapeutics involves noncovalent complexation with cell penetrating peptides (CPPs) which are able to penetrate the cell membranes of mammals. Although a number of CPPs have been discovered, our understanding of their complexation and translocation of siRNA is as yet insufficient. Here, we report on computational studies comparing the binding affinities of CPPs with siRNA, considering a variety of CPPs. Specifically, seventeen CPPs from three different categories, cationic, amphipathic, and hydrophobic CPPs, were studied. Molecular mechanics were used to minimize structures, while molecular docking calculations were used to predict the orientation and favorability of sequentially binding multiple peptides to siRNA. Binding scores from docking calculations were highest for amphipathic peptides over cationic and hydrophobic peptides. Results indicate that initial complexation of peptides will likely occur along the major groove of the siRNA, driven by electrostatic interactions. Subsequent binding of CPPs is likely to occur in the minor groove and later on bind randomly, to siRNA or previously bound CPPs, through hydrophobic interactions. However, hydrophobic CPPs do not show this binding pattern. Ultimately binding yields a positively charged nanoparticle capable of noninvasive cellular import of therapeutic molecules. PMID:28321253

  8. Mapping Optimal Charge Density and Length of ROMP-Based PTDMs for siRNA Internalization.

    PubMed

    Caffrey, Leah M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

    2016-10-10

    A fundamental understanding of how polymer structure impacts internalization and delivery of biologically relevant cargoes, particularly small interfering ribonucleic acid (siRNA), is of critical importance to the successful design of improved delivery reagents. Herein we report the use of ring-opening metathesis polymerization (ROMP) methods to synthesize two series of guanidinium-rich protein transduction domain mimics (PTDMs): one based on an imide scaffold that contains one guanidinium moiety per repeat unit, and another based on a diester scaffold that contains two guanidinium moieties per repeat unit. By varying both the degree of polymerization and, in effect, the relative number of cationic charges in each PTDM, the performances of the two ROMP backbones for siRNA internalization were evaluated and compared. Internalization of fluorescently labeled siRNA into Jurkat T cells demonstrated that fluorescein isothiocyanate (FITC)-siRNA internalization had a charge content dependence, with PTDMs containing approximately 40 to 60 cationic charges facilitating the most internalization. Despite this charge content dependence, the imide scaffold yielded much lower viabilities in Jurkat T cells than the corresponding diester PTDMs with similar numbers of cationic charges, suggesting that the diester scaffold is preferred for siRNA internalization and delivery applications. These developments will not only improve our understanding of the structural factors necessary for optimal siRNA internalization, but will also guide the future development of optimized PTDMs for siRNA internalization and delivery.

  9. New Type of BACE1 siRNA Delivery to Cells

    PubMed Central

    Jabłkowski, Maciej; Szemraj, Maciej; Oszajca, Katarzyna; Janiszewska, Grażyna; Bartkowiak, Jacek; Szemraj, Janusz

    2014-01-01

    Background Small interfering RNA (siRNA) gene therapy is a new molecular approach in the search for an efficient therapy for Alzheimer disease (AD), based on the principle of RNA interference. Reducing BACE activity can have great therapeutic potential for the treatment of AD. In this study, receptor-mediated delivery was used to deliver opioid peptide-conjugated BACE 1 to INR-32 human neuroblastoma cells. Material/Methods An INR-32 human neuroblastoma cell line was stably transfected to express the APP cDNA coding fragment containing the predicted sites for cleavage by α, β, or γ-secretase. This was then treated with BACE 1 siRNA to silence BACE gene expression. BACE gene transcription and translation was determined using BACE-1 siRNA cross-linked with opioid peptide, together with RT-PCR, Western blot analysis, and ELISA. Results Receptor-mediated delivery was used to introduce BACE1 siRNA to the APP – INR 32 human neuroblastoma cells. Decreased BACE mRNA and protein expression were observed after the cells were transfected with BACE1 siRNA. Conclusions Delivery of BACE1 siRNA appears to specifically reduce the cleavage of APP by inhibiting BACE1 activity. PMID:25491230

  10. Design of a Multicomponent Peptide-Woven Nanocomplex for Delivery of siRNA

    PubMed Central

    Jun, Eunsung; Kim, Soyoun; Kim, Jong-Ho; Cha, Kiweon; So, In-Seop; Son, Hye-Nam; Lee, Byung-Heon; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, Sang Yoon; Kim, In-San

    2015-01-01

    We developed and tested a multicomponent peptide-woven siRNA nanocomplex (PwSN) comprising different peptides designed for efficient cellular targeting, endosomal escape, and release of siRNA. To enhance tumor-specific cellular uptake, we connected an interleukin-4 receptor-targeting peptide (I4R) to a nine-arginine peptide (9r), yielding I4R-9r. To facilitate endosomal escape, we blended endosomolytic peptides into the I4R-9r to form a multicomponent nanocomplex. Lastly, we modified 9r peptides by varying the number and positions of positive charges to obtain efficient release of siRNA from the nanocomplex in the cytosol. Using this step-wise approach for overcoming the biological challenges of siRNA delivery, we obtained an optimized PwSN with significant biological activity in vitro and in vivo. Interestingly, surface plasmon resonance analyses and three-dimensional peptide models demonstrated that our designed peptide adopted a unique structure that was correlated with faster complex disassembly and a better gene-silencing effect. These studies further elucidate the siRNA nanocomplex delivery pathway and demonstrate the applicability of our stepwise strategy to the design of siRNA carriers capable of overcoming multiple challenges and achieving efficient delivery. PMID:25705892

  11. Delivery of siRNA into the cytoplasm by liposomal bubbles and ultrasound.

    PubMed

    Negishi, Yoichi; Endo, Yoko; Fukuyama, Tetsuya; Suzuki, Ryo; Takizawa, Tomoko; Omata, Daiki; Maruyama, Kazuo; Aramaki, Yukihiko

    2008-12-08

    Small interfering RNA (siRNA) is expected to be a novel therapeutic tool, however, its utilization has been limited by inefficient delivery systems. Recently, we have developed novel polyethyleneglycol modified liposomes (Bubble liposomes; BL) entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated whether the BL were suitable for the delivery of siRNA. BL efficiently delivered siRNA with only 10 s of exposure to US in vitro. Specific gene silencing effects could be achieved well even in the presence of serum or with the disruption of endocytosis. We suggest that siRNA is directly introduced into the cytoplasm by the BL and US and the mechanism enables effective transfection within a short time and in the presence of high serum. Transfection of siRNA into the tibialis muscles with BL and US was also performed. The gene-silencing effect could be sustained for more than 3 weeks. Thus, BL could be a useful siRNA delivery tool in vitro and in vivo.

  12. Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers

    PubMed Central

    2012-01-01

    Controlling gene expression via small interfering RNA (siRNA) has opened the doors to a plethora of therapeutic possibilities, with many currently in the pipelines of drug development for various ocular diseases. Despite the potential of siRNA technologies, barriers to intracellular delivery significantly limit their clinical efficacy. However, recent progress in the field of drug delivery strongly suggests that targeted manipulation of gene expression via siRNA delivered through nanocarriers can have an enormous impact on improving therapeutic outcomes for ophthalmic applications. Particularly, synthetic nanocarriers have demonstrated their suitability as a customizable multifunctional platform for the targeted intracellular delivery of siRNA and other hydrophilic and hydrophobic drugs in ocular applications. We predict that synthetic nanocarriers will simultaneously increase drug bioavailability, while reducing side effects and the need for repeated intraocular injections. This review will discuss the recent advances in ocular siRNA delivery via non-viral nanocarriers and the potential and limitations of various strategies for the development of a ‘universal’ siRNA delivery system for clinical applications. PMID:22686441

  13. siRNA Versus miRNA as Therapeutics for Gene Silencing

    PubMed Central

    Lam, Jenny K W; Chow, Michael Y T; Zhang, Yu; Leung, Susan W S

    2015-01-01

    Discovered a little over two decades ago, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNAs with important roles in gene regulation. They have recently been investigated as novel classes of therapeutic agents for the treatment of a wide range of disorders including cancers and infections. Clinical trials of siRNA- and miRNA-based drugs have already been initiated. siRNAs and miRNAs share many similarities, both are short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), yet their mechanisms of action and clinical applications are distinct. The major difference between siRNAs and miRNAs is that the former are highly specific with only one mRNA target, whereas the latter have multiple targets. The therapeutic approaches of siRNAs and miRNAs are therefore very different. Hence, this review provides a comparison between therapeutic siRNAs and miRNAs in terms of their mechanisms of action, physicochemical properties, delivery, and clinical applications. Moreover, the challenges in developing both classes of RNA as therapeutics are also discussed. PMID:26372022

  14. [siRNAs with high specificity to the target: a systematic design by CRM algorithm].

    PubMed

    Alsheddi, T; Vasin, L; Meduri, R; Randhawa, M; Glazko, G; Baranova, A

    2008-01-01

    'Off-target' silencing effect hinders the development of siRNA-based therapeutic and research applications. Common solution to this problem is an employment of the BLAST that may miss significant alignments or an exhaustive Smith-Waterman algorithm that is very time-consuming. We have developed a Comprehensive Redundancy Minimizer (CRM) approach for mapping all unique sequences ("targets") 9-to-15 nt in size within large sets of sequences (e.g. transcriptomes). CRM outputs a list of potential siRNA candidates for every transcript of the particular species. These candidates could be further analyzed by traditional "set-of-rules" types of siRNA designing tools. For human, 91% of transcripts are covered by candidate siRNAs with kernel targets of N = 15. We tested our approach on the collection of previously described experimentally assessed siRNAs and found that the correlation between efficacy and presence in CRM-approved set is significant (r = 0.215, p-value = 0.0001). An interactive database that contains a precompiled set of all human siRNA candidates with minimized redundancy is available at http://129.174.194.243. Application of the CRM-based filtering minimizes potential "off-target" silencing effects and could improve routine siRNA applications.

  15. Effective cytoplasmic release of siRNA from liposomal carriers by controlling the electrostatic interaction of siRNA with a charge-invertible peptide, in response to cytoplasmic pH

    NASA Astrophysics Data System (ADS)

    Itakura, Shoko; Hama, Susumu; Matsui, Ryo; Kogure, Kentaro

    2016-05-01

    Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is effectively released via electrostatic repulsion of siRNA with negatively charged SAPSP at cytoplasmic pH (7.4). The condensed complex of siRNA and positively-charged SAPSP at acidic pH (siRNA/SAPSP) was found to result in almost complete release of siRNA upon charge inversion of SAPSP at pH 7.4, with the resultant negatively-charged SAPSP having no undesirable interactions with endogenous mRNA. Moreover, liposomes encapsulating siRNA/SAPSP demonstrated knockdown efficiencies comparable to those of commercially available siRNA carriers. Taken together, SAPSP may be very useful as a siRNA condenser, as it facilitates effective cytoplasmic release of siRNA, and subsequent induction of specific RNAi effects.Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is

  16. Extreme Heat Guidebook

    EPA Pesticide Factsheets

    The 'Climate Change and Extreme Heat: What You Can Do to Prepare' handbook explains the connection between climate change and extreme heat events, and outlines actions citizens can take to protect their health during extreme heat.

  17. Magnetic nanoparticle and magnetic field assisted siRNA delivery in vitro.

    PubMed

    Mykhaylyk, Olga; Sanchez-Antequera, Yolanda; Vlaskou, Dialechti; Cerda, Maria Belen; Bokharaei, Mehrdad; Hammerschmid, Edelburga; Anton, Martina; Plank, Christian

    2015-01-01

    This chapter describes how to design and conduct experiments to deliver siRNA to adherent cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles (MNPs). These magnetic complexes are targeted to the cell surface by the application of a gradient magnetic field. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected MNPs, phospholipids, and siRNAs are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres (microbubbles). Methods are described how to accomplish the synthesis of magnetic nanoparticles for magnetofection and how to test the association of siRNA with the magnetic components of the transfection vector. A simple method is described to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Procedures are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA as well as magnetic microbubbles for magnetofection and downregulation of the target gene expression analysis with account for the toxicity determined using an MTT-based respiration activity test. A modification of the magnetic transfection triplexes with INF-7, fusogenic peptide, is described resulting in reporter gene silencing improvement in HeLa, Caco-2, and ARPE-19 cells. The methods described can also be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized siRNA transfection by magnetofection in any cell type.

  18. Chitosan Hydrogel as siRNA vector for prolonged gene silencing

    PubMed Central

    2014-01-01

    Background The periodontitis is one of the most prevalent diseases with alveolar resorption in adult people and is the main cause of the tooth loss. To investigate the possibility for protecting the loss of alveolar bone in periodontal diseases, a RNAi-based therapeutic strategy is applied for silencing RANK signaling using thermosensitive chitosan hydrogel as siRNA reservoir and vector. Results The thermosensitive chitosan hydrogel was formed from solution (PH = 7.2, at 4°C) at 37°C within 8 minutes. The degradation rates of hydrogel were ~50% and 5% (W remaining/W beginning) in the presence and absence of lysozyme, respectively, over a period of 20 days. The concurrent cumulative in vitro release of Cy3-labeled siRNA from the hydrogel was 50% and 17% over 14 days, with or without lysozyme digestion, respectively. High cell viability (>88%) was maintained for cells treated with hydrogel loaded with RANK specific siRNA and RANK knockdown was prolonged for up to 9 days when cells were incubated with siRNA/hydrogel complex. In vivo release of siRNA was investigated in a subcutaneous delivery setup in mice. The fluorescent signal from siRNA within hydrogel was remained for up to 14 days compared to less than one day for siRNA alone. Conclusions Chitosan hydrogel can potentially serve as a suitable reservoir and vector for local sustained delivery of siRNA in potential therapy. PMID:24946934

  19. PEGylation of lipoplexes: The right balance between cytotoxicity and siRNA effectiveness.

    PubMed

    Lechanteur, Anna; Furst, Tania; Evrard, Brigitte; Delvenne, Philippe; Hubert, Pascale; Piel, Géraldine

    2016-10-10

    The delivery of small interfering RNA (siRNA) is an attractive therapeutic approach to treat several pathologies, such as viral infections or cancers. However, the stability and the efficacy of these biotherapies are still a major obstacle to their use. Cationic liposomes (DOTAP/Chol/DOPE 1/0.75/0.5M ratio) have been complexed to siRNA (lipoplexes) in order to be administrated by the vaginal route, in the context of HPV16 induced cervical preneoplastic lesions. To overcome the constraint of the cervico-vaginal mucus, PEGylation is required to allow the diffusion of lipoplexes through it. Thereby, PEGylated lipoplexes coated with three types of polyethylene glycol (PEG) as DSPE-PEG2000, DSPE-PEG750 or C8-PEG2000-Ceramide (Ceramide-PEG2000) at different densities have been developed and characterized. PEGylated lipoplexes were successfully prepared and showed a hydrodynamic diameter around 200nm, appropriate for vaginal application. In vitro assays on HPV16 positive cell lines revealed that a positive charge of PEGylated lipoplexes allows a higher mRNA knockdown by siRNA. However, the cationic property is also associated to cytotoxicity. The addition of a high percentage of PEG prevented this toxicity but seemed also to reduce siRNA endosomal escape, probably by steric hindrance. The decreasing of PEG density of Ceramide-PEG2000 to 20% allows the release of siRNA and in consequence, biological activities, contrarily to DSPE-PEG. These results suggest that Ceramide-PEG is more appropriate for siRNA delivery compared to DSPE-PEG. In conclusion, the right balance between cytotoxicity and siRNA effectiveness has been found with the transfection of lipoplexes coated with 20% of Ceramide-PEG2000. This new nanovector could have a high potential against multiple mucosal diseases, such as human papillomavirus-induced genital lesions.

  20. Thermo-sensitive nanoparticles for triggered release of siRNA.

    PubMed

    Yang, Zheng; Cheng, Qiang; Jiang, Qian; Deng, Liandong; Liang, Zicai; Dong, Anjie

    2015-01-01

    Efficient delivery of small interfering RNA (siRNA) is crucially required for cancer gene therapy. Herein, a thermo-sensitive copolymer with a simple structure, poly (ethylene glycol) methyl ether acrylate-b-poly (N-isopropylacrylamide) (mPEG-b-PNIPAM) was developed. A novel kind of thermo-sensitive nanoparticles (DENPs) was constructed for the cold-shock triggered release of siRNA by double emulsion-solvent evaporation method using mPEG-b-PNIPAM and a cationic lipid, 3β [N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol [DC-Chol]. DENPs were observed by transmission electron microscopy and dynamical light scattering before and after 'cold shock' treatment. The encapsulation efficiency (EE) of siRNA in DENPs, which was measured by fluorescence spectrophotometer was 96.8% while it was significantly reduced to be 23.2% when DC-Chol was absent. DENPs/siRNA NPs exhibited a thermo-sensitive siRNA release character that the cumulatively released amount of siRNA from cold shock was approximately 2.2 folds higher after 7 days. In vitro luciferase silencing experiments indicated that DENPs showed potent gene silencing efficacy in HeLa-Luc cells (HeLa cells steadily expressed luciferase), which was further enhanced by a cold shock. Furthermore, MTT assay showed that cell viability with DENPs/siRNA up to 200 nM remained above 80%. We also observed that most of siRNA was accumulated in kidney mediated by DENPs instead of liver and spleen in vivo experiments. Thus, DENPs as a cold shock responsive quick release model for siRNA or hydrophilic macromolecules delivery provide a new way to nanocarrier design and clinic therapy.

  1. Flare Plasma Iron Abundance

    NASA Technical Reports Server (NTRS)

    Dennis, Brian R.; Dan, Chau; Jain, Rajmal; Schwartz, Richard A.; Tolbert, Anne K.

    2008-01-01

    The equivalent width of the iron-line complex at 6.7 keV seen in flare X-ray spectra suggests that the iron abundance of the hottest plasma at temperatures >approx.10 MK may sometimes be significantly lower than the nominal coronal abundance of four times the photospheric value that is commonly assumed. This conclusion is based on X-ray spectral observations of several flares seen in common with the Ramaty High Energy Solar Spectroscopic Imager (RHESSI) and the Solar X-ray Spectrometer (SOXS) on the second Indian geostationary satellite, GSAT-2. The implications of this will be discussed as it relates to the origin of the hot flare plasma - either plasma already in the corona that is directly heated during the flare energy release process or chromospheric plasma that is heated by flare-accelerated particles and driven up into the corona. Other possible explanations of lower-than-expected equivalent widths of the iron-line complex will also be discussed.

  2. The Origin of Element Abundance Variations in Solar Energetic Particles

    NASA Astrophysics Data System (ADS)

    Reames, Donald V.

    2016-08-01

    Abundance enhancements, during acceleration and transport in both gradual and impulsive solar energetic particle (SEP) events, vary approximately as power laws in the mass-to-charge ratio [A/Q] of the ions. Since the Q-values depend upon the electron temperature of the source plasma, this has allowed a determination of this temperature from the pattern of element-abundance enhancements and a verification of the expected inverse-time dependence of the power of A/Q for diffusive transport of ions from the SEP events, with scattering mean free paths found to be between 0.2 and 1 AU. SEP events derived from plasma of different temperatures map into different regions in typical cross-plots of abundances, spreading the distributions. In comparisons of SEP events with temperatures above 2 MK, impulsive events show much broader non-thermal variation of abundances than do gradual events. The extensive shock waves accelerating ions in gradual events may average over much of an active region where numerous but smaller magnetic reconnections, "nanojets", produce suprathermal seed ions, thus averaging over varying abundances, while an impulsive SEP event only samples one local region of abundance variations. Evidence for a reference He/O-abundance ratio of 91, rather than 57, is also found for the hotter plasma. However, while this is similar to the solar-wind abundance of He/O, the solar-wind abundances otherwise provide an unacceptably poor reference for the SEP-abundance enhancements, generating extremely large errors.

  3. Degradable Dextran Nanopolymer as a Carrier for Choline Kinase (ChoK) siRNA Cancer Therapy

    PubMed Central

    Chen, Zhihang; Krishnamachary, Balaji; Bhujwalla, Zaver M.

    2016-01-01

    Although small interfering RNA (siRNA) therapy has proven to be a specific and effective treatment in cells, the delivery of siRNA is a challenge for the applications of siRNA therapy. We present a degradable dextran with amine groups as an siRNA nano-carrier. In our nano-carrier, the amine groups are conjugated to the dextran platform through the acetal bonds, which are acid sensitive. Therefore this siRNA carrier is stable in neutral and basic conditions, while the amine groups can be cleaved and released from dextran platform under weak acid conditions (such as in endosomes). The cleavage and release of amine groups can reduce the toxicity of cationic polymer and enhance the transfection efficiency. We successfully applied this nano-carrier to deliver choline kinase (ChoK) siRNA for ChoK inhibition in cells.

  4. Photochemical Regulation of Gene Expression Using Caged siRNAs with Single Terminal Vitamin E Modification.

    PubMed

    Ji, Yuzhuo; Yang, Jiali; Wu, Li; Yu, Lijia; Tang, Xinjing

    2016-02-05

    Caged siRNAs with a single photolabile linker and/or vitamin E (vitE) modification at the 5' terminal were rationally designed and synthesized. These virtually inactive caged siRNAs were successfully used to photoregulate both firefly luciferase and GFP gene expression in cells with up to an 18.6-fold enhancement of gene silencing activity, which represents one of the best reported photomodulation of gene silencing efficiencies to date. siRNA tracking and vitE competition experiments indicated that the inactivity of vitE-modified siRNAs was not due to the bulky moiety of vitE; rather, the involvement of vitE-binding proteins has a large contribution to caged siRNA inactivation by preventing the dissociation of siRNA/lipo complexes and/or siRNA release. Further patterning experiments revealed the ability to spatially regulate gene expression through simple light irradiation.

  5. Highly efficient siRNA delivery system into human and murine cells using single-wall carbon nanotubes.

    PubMed

    Ladeira, M S; Andrade, V A; Gomes, E R M; Aguiar, C J; Moraes, E R; Soares, J S; Silva, E E; Lacerda, R G; Ladeira, L O; Jorio, A; Lima, P; Leite, M Fatima; Resende, R R; Guatimosim, S

    2010-09-24

    Development of RNA interference (RNAi) technology utilizing short interfering RNA sequences (siRNA) has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA coiling into carboxyl-functionalized single-wall carbon nanotubes (SWCNTs). The CNT-siRNA delivery system successfully demonstrates nonspecific toxicity and transfection efficiency greater than 95%. This approach offers the potential for siRNA delivery into different types of cells, including hard-to-transfect cells, such as neuronal cells and cardiomyocytes. We also tested the CNT-siRNA system in a non-metastatic human hepatocellular carcinoma cell line (SKHep1). In all types of cells used in this work the CNT-siRNA delivery system showed high efficiency and apparent no side effects for various in vitro applications.

  6. Atelocollagen-mediated Systemic Delivery Prevents Immunostimulatory Adverse Effects of siRNA in Mammals

    PubMed Central

    Inaba, Shinichiro; Nagahara, Shunji; Makita, Naoki; Tarumi, Yuzo; Ishimoto, Takuji; Matsuo, Seiichi; Kadomatsu, Kenji; Takei, Yoshifumi

    2012-01-01

    Short interfering RNA (siRNA) is a potent activator of the mammalian innate immune system. When considering possible clinical applications of siRNA for humans, the adverse immunostimulatory effects must also be taken into account. Here, we show that atelocollagen-mediated systemic delivery of siRNA without chemical modifications did not cause any immunostimulation in both animals and human peripheral blood mononuclear cells (PBMCs), even if the siRNA harbored an interferon (IFN)-inducible sequence. In contrast, systemic delivery of immunostimulatory RNA (isRNA)-mediated by a cationic lipid (such as Invivofectamine) induced potent type-I IFNs and inflammatory cytokines. Regarding the mechanism by which the isRNA/atelocollagen complex avoided adverse effects on immunostimulation, we revealed that this complex was not incorporated into PBMCs. On the other hand, Invivofectamine delivered isRNA into PBMCs. The use of either atelocollagen or Invivofectamine as a vehicle elicited significant and undistinguishable therapeutic effects in a contact hypersensitivity (CHS) inflammatory model mouse, when we intravenously injected the siRNA targeting monocyte chemoattractant protein-1 as the complex. For the goal of realizing siRNA-based medicines for humans, atelocollagen is an excellent and promising delivery vehicle, and it has the useful advantage of evading detection by the “radar” of innate immunity. PMID:22031237

  7. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling.

    PubMed

    Sahay, Gaurav; Querbes, William; Alabi, Christopher; Eltoukhy, Ahmed; Sarkar, Sovan; Zurenko, Christopher; Karagiannis, Emmanouil; Love, Kevin; Chen, Delai; Zoncu, Roberto; Buganim, Yosef; Schroeder, Avi; Langer, Robert; Anderson, Daniel G

    2013-07-01

    Despite efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain unclear. Here we examine cellular uptake of short interfering RNA (siRNA) delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy. We also employed defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR and cathepsins. siRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann-Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes and lysosomes, and increased gene silencing of the target gene. Our data suggest that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.

  8. Hypoxia-responsive ionizable liposome delivery siRNA for glioma therapy

    PubMed Central

    Liu, Hong-Mei; Zhang, Ya-Fei; Xie, Yan-Dong; Cai, Yi-Fan; Li, Bai-Yang; Li, Wen; Zeng, Ling-Yu; Li, Yu-Ling; Yu, Ru-Tong

    2017-01-01

    Here, we report the hypoxia-responsive ionizable liposomes to deliver small interference RNA (siRNA) anticancer drugs, which can selectively enhance cellular uptake of the siRNA under hypoxic and low-pH conditions to cure glioma. For this purpose, malate dehydrogenase lipid molecules were synthesized, which contain nitroimidazole groups that impart hypoxia sensitivity and specificity as hydrophobic tails, and tertiary amines as hydrophilic head groups. These malate dehydrogenase molecules, together with DSPE-PEG2000 and cholesterol, were self-assembled into O′1,O1-(3-(dimethylamino)propane-1,2-diyl) 16-bis(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) di(hexadecanedioate) liposomes (MLP) to encapsulate siRNA through electrostatic interaction. Our study showed that the MLP could deliver polo-like kinase 1 siRNA (siPLK1) into glioma cells and effectively enhance the cellular uptake of MLP/siPLK1 because of increased positive charges induced by hypoxia and low pH. Moreover, MLP/siPLK1 was shown to be very effective in inhibiting the growth of glioma cells both in vitro and in vivo. Therefore, the MLP is a promising siRNA delivery system for tumor therapy. PMID:28223799

  9. Improved siRNA Delivery Efficiency via Solvent-Induced Condensation of Micellar Nanoparticles.

    PubMed

    Wu, Juan; Qu, Wei; Williford, John-Michael; Ren, Yong; Jiang, Xuesong; Jiang, Xuan; Pan, Deng; Mao, Hai-Quan; Luijten, Erik

    2017-03-07

    Efficient delivery of siRNA remains one of the primary challenges of RNA interference therapy. PEGylated polycationic carriers have been widely used for the condensation of DNA and RNA molecules into complex-core micelles. The PEG corona of such nanoparticles can significantly improve their colloidal stability in serum, but PEGylation of the carriers also reduces their condensation capacity, hindering the generation of micellar particles with sufficient complex stability. This presents a particularly significant challenge for packaging siRNA into complex micelles, as it has a much smaller size and more rigid chain structure than DNA plasmids. Here, we report a new method to enhance the condensation of siRNA with PEGylated linear polyethylenimine (lPEI) using organic solvent and to prepare smaller siRNA nanoparticles with a more extended PEG corona and consequently higher stability. As a proof of principle, we have demonstrated the improved gene knockdown efficiency resulting from the reduced siRNA micelle size in mouse liver following intravenous administration.

  10. Synthesis of folate-functionalized RAFT polymers for targeted siRNA delivery.

    PubMed

    Benoit, Danielle S W; Srinivasan, Selvi; Shubin, Andrew D; Stayton, Patrick S

    2011-07-11

    Receptor-mediated, cell-specific delivery of siRNA enables silencing of target genes in specific tissues, opening the door to powerful therapeutic options for a multitude of diseases. However, the development of delivery systems capable of targeted and effective siRNA delivery typically requires multiple steps and the use of sophisticated, orthogonal chemistries. Previously, we developed diblock copolymers consisting of dimethaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-co-propylacrylic acid as potent siRNA delivery systems that protect siRNA from enzymatic degradation and enable its cytosolic delivery through pH-responsive, endosomolytic behavior. (1, 2) These architectures were polymerized using a living radical polymerization method, specifically reversible addition-fragmentation chain transfer (RAFT) polymerization, which employs a chain transfer agent (CTA) to modulate the rate of reaction, resulting in polymers with low polydispersity and telechelic chain ends reflecting the chemistry of the CTA. Here we describe the straightforward, facile synthesis of a folate receptor-targeted diblock copolymer siRNA delivery system because the folate receptor is an attractive target for tumor-selective therapies as a result of its overexpression in a number of cancers. Specifically, we detail the de novo synthesis of a folate-functionalized CTA, use the folate-CTA for controlled polymerizations of diblock copolymers, and demonstrate efficient, specific cellular folate receptor interaction and in vitro gene knockdown using the folate-functionalized polymer.

  11. Efficient inhibition of fibroblast proliferation and collagen expression by ERK2 siRNAs

    SciTech Connect

    Li, Fengfeng; Fan, Cunyi; Cheng, Tao; Jiang, Chaoyin; Zeng, Bingfang

    2009-05-01

    Transforming growth factor-{beta}1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p < 0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.

  12. Chapter 17 - Engineering cationic liposome siRNA complexes for in vitro and in vivo delivery.

    PubMed

    Podesta, Jennifer E; Kostarelos, Kostas

    2009-01-01

    RNA interference, the sequence-specific silencing of gene expression by introduction of short interfering RNA (siRNA) is a powerful tool that that the potential to act as a therapeutic agent and the advantage of decreasing toxic effects on normal tissue sometimes seen with conventional treatments i.e. small molecule inhibitors. Naked, unmodified siRNA is poorly taken up by cells and is subject to degradation when exposed to blood proteins during systemic administration. It has also been shown to produce non-specific immune response as well as having the potential to generate 'off-target' effects. Therefore there is a requirement for a delivery system to not only protect the siRNA and facilitate its uptake, but additionally to offer the potential for targeted delivery with an aim of exploiting the high specificity afforded by RNA interference. Cationic liposomes are the most studied, non-viral delivery system used for nucleic acid delivery. As such, the use of cationic liposomes is promising for siRNA for delivery. Furthermore, polyethylene glycol (PEG) can be incorporated into the liposome formulation to create sterically stabilized or 'stealth' liposomes. Addition of PEG can reduce recognition by the reticuloendothelial system (RES) thereby prolonging circulation time. Here we describe a methodology for the complexation of siRNA with cationic liposomes and PEGylated liposomes using two protocols: mixing and encapsulation. Moreover, the different formulations are compared head to head to demonstrate their efficacy for gene silencing.

  13. Silencing Myostatin Using Cholesterol-conjugated siRNAs Induces Muscle Growth

    PubMed Central

    Khan, Tayeba; Weber, Hans; DiMuzio, Jillian; Matter, Andrea; Dogdas, Belma; Shah, Tosha; Thankappan, Anil; Disa, Jyoti; Jadhav, Vasant; Lubbers, Laura; Sepp-Lorenzino, Laura; Strapps, Walter R; Tadin-Strapps, Marija

    2016-01-01

    Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85–95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes. PMID:27483025

  14. CDE-1 affects chromosome segregation through uridylation of CSR-1-bound siRNAs.

    PubMed

    van Wolfswinkel, Josien C; Claycomb, Julie M; Batista, Pedro J; Mello, Craig C; Berezikov, Eugene; Ketting, René F

    2009-10-02

    We have studied the function of a conserved germline-specific nucleotidyltransferase protein, CDE-1, in RNAi and chromosome segregation in C. elegans. CDE-1 localizes specifically to mitotic chromosomes in embryos. This localization requires the RdRP EGO-1, which physically interacts with CDE-1, and the Argonaute protein CSR-1. We found that CDE-1 is required for the uridylation of CSR-1 bound siRNAs, and that in the absence of CDE-1 these siRNAs accumulate to inappropriate levels, accompanied by defects in both meiotic and mitotic chromosome segregation. Elevated siRNA levels are associated with erroneous gene silencing, most likely through the inappropriate loading of CSR-1 siRNAs into other Argonaute proteins. We propose a model in which CDE-1 restricts specific EGO-1-generated siRNAs to the CSR-1 mediated, chromosome associated RNAi pathway, thus separating it from other endogenous RNAi pathways. The conserved nature of CDE-1 suggests that similar sorting mechanisms may operate in other animals, including mammals.

  15. Multifunctional nanocarrier based on clay nanotubes for efficient intracellular siRNA delivery and gene silencing.

    PubMed

    Wu, Hui; Shi, Yinfeng; Huang, Chusen; Zhang, Yang; Wu, Jiahui; Shen, Hebai; Jia, Nengqin

    2014-04-01

    RNA interference-mediated gene silencing relating to disease has recently emerged as a powerful method in gene therapy. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. Halloysites are cheap and naturally available aluminosilicate clay nanotubes with high mechanical strength and biocompatibility. In this study, a novel multifunctional nanocarrier based on functionalized halloysite nanotubes (f-HNTs) has been developed via electrostatic layer-by-layer assembling approach for loading and intracellular delivery of therapeutic antisurvivin siRNA and simultaneously tracking their intracellular transport, in which PEI-modified HNTs are used as gene vector, antisurvivin siRNA as gene therapeutic agent, and mercaptoacetic acid-capped CdSe quantum dots as fluorescent labeling probes. The successful assembly of the f-HNTs-siRNA complexes was systematically characterized by transmission electron microscopy (TEM), UV-visible spectrophotometry, Zeta potential measurement, fluorescence spectrophotometry, and electrochemical impedance spectroscopy. Confocal microscopy, biological TEM, and flow cytometry studies revealed that the complexes enabled the efficient intracellular delivery of siRNA for cell-specific gene silencing. MTT assays exhibited that the complexes can enhance antitumor activity. Furthermore, Western blot analysis showed that f-HNTs-mediated siRNA delivery effectively knocked down gene expression of survivin and thereby decreased the levels of target proteins of PANC-1 cells. Therefore, this study suggested that the synthesized f-HNTs were a new effective drug delivery system for potential application in cancer gene therapy.

  16. Mechanisms of Nanoparticle Mediated siRNA Transfection by Melittin-Derived Peptides

    PubMed Central

    Hou, Kirk K.; Pan, Hua; Ratner, Lee; Schlesinger, Paul H.; Wickline, Samuel A.

    2014-01-01

    Traditional peptide-mediated siRNA transfection via peptide transduction domains exhibits limited cytoplasmic delivery of siRNA due to endosomal entrapment. This work overcomes these limitations with the use of membrane-destabilizing peptides derived from melittin for the knockdown of NFkB signaling in a model of adult T-Cell leukemia/lymphoma. While the mechanism of siRNA delivery into the cytoplasmic compartment by peptide transduction domains has not been well studied, our analysis of melittin derivatives indicates that concurrent nanocomplex disassembly and peptide-mediated endosomolysis are crucial to siRNA transfection. Importantly, in the case of the most active derivative, p5RHH, this process is initiated by acidic pH, indicating that endosomal acidification after macropinocytosis can trigger siRNA release into the cytoplasm. These data provide general principles regarding nanocomplex response to endocytosis which may guide the development of peptide/siRNA nanocomplex-based transfection. PMID:24053333

  17. Oxygen abundance and convection

    NASA Astrophysics Data System (ADS)

    Van't Veer, C.; Cayrel, R.

    The triplet IR lines of O I near 777 nm are computed with the Kurucz's code, modified to accept several convection models. The program has been run with the MLT algorithm, with l/H = 1.25 and 0.5, and with the Canuto-Mazzitelli and Canuto-Goldman-Mazzitelli approaches, on a metal-poor turnoff-star model atmosphere with Teff=6200 K, log g = 4.3, [Fe/H]= -1.5. The results show that the differences in equivalent widths for the 4 cases do not exceed 2 per cent (0.3 mA). The convection treatment is therefore not an issue for the oxygen abundance derived from the permitted lines.

  18. Unique Gene-Silencing and Structural Properties of 2;#8242;-Fluoro-Modified siRNAs

    SciTech Connect

    Manoharan, Muthiah; Akinc, Akin; Pandey, Rajendra K.; Qin, June; Hadwiger, Philipp; John, Matthias; Mills, Kathy; Charisse, Klaus; Maier, Martin A.; Nechev, Lubomir; Greene, Emily M.; Pallan, Pradeep S.; Rozners, Eriks; Rajeev, Kallanthottathil G.; Egli, Martin

    2015-10-15

    With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2'-deoxy-2'-fluoro (2'-F) modification is unique. Furthermore, the 2'-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response.

  19. Delivery of siRNA using ternary complexes containing branched cationic peptides: the role of peptide sequence, branching and targeting.

    PubMed

    Kudsiova, Laila; Welser, Katharina; Campbell, Frederick; Mohammadi, Atefeh; Dawson, Natalie; Cui, Lili; Hailes, Helen C; Lawrence, M Jayne; Tabor, Alethea B

    2016-03-01

    Ternary nanocomplexes, composed of bifunctional cationic peptides, lipids and siRNA, as delivery vehicles for siRNA have been investigated. The study is the first to determine the optimal sequence and architecture of the bifunctional cationic peptide used for siRNA packaging and delivery using lipopolyplexes. Specifically three series of cationic peptides of differing sequence, degrees of branching and cell-targeting sequences were co-formulated with siRNA and vesicles prepared from a 1 : 1 molar ratio of the cationic lipid DOTMA and the helper lipid, DOPE. The level of siRNA knockdown achieved in the human alveolar cell line, A549-luc cells, in both reduced serum and in serum supplemented media was evaluated, and the results correlated to the nanocomplex structure (established using a range of physico-chemical tools, namely small angle neutron scattering, transmission electron microscopy, dynamic light scattering and zeta potential measurement); the conformational properties of each component (circular dichroism); the degree of protection of the siRNA in the lipopolyplex (using gel shift assays) and to the cellular uptake, localisation and toxicity of the nanocomplexes (confocal microscopy). Although the size, charge, structure and stability of the various lipopolyplexes were broadly similar, it was clear that lipopolyplexes formulated from branched peptides containing His-Lys sequences perform best as siRNA delivery agents in serum, with protection of the siRNA in serum balanced against efficient release of the siRNA into the cytoplasm of the cell.

  20. Synthesis and evaluation of degradable polyurea block copolymers as siRNA delivery agents.

    PubMed

    Cass, Peter; Knower, Warren; Hinton, Tracey; Shi, Shuning; Grusche, Felix; Tizard, Mark; Gunatillake, Pathiraja

    2013-09-01

    Chain extension by diisocyanate condensation provides a versatile and convenient means for preparing block copolymers. We have utilized this chemistry to prepare reducible multiblock polycations for siRNA delivery. This approach, an alternative to oxidative coupling, was suitable for preparing multiblock polycations with defined molecular weight and architecture. The polymer, PEG-b-multi-(polyhexylurea-co-oligo-L-lysine)-b-PEG, was capable of electrostatically condensing siRNA to form nano-sized polyplexes across a broad compositional range. We demonstrated that the polyplexes enter the cells via endocytosis and interact with the endosome membrane leading to destabilization and hence endosome escape. Another feature of these polymers is their multiple intra-chain disulfide linkages. This enables weakening of the polyplex via chain scission within the cytosol's reductive environment. In addition to the controlled preparation of the polymer, the polyplexes were capable of delivering siRNA in vitro to silence greater than 50% green fluorescent protein expression with negligible toxicity.

  1. Carbon nanotube-mediated siRNA delivery for gene silencing in cancer cells

    NASA Astrophysics Data System (ADS)

    Hong, Tu; Guo, Honglian; Xu, Yaqiong

    2011-10-01

    Small interfering RNA (siRNA) is potentially a promising tool in influencing gene expression with a high degree of target specificity. However, its poor intracellular uptake, instability in vivo, and non-specific immune stimulations impeded its effect in clinical applications. In this study, carbon nanotubes (CNTs) functionalized with two types of phospholipid-polyethylene glycol (PEG) have shown capabilities to stabilize siRNA in cell culture medium during the transfection and efficiently deliver siRNA into neuroblastoma and breast cancer cells. Moreover, the intrinsic optical properties of CNTs have been investigated through absorption and fluorescence measurements. We have found that the directly-functionalized groups play an important role on the fluorescence imaging of functionalized CNTs. The unique fluorescence imaging and high delivery efficiency make CNTs a promising material to deliver drugs and evaluate the treatment effect simultaneously.

  2. Selective Modification of HK Peptides Enhances siRNA Silencing of Tumor Targets In Vivo

    PubMed Central

    Chou, Szu-Ting; Leng, Qixin; Scaria, Puthupparampil; Woodle, Martin; Mixson, A. James

    2011-01-01

    Our research has focused on systemic delivery of small interference RNA (siRNA) by branched peptides composed of histidine and lysine, called HK peptides. After studying several HK peptides, one four-branched peptide, H3K(+H)4b, with a predominant repeating pattern of -HHHK-, was found to be an effective carrier of siRNA. Although the unmodified H3K(+H)4b carrier of siRNA targeting an oncogene was previously shown to have promise in a tumor-bearing mouse model, we sought to develop a more effective HK carrier of siRNA in the current study. Our primary goal was to determine whether different ligand (cyclic RGD)-pegylation patterns on the H3K(+H)4b peptide affect siRNA delivery in vitro and in vivo. We compared the unmodified H3K(+H)4b with two modified H3K(+H)4b peptides for their ability to deliver siRNA in a tumor-bearing mouse model; one modified HK peptide, (RGD-PEG)4-H3K(+H)4b, had four cRGD-PEG conjugated to each molecule, while the other peptide, (RGD-PEG)-H3K(+H)4b, had one cRGD-PEG per molecule. Although the modified HK peptides by themselves did not form stable nanoplexes with siRNA, combination of a highly charged unmodified HK peptide, H2K4b, with either of the modified HK peptides did form stable siRNA nanoparticles. For in vitro experiments with MDA-MB-435 cells that expressed luciferase, the H3K(+H)4b siRNA nanoplexes targeting luciferase decreased its activity by 90% compared with negligible down-regulation by the modified H3K(+H)4b nanoplexes (P<0.01). In contrast, the two modified H3K(+H)4b siRNA nanoplexes administered intravenously were more effective than the H3K(+H)4b nanoplexes in silencing luciferase in a tumor xenograft model. The luciferase activity in tumor lysates of mice administered H3K(+H)4b, (RGD-PEG)-H3K(+H)4b, and (RGD-PEG)4-H3K(+H)4b nanoplexes decreased by 18%, 35%, and 75%, respectively. Thus, the siRNA nanoplex incorporating the highly modified peptide, (RGD-PEG)4-H3K(+H)4b, was the most effective at silencing its target in vivo

  3. Exosomes as nanocarriers for siRNA delivery: paradigms and challenges

    PubMed Central

    Shahabipour, Fahimeh; Banach, Maciej

    2016-01-01

    Exosomes are nano-sized vesicles that facilitate intercellular communications through carrying genetic materials and functional biomolecules. Owing to their unique size and structure, exosomes have emerged as a useful tool to overcome the limitations of siRNA delivery. The use of exosomes as siRNA delivery vehicles lacks certain disadvantages of the existing foreign delivery systems such as viruses, polycationic polymers and liposomes, and introduces several advantages including inherent capacity to pass through biological barriers and escape from phagocytosis by the reticuloendothelial system, as well as being biocompatible, non-toxic, and immunologically inert. Different strategies have been employed to harness exosome-based delivery systems, including surface modification with targeting ligands, and using exosome-display technology, virus-modified exosomes, and exosome-mimetic vesicles. The present review provides a capsule summary of the recent advances and current challenges in the field of exosome-mediated siRNA delivery. PMID:27904525

  4. Capella: Structure and Abundances

    NASA Technical Reports Server (NTRS)

    Brickhouse, Nancy S.

    1999-01-01

    This grant covers the analysis of EUVE spectra of the cool star binary system Capella. This project has also required the analysis of simultaneous Advanced Satellite for Cosmology and Astrophysics (ASCA) data. The ASCA spectrum of Capella could not be fit with standard models; by imposing models based on strong lines observed with EUVE, a problem wavelength region was identified. Correcting the problem required calculations of atomic collision strengths of higher principal quantum number than had ever been calculated. With these new models applied to the ASCA spectrum, better fits were obtained. Findings are that: (1) ASCA and EUVE spectra are both dominated by a region at 6 x 10(exp 6) K. (2) The high energy cut-off of the ASCA spectrum is consistent with emission from the highest ionization stages of EUVE, namely Fe XXIV. (3) EUVE requires a continuous emission measure distribution with more than two temperatures. (4) The ASCA spectra are of such high statistical significance that systematic uncertainties dominate, including atomic physics issues and calibration issues. (5) While the ASCA spectral fits achieve lower Chi(exp 2 with two-temperature fits, the EUVE-derived emission measure distribution models are also consistent with the spectra. (6) The Fe/H ratio obtained from the ASCA fit is within 20 % of the Fe/H abundance obtained from the summed spectra of Capella over 5 EUVE pointings, as well as the 1996 EUVE data. This result confirms our claims that quasi-continua composed of weak emission lines in the short wavelength spectrometer of EUVE are not major contributors to the measured Capella continuum. Other abundance ratios are also determined from the ASCA data, using models derived with EUVE. Si, Si, and Mg appear to be close to solar photospheric values, while the ratio of Ne/Fe is three to four times lower than solar photospheric values. Whether there is a general First Ionization Potential (FIP) effect or a specific neon anomaly cannot be determined

  5. Targeted delivery of siRNA to macrophages for anti-inflammatory treatment.

    PubMed

    Kim, Sang-Soo; Ye, Chunting; Kumar, Priti; Chiu, Isaac; Subramanya, Sandesh; Wu, Haoquan; Shankar, Premlata; Manjunath, N

    2010-05-01

    Inflammation mediated by tumor necrosis factor-alpha (TNF-alpha) and the associated neuronal apoptosis characterizes a number of neurologic disorders. Macrophages and microglial cells are believed to be the major source of TNF-alpha in the central nervous system (CNS). Here, we show that suppression of TNF-alpha by targeted delivery of small interfering RNA (siRNA) to macrophage/microglial cells dramatically reduces lipopolysaccharide (LPS)-induced neuroinflammation and neuronal apoptosis in vivo. Because macrophage/microglia express the nicotinic acetylcholine receptor (AchR) on their surface, we used a short AchR-binding peptide derived from the rabies virus glycoprotein (RVG) as a targeting ligand. This peptide was fused to nona-D-arginine residues (RVG-9dR) to enable siRNA binding. RVG-9dR was able to deliver siRNA to induce gene silencing in macrophages and microglia cells from wild type, but not AchR-deficient mice, confirming targeting specificity. Treatment with anti-TNF-alpha siRNA complexed to RVG-9dR achieved efficient silencing of LPS-induced TNF-alpha production by primary macrophages and microglia cells in vitro. Moreover, intravenous injection with RVG-9dR-complexed siRNA in mice reduced the LPS-induced TNF-alpha levels in blood as well as in the brain, leading to a significant reduction in neuronal apoptosis. These results demonstrate that RVG-9dR provides a tool for siRNA delivery to macrophages and microglia and that suppression of TNF-alpha can potentially be used to suppress neuroinflammation in vivo.

  6. Knockdown of Antiapoptotic Genes in Breast Cancer Cells by siRNA Loaded Into Hybrid Nanoparticles.

    PubMed

    Mello Júnior, Leônidas; Rosa E Souza, Gabriela; Winter, Evelyn; Henrique Silva, Adny; Pittella, Frederico; Creczynski-Pasa, Tânia Beatriz

    2017-02-23

    Tumorigenesis is related to an imbalance in controlling mechanisms of apoptosis. Expression of the genes BCL-2 and BCL-xL results in promotion of cell survival by inhibiting apoptosis. Thus, a novel approach to suppress antiapoptotic genes is the use of small interfering RNA (siRNA) in cancer cells. However, there are some limitations for the application of siRNA such as low bioavailability, requiring vectors as a strategy to achieve the nucleic acid transfection. In this study formulations containing CaP-siRNA-PEG-polyanion hybrid nanoparticles were developed to enhance siRNA delivery to cultured human breast cancer cells (MCF-7) in order to evaluate if the silencing of antiapoptotic genes BCL-2 and BCL-xL by siRNA would succeed in increasing cancer cells death. After 48h of incubation the expression of BCL-2 and BCL-xL genes decreased to 49% and 23%, respectively. The formulation proved to be toxic to cancer cells at concentration of 200 nM siRNA after 72h of incubation. As the targeted proteins are related to the resistance to chemotherapeutic drugs, the nanocarriers systems were also tested in the presence of doxorubicin (DOX). The results showed a significant reduction in CC50 of DOX, for both targets. In addition, an increase in apoptotic cell counts for both incubations conditions was observed as well. In conclusion, silencing antiapoptotic genes such as BCL-2 and BCL-xL through the use of siRNA carried by hybrid nanoparticles showed to be effective in vitro, and presents a promising strategy for pre-clinical analysis, especially when combined with DOX against breast cancer.

  7. RGD-based active targeting of novel polycation liposomes bearing siRNA for cancer treatment.

    PubMed

    Yonenaga, Norihito; Kenjo, Eriya; Asai, Tomohiro; Tsuruta, Atsushi; Shimizu, Kosuke; Dewa, Takehisa; Nango, Mamoru; Oku, Naoto

    2012-06-10

    For the purpose of systemic delivery of siRNA, we previously developed polycation liposomes (PCLs) containing dicetylphosphate-tetraethylenepentamine (DCP-TEPA) as an effective siRNA carrier. In the present study, to endow these PCLs (TEPA-PCL) actively target cancer cells and angiogenic vessels, we decorated the PCLs with cyclic RGD, by using cyclic RGD-grafted distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and investigated the usefulness of this type of carrier (RGD-PEG-PCL) for active targeting. Firstly, the gene-silencing efficacy of siRNA for luciferase (siLuc2) formulated in RGD-PEG-PCL (RGD-PEG-PCL/siLuc2) was examined in vitro by using B16F10-luc2 murine melanoma cells stably expressing the luciferase 2 gene, where the siRNA was grafted with cholesterol at the 3'-end of the sense strand (siRNA-C) for the stable association of the siRNA with the PCL. RGD-PEG-PCL/siLuc2 showed high knockdown efficiency compared with siLuc2 formulated in PEGylated TEPA-PCL without cyclic RGD (PEG-PCL). Next, the gene-silencing efficacy of RGD-PEG-PCL/siLuc2 was examined in vivo by use of B16F10-luc2 lung metastatic model mice. The intravenous injection of RGD-PEG-PCL/siLuc2 showed high knockdown efficiency against metastatic B16F10-luc2 tumors in the lungs of the mice, as assessed with an in vivo imaging system. These data strongly suggest that systemic and active targeting siRNA delivery using RGD-PEG-PCL is useful for cancer RNAi therapy.

  8. Surface engineering of gold nanoparticles for in vitro siRNA delivery

    NASA Astrophysics Data System (ADS)

    Zhao, Enyu; Zhao, Zhixia; Wang, Jiancheng; Yang, Chunhui; Chen, Chengjun; Gao, Lingyan; Feng, Qiang; Hou, Wenjie; Gao, Mingyuan; Zhang, Qiang

    2012-07-01

    Cellular uptake, endosomal/lysosomal escape, and the effective dissociation from the carrier are a series of hurdles for specific genes to be delivered both in vitro and in vivo. To construct siRNA delivery systems, poly(allylamine hydrochloride) (PAH) and siRNA were alternately assembled on the surface of 11.8 +/- 0.9 nm Au nanoparticles (GNP), stabilized by denatured bovine serum albumin, by the ionic layer-by-layer (LbL) self-assembly method. By manipulating the outmost PAH layer, GNP-PAH vectors with different surface electric potentials were prepared. Then, the surface potential-dependent cytotoxicity of the resultant GNP-PAH particles was evaluated via sulforhodamine B (SRB) assay, while the surface potential-dependent cellular uptake efficiency was quantitatively analyzed by using the flow cytometry method based on carboxyfluorescein (FAM)-labeled siRNA. It was revealed that the GNP-PAH particles with surface potential of +25 mV exhibited the optimal cellular uptake efficiency and cytotoxicity for human breast cancer MCF-7 cells. Following these results, two more positively charged polyelectrolytes with different protonating abilities in comparison with PAH, i.e., polyethylenimine (PEI), and poly(diallyl dimethyl ammonium chloride) (PDDA), were chosen to fabricate similarly structured vectors. Confocal fluorescence microscopy studies indicated that siRNA delivered by GNP-PAH and GNP-PEI systems was better released than that delivered by the GNP-PDDA system. Further flow cytometric assays based on immunofluorescence staining of the epidermal growth factor receptor (EGFR) revealed that EGFR siRNA delivered by GNP-PAH and GNP-PEI exhibited similar down-regulation effects on EGFR expression in MCF-7 cells. The following dual fluorescence flow cytometry assays by co-staining phosphatidylserine and DNA suggested the EGFR siRNA delivered by GNP-PAH exhibited an improved silencing effect in comparison with that delivered by the commercial transfection reagent

  9. Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.

    PubMed

    Nadithe, Venkatareddy; Liu, Runhui; Killinger, Bryan A; Movassaghian, Sara; Kim, Na Hyung; Moszczynska, Anna B; Masters, Kristyn S; Gellman, Samuel H; Merkel, Olivia M

    2015-02-02

    Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20-65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.

  10. Targeted Sterically Stabilized Phospholipid siRNA Nanomedicine for Hepatic and Renal Fibrosis

    PubMed Central

    Khaja, Fatima; Jayawardena, Dulari; Kuzmis, Antonina; Önyüksel, Hayat

    2016-01-01

    Since its discovery, small interfering RNA (siRNA) has been considered a potent tool for modulating gene expression. It has the ability to specifically target proteins via selective degradation of messenger RNA (mRNA) not easily accessed by conventional drugs. Hence, RNA interference (RNAi) therapeutics have great potential in the treatment of many diseases caused by faulty protein expression such as fibrosis and cancer. However, for clinical application siRNA faces a number of obstacles, such as poor in vivo stability, and off-target effects. Here we developed a unique targeted nanomedicine to tackle current siRNA delivery issues by formulating a biocompatible, biodegradable and relatively inexpensive nanocarrier of sterically stabilized phospholipid nanoparticles (SSLNPs). This nanocarrier is capable of incorporating siRNA in its core through self-association with a novel cationic lipid composed of naturally occuring phospholipids and amino acids. This overall assembly protects and delivers sufficient amounts of siRNA to knockdown over-expressed protein in target cells. The siRNA used in this study, targets connective tissue growth factor (CTGF), an important regulator of fibrosis in both hepatic and renal cells. Furthermore, asialoglycoprotein receptors are targeted by attaching the galactosamine ligand to the nanocarries which enhances the uptake of nanoparticles by hepatocytes and renal tubular epithelial cells, the major producers of CTGF in fibrosis. On animals this innovative nanoconstruct, small interfering RNA in sterically stabilized phospholipid nanoparticles (siRNA-SSLNP), showed favorable pharmacokinetic properties and accumulated mostly in hepatic and renal tissues making siRNA-SSLNP a suitable system for targeting liver and kidney fibrotic diseases.

  11. Actinide abundances in ordinary chondrites

    NASA Technical Reports Server (NTRS)

    Hagee, B.; Bernatowicz, T. J.; Podosek, F. A.; Johnson, M. L.; Burnett, D. S.

    1990-01-01

    Measurements of actinide and light REE (LREE) abundances and of phosphate abundances in equilibrated ordinary chondrites were obtained and were used to define the Pu abundance in the solar system and to determine the degree of variation of actinide and LREE abundances. The results were also used to compare directly the Pu/U ratio with the earlier obtained ratio determined indirectly, as (Pu/Nd)x(Nd/U), assuming that Pu behaves chemically as a LREE. The data, combined with high-accuracy isotope-dilution data from the literature, show that the degree of gram-scale variability of the Th, U, and LREE abundances for equilibrated ordinary chondrites is a factor of 2-3 for absolute abundances and up to 50 percent for relative abundances. The observed variations are interpreted as reflecting the differences in the compositions and/or proportions of solar nebula components accreted to ordinary chondrite parent bodies.

  12. Amylose-Based Cationic Star Polymers for siRNA Delivery

    PubMed Central

    Nishimura, Tomoki; Umezaki, Kaori; Mukai, Sada-atsu; Sawada, Shin-ichi; Akiyoshi, Kazunari

    2015-01-01

    A new siRNA delivery system using a cationic glyco-star polymer is described. Spermine-modified 8-arm amylose star polymer (with a degree of polymerization of approximately 60 per arm) was synthesized by chemoenzymatic methods. The cationic star polymer effectively bound to siRNA and formed spherical complexes with an average hydrodynamic diameter of 230 nm. The cationic 8-arm star polymer complexes showed superior cellular uptake characteristics and higher gene silencing effects than a cationic 1-arm polymer. These results suggest that amylose-based star polymers are a promising nanoplatform for glycobiomaterials. PMID:26539548

  13. Clustering siRNA conjugates for MMP-responsive therapeutics in chronic wounds of diabetic animals

    NASA Astrophysics Data System (ADS)

    Kim, Hye Sung; Son, Young Ju; Yoo, Hyuk Sang

    2016-07-01

    The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was efficiently endocytosed by cells. An in vivo fluorescence resonance energy transfer (FRET) study also revealed that the clusters were effectively dissociated in MMP-rich environments of dorsal wounds in diabetic animals. In addition, diabetic ulcers treated with the clusters showed a faster wound closure rate and the recovered tissue expressed a larger amount of cytokeratin along with a lower expression level of MMP-2 compared to the other groups.The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was

  14. Pmp22 mutant allele-specific siRNA alleviates demyelinating neuropathic phenotype in vivo.

    PubMed

    Lee, Ji-Su; Chang, Eun Hyuk; Koo, Ok Jae; Jwa, Dong Hwan; Mo, Won Min; Kwak, Geon; Moon, Hyo Won; Park, Hwan Tae; Hong, Young Bin; Choi, Byung-Ok

    2017-04-01

    Charcot-Marie-Tooth disease (CMT) is a genetic disorder that can be caused by aberrations in >80 genes. CMT has heterogeneous modes of inheritance, including autosomal dominant, autosomal recessive, X-linked dominant, and X-linked recessive. Over 95% of cases are dominantly inherited. In this study, we investigated whether regulation of a mutant allele by an allele-specific small interfering RNA (siRNA) can alleviate the demyelinating neuropathic phenotype of CMT. We designed 19 different allele-specific siRNAs for Trembler J (Tr-J) mice harboring a naturally occurring mutation (Leu16Pro) in Pmp22. Using a luciferase assay, we identified an siRNA that specifically and selectively reduced the expression level of the mutant allele and reversed the low viability of Schwann cells caused by mutant Pmp22 over-expression in vitro. The in vivo efficacy of the allele-specific siRNA was assessed by its intraperitoneal injection to postnatal day 6 of Tr-J mice. Administration of the allele-specific siRNA to Tr-J mice significantly enhanced motor function and muscle volume, as assessed by the rotarod test and magnetic resonance imaging analysis, respectively. Increases in motor nerve conduction velocity and compound muscle action potentials were also observed in the treated mice. In addition, myelination, as evidenced by toluidine blue staining and electron microscopy, was augmented in the sciatic nerves of the mice after allele-specific siRNA treatment. After validating suppression of the Pmp22 mutant allele at the mRNA level in the Schwann cells of Tr-J mice, we observed increased expression levels of myelinating proteins such as myelin basic protein and myelin protein zero. These data indicate that selective suppression of the Pmp22 mutant allele by non-viral delivery of siRNA alleviates the demyelinating neuropathic phenotypes of CMT in vivo, implicating allele-specific siRNA treatment as a potent therapeutic strategy for dominantly inherited peripheral neuropathies.

  15. Capella: Structure and Abundances

    NASA Technical Reports Server (NTRS)

    Brickhouse, Nancy S.

    1999-01-01

    This grant covers the analysis of ASCA spectra of the cool star binary system Capella. This project has also required the analysis of simultaneous EUVE data. The ASCA spectrum of Capella could not be fit with standard models; by imposing models based on strong lines observed with EUVE, a problem wavelength region was identified. Correcting the problem required calculations of atomic collision strengths of higher principal quantum number than had ever been calculated, resulting in a paper in process by Liedahl and Brickhouse. With these new models applied to the ASCA spectrum, better fits were obtained. While solar abundance ratios are generally consistent with the ASCA data, the ratio of Ne/Fe is three to four times lower than solar photospheric values. Whether there is a general First Ionization Potential (FIP) effect or a specific neon anomaly cannot be determined from these data. Detailed discussion has been provided to NASA in the most recent annual report (1997). Two poster presentations have been made regarding modeling requirements. A substantial paper is in the final revision form, following review by six co-authors. The results of this work have wide implications, since the newly calculated emission lines almost certainly contribute to other problems in fitting not only other stellar spectra, but also composite supernova remnants, galaxies, and cooling flow clusters of galaxies. Furthermore, Liedahl and Brickhouse have identified other species for which lines of a similar nature (high principal quantum number) will contribute significant flux. For moderate resolution X-ray spectra, lines left out of the models in relatively isolated bands, will be attributed to continuum flux by spectral fitting engines, causing errors in line-to-continuum ratios. Thus addressing the general theoretical problem is of crucial importance.

  16. Development of RNAi technology for targeted therapy--a track of siRNA based agents to RNAi therapeutics.

    PubMed

    Zhou, Yinjian; Zhang, Chunling; Liang, Wei

    2014-11-10

    RNA interference (RNAi) was intensively studied in the past decades due to its potential in therapy of diseases. The target specificity and universal treatment spectrum endowed siRNA advantages over traditional small molecules and protein drugs. However, barriers exist in the blood circulation system and the diseased tissues blocked the actualization of RNAi effect, which raised function versatility requirements to siRNA therapeutic agents. Appropriate functionalization of siRNAs is necessary to break through these barriers and target diseased tissues in local or systemic targeted application. In this review, we summarized that barriers exist in the delivery process and popular functionalized technologies for siRNA such as chemical modification and physical encapsulation. Preclinical targeted siRNA delivery and the current status of siRNA based RNAi therapeutic agents in clinical trial were reviewed and finally the future of siRNA delivery was proposed. The valuable experience from the siRNA agent delivery study and the RNAi therapeutic agents in clinical trial paved ways for practical RNAi therapeutics to emerge early.

  17. Conjugation of Palmitic Acid Improves Potency and Longevity of siRNA Delivered via Endosomolytic Polymer Nanoparticles

    PubMed Central

    Sarett, Samantha M.; Kilchrist, Kameron V.; Miteva, Martina; Duvall, Craig L.

    2015-01-01

    Clinical translation of siRNA therapeutics has been limited by the inability to effectively overcome the rigorous delivery barriers associated with intracellular-acting biologics. Here, in order to address both potency and longevity of siRNA gene silencing, pH-responsive micellar nanoparticle (NP) carriers loaded with siRNA conjugated to palmitic acid (siRNA-PA) were investigated as a combined approach to improve siRNA endosomal escape and stability. Conjugation to hydrophobic PA improved NP loading efficiency relative to unmodified siRNA, enabling complete packaging of siRNA-PA at a lower polymer:siRNA ratio. PA conjugation also increased intracellular uptake of the nucleic acid cargo by 35-fold and produced a 3.1-fold increase in intracellular half-life. The higher uptake and improved retention of siRNA-PA NPs correlated to a 2- and 11-fold decrease in gene silencing IC50 in comparison to siRNA NPs in fibroblasts and mesenchymal stem cells, respectively, for both the model gene luciferase and the therapeutically relevant gene PHD2. PA conjugation also significantly increased longevity of silencing activity following a single treatment, as observed in fibroblasts. Thus, conjugation of PA to siRNA paired with endosomolytic NPs is a promising approach to enhance the functional efficacy of siRNA in tissue regenerative and other applications. PMID:25641816

  18. Codelivery of mTERT siRNA and paclitaxel by chitosan-based nanoparticles promoted synergistic tumor suppression.

    PubMed

    Wei, Wei; Lv, Pi-Ping; Chen, Xiao-Ming; Yue, Zhan-Guo; Fu, Qiang; Liu, Shi-Ying; Yue, Hua; Ma, Guang-Hui

    2013-05-01

    Clinical applications of siRNA are being hindered by poor intracellular uptake and enzymatic degradation. To address these problems, we devised an oral delivery system for telomerase reverse transcriptase siRNA using N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride (HTCC) nanoparticles (HNP). Both the porous structure and the positive charge of HNP facilitated siRNA encapsulation. The outer coating of HTCC not only protected siRNA from enzymatic degradation, but also improved siRNA permeability in intestine tract. In vivo and in vitro experiments proved that HNP could effectively deliver siRNA to lesion site and further into tumor cells. On the basis of confirming the antitumor activity of HNP:siRNA, we continued to encapsulate a hydrophobic chemotherapeutic drug-paclitaxel (PTX) into HNP to form a "two-in-one" nano-complex (HNP:siRNA/PTX). We demonstrated that HNP:siRNA/PTX could simultaneously ferry siRNA and PTX into tumor cells and increase drug concentration, which, in particular, was much more effective in tumor suppression than that of traditional cocktail therapy. These results suggested that the HNP, as a powerful delivery system for both siRNA and chemotherapeutic drug, would have a far-reaching application in human cancer therapy.

  19. De novo reconstruction of consensus master genomes of plant RNA and DNA viruses from siRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In antiviral defense, plants produce massive quantities of 21-24 nucleotide siRNAs. Here we demonstrate that the complete genomes of DNA and RNA viruses and viroids can be reconstructed by deep sequencing and de novo assembly of viral/viroid siRNAs from experimentally- and naturally-infected plants....

  20. Measuring Solar Abundances with Seismology

    NASA Astrophysics Data System (ADS)

    Mussack, K.; Gough, D.

    2009-12-01

    The revision of the photospheric abundances proferred by Asplund et al. (2005) has rendered opacity theory inconsistent with the seismologically determined opacity through the Sun. This highlights the need for a direct seismological measurement of solar abundances. Here we describe the technique used to measure abundances with seismology, examine our ability to detect differences between solar models using this technique, and discuss its application in the Sun.

  1. Extreme Scale Visual Analytics

    SciTech Connect

    Wong, Pak C.; Shen, Han-Wei; Pascucci, Valerio

    2012-05-08

    Extreme-scale visual analytics (VA) is about applying VA to extreme-scale data. The articles in this special issue examine advances related to extreme-scale VA problems, their analytical and computational challenges, and their real-world applications.

  2. RVG-peptide-linked trimethylated chitosan for delivery of siRNA to the brain.

    PubMed

    Gao, Yikun; Wang, Zhan-You; Zhang, Jinghai; Zhang, Youxi; Huo, Hong; Wang, Tianyi; Jiang, Tongying; Wang, Siling

    2014-03-10

    In this work, a peptide derived from the rabies virus glycoprotein (RVG) was linked to siRNA/trimethylated chitosan (TMC) complexes through bifunctional PEG for efficient brain-targeted delivery of siRNA. The physiochemical properties of the complexes, such as siRNA complexing ability, size and ζ potential, morphology, serum stability, and cytotoxicity, were investigated prior to studying the cellular uptake, in vitro gene silencing efficiency, and in vivo biodistribution. The RVG-peptide-linked siRNA/TMC-PEG complexes showed increased serum stability, negligible cytotoxicity, and higher cellular uptake than the unmodified siRNA/TMC-mPEG complexes in acetylcholine receptor positive Neuro2a cells. The potent knockdown of BACE1, a therapeutic target in Alzheimer's disease, demonstrated the gene silencing efficiency. In vivo imaging analysis showed significant accumulation of Cy5-siRNA in the isolated brain of mice injected with RVG-peptide-linked complexes. Therefore, the RVG-peptide-linked TMC-PEG developed in this study can be used as a potential carrier for delivery of siRNA to the brain.

  3. Magnetic Core-Shell Silica Nanoparticles with Large Radial Mesopores for siRNA Delivery.

    PubMed

    Xiong, Lin; Bi, Jingxu; Tang, Youhong; Qiao, Shi-Zhang

    2016-09-01

    A novel type of magnetic core-shell silica nanoparticles is developed for small interfering RNA (siRNA) delivery. These nanoparticles are fabricated by coating super-paramagnetic magnetite nanocrystal clusters with radial large-pore mesoporous silica. The amine functionalized nanoparticles have small particle sizes around 150 nm, large radial mesopores of 12 nm, large surface area of 411 m(2) g(-1) , high pore volume of 1.13 cm(3) g(-1) and magnetization of 25 emu g(-1) . Thus, these nanoparticles possess both high loading capacity of siRNA (2 wt%) and strong magnetic response under an external magnetic field. An acid-liable coating composed of tannic acid can further protect the siRNA loaded in these nanoparticles. The coating also increases the dispersion stability of the siRNA-loaded carrier and can serve as a pH-responsive releasing switch. Using the magnetic silica nanoparticles with tannic acid coating as carriers, functional siRNA has been successfully delivered into the cytoplasm of human osteosarcoma cancer cells in vitro. The delivery is significantly enhanced with the aid of the external magnetic field.

  4. RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model.

    PubMed

    Lee, Tae Jin; Haque, Farzin; Shu, Dan; Yoo, Ji Young; Li, Hui; Yokel, Robert A; Horbinski, Craig; Kim, Tae Hyong; Kim, Sung-Hak; Kwon, Chang-Hyuk; Nakano, Ichiro; Kaur, Balveen; Guo, Peixuan; Croce, Carlo M

    2015-06-20

    Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues.

  5. Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing.

    PubMed

    Rabbani, Piul S; Zhou, Anna; Borab, Zachary M; Frezzo, Joseph A; Srivastava, Nikita; More, Haresh T; Rifkin, William J; David, Joshua A; Berens, Samuel J; Chen, Raymond; Hameedi, Sophia; Junejo, Muhammad H; Kim, Camille; Sartor, Rita A; Liu, Che F; Saadeh, Pierre B; Montclare, Jin K; Ceradini, Daniel J

    2017-04-03

    Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC). Addition of a cationic engineered supercharged coiled-coil protein (CSP) in a 10:1:1 ratio of DNC:CSP:siRNA produced a stable lipoproteoplex (LPP) nanoparticle, with optimal siRNA complexation, minimal cytotoxicity, and increased transfection efficacy. In a humanized murine diabetic wound healing model, our optimized LPP formulation successfully delivered siRNA targeted against Keap1, key repressor of Nrf2 which is a central regulator of redox mechanisms. Application of LPP complexing siKeap1 restored Nrf2 antioxidant function, accelerated diabetic tissue regeneration, and augmented reduction-oxidation homeostasis in the wound environment. Our topical LPP delivery system can readily be translated into clinical use for the treatment of diabetic wounds and can be extended to other cutaneous diseases with genetic components.

  6. Dual-Functional Nanoparticles Targeting CXCR4 and Delivering Antiangiogenic siRNA Ameliorate Liver Fibrosis.

    PubMed

    Liu, Chun-Hung; Chan, Kun-Ming; Chiang, Tsaiyu; Liu, Jia-Yu; Chern, Guann-Gen; Hsu, Fu-Fei; Wu, Yu-Hsuan; Liu, Ya-Chi; Chen, Yunching

    2016-07-05

    The progression of liver fibrosis, an intrinsic response to chronic liver injury, is associated with hepatic hypoxia, angiogenesis, abnormal inflammation, and significant matrix deposition, leading to the development of cirrhosis and hepatocellular carcinoma (HCC). Due to the complex pathogenesis of liver fibrosis, antifibrotic drug development has faced the challenge of efficiently and specifically targeting multiple pathogenic mechanisms. Therefore, CXCR4-targeted nanoparticles (NPs) were formulated to deliver siRNAs against vascular endothelial growth factor (VEGF) into fibrotic livers to block angiogenesis during the progression of liver fibrosis. AMD3100, a CXCR4 antagonist that was incorporated into the NPs, served dual functions: it acted as a targeting moiety and suppressed the progression of fibrosis by inhibiting the proliferation and activation of hepatic stellate cells (HSCs). We demonstrated that CXCR4-targeted NPs could deliver VEGF siRNAs to fibrotic livers, decrease VEGF expression, suppress angiogenesis and normalize the distorted vessels in the fibrotic livers in the carbon tetrachloride (CCl4) induced mouse model. Moreover, blocking SDF-1α/CXCR4 by CXCR4-targeted NPs in combination with VEGF siRNA significantly prevented the progression of liver fibrosis in CCl4-treated mice. In conclusion, the multifunctional CXCR4-targeted NPs delivering VEGF siRNAs provide an effective antifibrotic therapeutic strategy.

  7. Hydrogel doped with nanoparticles for local sustained release of siRNA in breast cancer.

    PubMed

    Segovia, Nathaly; Pont, Maria; Oliva, Nuria; Ramos, Victor; Borrós, Salvador; Artzi, Natalie

    2015-01-28

    Of all the much hyped and pricy cancer drugs, the benefits from the promising siRNA small molecule drugs are limited. Lack of efficient delivery vehicles that would release the drug locally, protect it from degradation, and ensure high transfection efficiency, precludes it from fulfilling its full potential. This work presents a novel platform for local and sustained delivery of siRNA with high transfection efficiencies both in vitro and in vivo in a breast cancer mice model. siRNA protection and high transfection efficiency are enabled by their encapsulation in oligopeptide-terminated poly(β-aminoester) (pBAE) nanoparticles. Sustained delivery of the siRNA is achieved by the enhanced stability of the nanoparticles when embedded in a hydrogel scaffold based on polyamidoamine (PAMAM) dendrimer cross-linked with dextran aldehyde. The combination of oligopeptide-terminated pBAE polymers and biodegradable hydrogels shows improved transfection efficiency in vivo even when compared with the most potent commercially available transfection reagents. These results highlight the advantage of using composite materials for successful delivery of these highly promising small molecules to combat cancer.

  8. Identification of siRNA delivery enhancers by a chemical library screen

    PubMed Central

    Gilleron, Jerome; Paramasivam, Prasath; Zeigerer, Anja; Querbes, William; Marsico, Giovanni; Andree, Cordula; Seifert, Sarah; Amaya, Pablo; Stöter, Martin; Koteliansky, Victor; Waldmann, Herbert; Fitzgerald, Kevin; Kalaidzidis, Yannis; Akinc, Akin; Maier, Martin A.; Manoharan, Muthiah; Bickle, Marc; Zerial, Marino

    2015-01-01

    Most delivery systems for small interfering RNA therapeutics depend on endocytosis and release from endo-lysosomal compartments. One approach to improve delivery is to identify small molecules enhancing these steps. It is unclear to what extent such enhancers can be universally applied to different delivery systems and cell types. Here, we performed a compound library screen on two well-established siRNA delivery systems, lipid nanoparticles and cholesterol conjugated-siRNAs. We identified fifty-one enhancers improving gene silencing 2–5 fold. Strikingly, most enhancers displayed specificity for one delivery system only. By a combination of quantitative fluorescence and electron microscopy we found that the enhancers substantially differed in their mechanism of action, increasing either endocytic uptake or release of siRNAs from endosomes. Furthermore, they acted either on the delivery system itself or the cell, by modulating the endocytic system via distinct mechanisms. Interestingly, several compounds displayed activity on different cell types. As proof of principle, we showed that one compound enhanced siRNA delivery in primary endothelial cells in vitro and in the endocardium in the mouse heart. This study suggests that a pharmacological approach can improve the delivery of siRNAs in a system-specific fashion, by exploiting distinct mechanisms and acting upon multiple cell types. PMID:26220182

  9. Combination of integrin siRNA and irradiation for breast cancer therapy

    SciTech Connect

    Cao Qizhen; Cai Weibo; Li Tianfang; Yang Yong; Chen Kai; Xing Lei; Chen Xiaoyuan . E-mail: shawchen@stanford.edu

    2006-12-22

    Up-regulation of integrin {alpha}{sub v}{beta}{sub 3} has been shown to play a key role in tumor angiogenesis and metastasis. In this study, we evaluated the role of integrin {alpha}{sub v}{beta}{sub 3} in breast cancer cell resistance to ionizing irradiation (IR) and tested the anti-tumor efficacy of combining integrin {alpha}{sub v} siRNA and IR. Colonogenic survival assay, cell proliferation, apoptosis, and cell cycle analysis were carried out to determine the treatment effect of siRNA, IR, or combination of both on MDA-MB-435 cells (integrin {alpha}{sub v}{beta}{sub 3}-positive). Integrin {alpha}{sub v}{beta}{sub 3}-negative MCF-7 cells exert more radiosensitivity than MDA-MB-435 cells. IR up-regulates integrin {alpha}{sub v}{beta}{sub 3} expression in MDA-MB-435 cells and integrin {alpha}{sub v} siRNA can effectively reduce both {alpha}{sub v} and {alpha}{sub v}{beta}{sub 3} integrin expression, leading to increased radiosensitivity. Integrin {alpha}{sub v} siRNA also promotes IR-induced apoptosis and enhances IR-induced G2/M arrest in cell cycle progression. This study, with further optimization, may provide a simple and highly efficient treatment strategy for breast cancer as well as other integrin {alpha}{sub v}{beta}{sub 3}-positive cancer types.

  10. Multifunctional Envelope-Type siRNA Delivery Nanoparticle Platform for Prostate Cancer Therapy.

    PubMed

    Xu, Xiaoding; Wu, Jun; Liu, Yanlan; Saw, Phei Er; Tao, Wei; Yu, Mikyung; Zope, Harshal; Si, Michelle; Victorious, Amanda; Rasmussen, Jonathan; Ayyash, Dana; Farokhzad, Omid C; Shi, Jinjun

    2017-03-28

    With the capability of specific silencing of target gene expression, RNA interference (RNAi) technology is emerging as a promising therapeutic modality for the treatment of cancer and other diseases. One key challenge for the clinical applications of RNAi is the safe and effective delivery of RNAi agents such as small interfering RNA (siRNA) to a particular nonliver diseased tissue (e.g., tumor) and cell type with sufficient cytosolic transport. In this work, we proposed a multifunctional envelope-type nanoparticle (NP) platform for prostate cancer (PCa)-specific in vivo siRNA delivery. A library of oligoarginine-functionalized and sharp pH-responsive polymers was synthesized and used for self-assembly with siRNA into NPs with the features of long blood circulation and pH-triggered oligoarginine-mediated endosomal membrane penetration. By further modification with ACUPA, a small molecular ligand specifically recognizing prostate-specific membrane antigen (PSMA) receptor, this envelope-type nanoplatform with multifunctional properties can efficiently target PSMA-expressing PCa cells and silence target gene expression. Systemic delivery of the siRNA NPs can efficiently silence the expression of prohibitin 1 (PHB1), which is upregulated in PCa and other cancers, and significantly inhibit PCa tumor growth. These results suggest that this multifunctional envelope-type nanoplatform could become an effective tool for PCa-specific therapy.

  11. Functionalized silicon quantum dots tailored for targeted siRNA delivery

    SciTech Connect

    Klein, S.; Zolk, O.; Fromm, M.F.; Schroedl, F.; Kryschi, C.

    2009-09-11

    For RNA interference (RNAi) mediated silencing of the ABCB1 gene in Caco-2 cells biocompatible luminescent silicon quantum dots (SiQDs) were developed to serve as self-tracking transfection tool for ABCB1 siRNA. While the 2-3 nm sized SiQD core exhibits green luminescence, the QD surfaces are completely saturated with covalently linked 2-vinylpyridine that may electrostatically bind siRNA. For down-regulating P-glycoprotein (Pgp) expression of the ABCB1 gene the SiQDs were complexed with siRNA. The cellular uptake and allocation of SiQD-siRNA complexes in Caco-2 cells were monitored using confocal laser scanning microscopy and transmission electron microscopy. The release of siRNA to the cytoplasm was verified through real-time PCR quantification of the reduced ABCB1 mRNA level. Additional evidence was obtained from time-resolved in situ fluorescence spectroscopic monitoring of the Pgp efflux dynamics in transfected Caco-2 cells which yielded significantly reduced transporter efficiencies for the Pgp substrate Rhodamine 123.

  12. Focused ultrasound for targeted delivery of siRNA and efficient knockdown of Htt expression.

    PubMed

    Burgess, Alison; Huang, Yuexi; Querbes, William; Sah, Dinah W; Hynynen, Kullervo

    2012-10-28

    RNA interference is a promising strategy for the treatment of Huntington's disease (HD) as it can specifically decrease the expression of the mutant Huntingtin protein (Htt). However, siRNA does not cross the blood-brain barrier and therefore delivery to the brain is limited to direct CNS delivery. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients. Focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48h following treatment with siRNA, the right (treated) and the left (control) striatum were dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain.

  13. HPV16E7-specific siRNA inhibits cell proliferation in CaSki cells.

    PubMed

    Li, Jun-guo; Li, Li; Zhang, Shui-Wen; Wei, Xiaoguang

    2015-03-01

    High-risk human papilloma virus (HPV) infection is the main cause for the genesis of cervical carcinomas. After infection, E6 and E7 genes of HPV were integrated to the genome of the cervical epithelium. Continued expression of the transforming oncoproteins E6 and E7 not only drives the neoplastic progression in cervical epithelium, but also plays an important role in maintaining the malignant phenotype of cervical cancer cells. The aim of this study is to explore the effects of liposomal transfection of HPV16E7 siRNA on the proliferation of cervical carcinoma cell line CaSki. The siRNA interfering HPV16E7 gene was synthesized and transfected into CaSki cells by liposome to observe the cell morphology changes under microscope. The cell proliferation index was detected by flow cytometry; HPV16E7 mRNA expression was determined by RT-PCR and its protein level was determined by Western blot. After transfection of the CaSki cell by siRNA, cell proliferation was inhibited significantly, and the expression of HPV16E7 mRNA and protein level of HPV16E7 decreased. HPV16E7 siRNA is able to inhibit growth of CaSki cells. HPV16E7 might become a new target for genetic therapy of cervical carcinoma.

  14. Comparing Gene Silencing and Physiochemical Properties in siRNA Bound Cationic Star-Polymer Complexes.

    PubMed

    Dearnley, Megan; Reynolds, Nicholas P; Cass, Peter; Wei, Xiaohu; Shi, Shuning; Mohammed, A Aalam; Le, Tam; Gunatillake, Pathiraja; Tizard, Mark L; Thang, San H; Hinton, Tracey M

    2016-11-14

    The translation of siRNA into clinical therapies has been significantly delayed by issues surrounding the delivery of naked siRNA to target cells. Here we investigate siRNA delivery by cationic acrylic polymers developed by Reversible Addition-Fragmentation chain Transfer (RAFT) mediated free radical polymerization. We investigated cell uptake and gene silencing of a series of siRNA-star polymer complexes both in the presence and absence of a protein "corona". Using a multidisciplinary approach including quantitative nanoscale mechanical-atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis we have characterized the nanoscale morphology, stiffness, and surface charge of the complexes with and without the protein corona. This is one of the first examples of a comprehensive physiochemical analysis of siRNA-polymer complexes being performed alongside in vitro biological assays, allowing us to describe a set of desirable physical features of cationic polymer complexes that promote gene silencing. Multifaceted studies such as this will improve our understanding of structure-function relationships in nanotherapeutics, facilitating the rational design of polymer-mediated siRNA delivery systems for novel treatment strategies.

  15. Intrathecal siRNA against GPNMB attenuates nociception in a rat model of neuropathic pain.

    PubMed

    Hou, Lili; Zhang, Yanfeng; Yang, Yong; Xiang, Kai; Tan, Qindong; Guo, Qulian

    2015-02-01

    Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1-GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a time-dependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain.

  16. RNA nanoparticle as a vector for targeted siRNA delivery into glioblastoma mouse model

    PubMed Central

    Lee, Tae Jin; Haque, Farzin; Shu, Dan; Yoo, Ji Young; Li, Hui; Yokel, Robert A.; Horbinski, Craig; Kim, Tae Hyong; Kim, Sung-Hak; Kwon, Chang-Hyuk; Nakano, Ichiro; Kaur, Balveen; Guo, Peixuan; Croce, Carlo M.

    2015-01-01

    Systemic siRNA administration to target and treat glioblastoma, one of the most deadly cancers, requires robust and efficient delivery platform without immunogenicity. Here we report newly emerged multivalent naked RNA nanoparticle (RNP) based on pRNA 3-way-junction (3WJ) from bacteriophage phi29 to target glioblastoma cells with folate (FA) ligand and deliver siRNA for gene silencing. Systemically injected FA-pRNA-3WJ RNPs successfully targeted and delivered siRNA into brain tumor cells in mice, and efficiently reduced luciferase reporter gene expression (4-fold lower than control). The FA-pRNA-3WJ RNP also can target human patient-derived glioblastoma stem cells, thought to be responsible for tumor initiation and deadly recurrence, without accumulation in adjacent normal brain cells, nor other major internal organs. This study provides possible application of pRNA-3WJ RNP for specific delivery of therapeutics such as siRNA, microRNA and/or chemotherapeutic drugs into glioblastoma cells without inflicting collateral damage to healthy tissues. PMID:25885522

  17. Polymers modified with double-tailed fluorous compounds for efficient DNA and siRNA delivery.

    PubMed

    He, Bingwei; Wang, Yitong; Shao, Naimin; Chang, Hong; Cheng, Yiyun

    2015-08-01

    Cationic polymers are widely used as gene carriers, however, these polymers are usually associated with low transfection efficacy and non-negligible toxicity. Fluorination on polymers significantly improves their performances in gene delivery, but a high density of fluorous chains must be conjugated on a single polymer. Here we present a new strategy to construct fluorinated polymers with minimal fluorous chains for efficient DNA and siRNA delivery. A double-tailed fluorous compound 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) was conjugated on dendrimers of different generations and low molecular weight polyethylenimine via a facile synthesis. The yielding products with average numbers of 1-2 conjugated CBT moieties showed much improved EGFP and luciferase transfection efficacy compared to unmodified polymers. In addition, these polymers show high siRNA delivery efficacy on different cell lines. Among the synthesized polymers, generation 1 (G1) dendrimer modified with an average number of 1.9 CBT moieties (G1-CBT1.9) shows the highest efficacy when delivering both DNA and siRNA and its efficacy approaches that of Lipofectamine 2000. G1-CBT1.9 also shows efficient gene silencing in vivo. All of the CBT-modified polymers exhibit minimal toxicity on the cells at their optimal transfection conditions. This study provides a new strategy to design efficient fluorous polymers for DNA and siRNA delivery.

  18. Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs

    SciTech Connect

    Park, Jeonghae; Nadeau, Peter; Zucali, James R.; Johnson, Calvin M.; Mergia, Ayalew . E-mail: mergiaa@mail.vetmed.ufl.edu

    2005-12-20

    Viral vectors available for gene therapy are either inefficient or suffer from safety concerns for human applications. Foamy viruses are non-pathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. In this report, we describe the use of simian foamy virus type 1 (SFV-1) vector to examine the efficacy of therapeutic genes. Hairpin short-interfering RNA (siRNA) that targets the simian immunodeficiency virus (SIV) rev/env was placed under the control of the PolIII U6 snRNA promoter for expression and screened for silencing target genes using cognate target-reporter fusions. We have identified an effective siRNA (designated R2) which reduces the rev and env gene expression by 89% and 95%, respectively. Using the simian foamy virus type 1 (SFV-1) based vector, we delivered the PolIII expressed R2 siRNA into cultured cells and challenged with SIV. The results show that the R2 siRNA is a potent inhibitor of SIV replication as determined by p27 expression and reverse transcriptase assays. Vectors based on a non-pathogenic SFV-1 vector may provide a safe and efficient alternative to currently available vectors, and the SIV model will help devise protocols for effective anti-HIV gene therapy.

  19. Nanoparticle-Mediated Systemic Delivery of siRNA for Treatment of Cancers and Viral Infections

    PubMed Central

    Draz, Mohamed Shehata; Fang, Binbin Amanda; Zhang, Pengfei; Hu, Zhi; Gu, Shenda; Weng, Kevin C.; Gray, Joe W.; Chen, Fanqing Frank

    2014-01-01

    RNA interference (RNAi) is an endogenous post-transcriptional gene regulatory mechanism, where non-coding, double-stranded RNA molecules interfere with the expression of certain genes in order to silence it. Since its discovery, this phenomenon has evolved as powerful technology to diagnose and treat diseases at cellular and molecular levels. With a lot of attention, short interfering RNA (siRNA) therapeutics has brought a great hope for treatment of various undruggable diseases, including genetic diseases, cancer, and resistant viral infections. However, the challenge of their systemic delivery and on how they are integrated to exhibit the desired properties and functions remains a key bottleneck for realizing its full potential. Nanoparticles are currently well known to exhibit a number of unique properties that could be strategically tailored into new advanced siRNA delivery systems. This review summarizes the various nanoparticulate systems developed so far in the literature for systemic delivery of siRNA, which include silica and silicon-based nanoparticles, metal and metal oxides nanoparticles, carbon nanotubes, graphene, dendrimers, polymers, cyclodextrins, lipids, hydrogels, and semiconductor nanocrystals. Challenges and barriers to the delivery of siRNA and the role of different nanoparticles to surmount these challenges are also included in the review. PMID:25057313

  20. Structural studies of the formation of lipoplexes between siRNA and selected bis-imidazolium gemini surfactants.

    PubMed

    Andrzejewska, W; Pietralik, Z; Skupin, M; Kozak, M

    2016-10-01

    Dicationic (gemini) surfactants are agents that can be used for the preparation of stable complexes of nucleic acids, particularly siRNA for therapeutic purposes. In this study, we demonstrated that bis-imidazolium gemini surfactants with variable lengths of dioxyalkyl linker groups (from dioxyethyl to dioxydodecyl) and dodecyl side chains are excellent for the complexation of siRNA. All of these compounds effectively complexed siRNA in a charge ratio range (p/n) of 1.5-10. The low resolution structure of siRNA oligomers was characterised by small angle scattering of synchrotron radiation (SR-SAXS) and ab initio modelling. The structures of the formed complexes were also analysed using SR-SAXS, circular dichroism studies and electrophoretic mobility tests. The most promising agents for complexation with siRNA were the surfactants that contained dioxyethyl and dioxyhexyl spacer groups.

  1. Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription

    NASA Astrophysics Data System (ADS)

    Jang, Mihue; Kim, Jong Hwan; Nam, Hae Yun; Kwon, Ick Chan; Ahn, Hyung Jun

    2015-08-01

    For therapeutic applications of siRNA, there are technical challenges with respect to targeted and systemic delivery. We here report a new siRNA carrier, RNAtr NPs, in a way that multiple tandem copies of RNA hairpins as a result of rolling circle transcription (RCT) can be readily adapted in tumour-targeted and systemic siRNA delivery. RNAtr NPs provide a means of condensing large amounts of multimeric RNA transcripts into the compact nanoparticles, especially without the aid of polycationic agents, and thus reduce the risk of immunogenicity and cytotoxicity by avoiding the use of synthetic polycationic reagents. This strategy allows the design of a platform technology for systemic delivery of siRNA to tumour sites, because RCT reaction, which enzymatically generates RNA polymers in multiple copy numbers at low cost, can lead to directly accessible routes to targeted and systemic delivery. Therefore, RNAtr NPs suggest great potentials as the siRNA therapeutics for cancer treatment.

  2. The antifibrotic effects of TGF-{beta}1 siRNA on hepatic fibrosis in rats

    SciTech Connect

    Lang, Qing; Liu, Qi; Xu, Ning; Qian, Ke-Li; Qi, Jing-Hu; Sun, Yin-Chun; Xiao, Lang; Shi, Xiao-Feng

    2011-06-10

    Highlights: {yields} We constructed CCL4 induced liver fibrosis model successfully. {yields} We proofed that the TGF-{beta}1 siRNA had a definite therapy effect to CCL4 induced liver fibrosis. {yields} The therapy effect of TGF-{beta}1 siRNA had dose-dependent. -- Abstract: Background/aims: Hepatic fibrosis results from the excessive secretion of matrix proteins by hepatic stellate cells (HSCs), which proliferate during fibrotic liver injury. Transforming growth factor (TGF)-{beta}1 is the dominant stimulus for extracellular matrix (ECM) production by stellate cells. Our study was designed to investigate the antifibrotic effects of using short interference RNA (siRNA) to target TGF-{beta}1 in hepatic fibrosis and its mechanism in rats exposed to a high-fat diet and carbon tetrachloride (CCL4). Methods: A total of 40 healthy, male SD (Sprague-Dawley) rats were randomly divided into five even groups containing of eight rats each: normal group, model group, TGF-{beta}1 siRNA 0.125 mg/kg treatment group, TGF-{beta}1 siRNA 0.25 mg/kg treatment group and TGF-{beta}1 siRNA negative control group (0.25 mg/kg). CCL4 and a high-fat diet were used for 8 weeks to induce hepatic fibrosis. All the rats were then sacrificed to collect liver tissue samples. A portion of the liver samples were soaked in formalin for Hematoxylin-Eosin staining, classifying the degree of liver fibrosis, and detecting the expression of type I and III collagen and TGF-{beta}1; the remaining liver samples were stored in liquid nitrogen to be used for detecting TGF-{beta}1 by Western blotting and for measuring the mRNA expression of type I and III collagen and TGF-{beta}1 by quantitative real-time polymerase chain reaction. Results: Comparing the TGF-{beta}1 siRNA 0.25 mg/kg treatment group to the model group, the TGF-{beta}1 siRNA negative control group and the TGF-{beta}1 siRNA 0.125 mg/kg treatment group showed significantly reduced levels of pathological changes, protein expression and the m

  3. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles

    PubMed Central

    Davis, Mark E.; Zuckerman, Jonathan E.; Choi, Chung Hang J.; Seligson, David; Tolcher, Anthony; Alabi, Christopher A.; Yen, Yun; Heidel, Jeremy D.; Ribas, Antoni

    2010-01-01

    Therapeutics that are designed to engage RNA interference (RNAi) pathways have the potential to provide new, major ways of imparting therapy to patients.1,2 Fire et al. first demonstrated that long, double stranded RNAs mediate RNAi in Caenorhabditis elegans,3 and Elbashir et al. opened the pathway to the use of RNAi for human therapy by showing that small interfering RNAs (siRNAs: ca. 21 base pair double stranded RNA) can elicit RNAi in mammalian cells without producing an interferon response.4 We are currently conducting the first-in-human Phase I clinical trial involving the systemic administration of siRNA to patients with solid cancers using a targeted, nanoparticle delivery system. Here we provide evidence of inducing an RNAi mechanism of action in a human from the delivered siRNA. Tumor biopsies from melanoma patients obtained after treatment reveal: (i) the presence of intracellularly-localized nanoparticles in amounts that correlate with dose levels of the nanoparticles administered (this is a first for systemically delivered nanoparticles of any kind), and (ii) reduction in both the specific mRNA (M2 subunit of ribonucleotide reductase (RRM2)) and the protein (RRM2) when compared to pre-dosing tissue. Most importantly, we detect the presence of an mRNA fragment that demonstrates siRNA mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles. These data when taken in total demonstrate that siRNA administered systemically to a human can produce a specific gene inhibition (reduction in mRNA and protein) by an RNAi mechanism of action. PMID:20305636

  4. Delivery of kinesin spindle protein targeting siRNA in solid lipid nanoparticles to cellular models of tumor vasculature

    SciTech Connect

    Ying, Bo; Campbell, Robert B.

    2014-04-04

    Highlights: • siRNA-lipid nanoparticles are solid particles not lipid bilayers with aqueous core. • High, but not low, PEG content can prevent nanoparticle encapsulation of siRNA. • PEG reduces cellular toxicity of cationic nanoparticles in vitro. • PEG reduces zeta potential while improving gene silencing of siRNA nanoparticles. • Kinesin spindle protein can be an effective target for tumor vascular targeting. - Abstract: The ideal siRNA delivery system should selectively deliver the construct to the target cell, avoid enzymatic degradation, and evade uptake by phagocytes. In the present study, we evaluated the importance of polyethylene glycol (PEG) on lipid-based carrier systems for encapsulating, and delivering, siRNA to tumor vessels using cellular models. Lipid nanoparticles containing different percentage of PEG were evaluated based on their physical chemical properties, density compared to water, siRNA encapsulation, toxicity, targeting efficiency and gene silencing in vitro. siRNA can be efficiently loaded into lipid nanoparticles (LNPs) when DOTAP is included in the formulation mixture. However, the total amount encapsulated decreased with increase in PEG content. In the presence of siRNA, the final formulations contained a mixed population of particles based on density. The major population which contains the majority of siRNA exhibited a density of 4% glucose, and the minor fraction associated with a decreased amount of siRNA had a density less than PBS. The inclusion of 10 mol% PEG resulted in a greater amount of siRNA associated with the minor fraction. Finally, when kinesin spindle protein (KSP) siRNA was encapsulated in lipid nanoparticles containing a modest amount of PEG, the proliferation of endothelial cells was inhibited due to the efficient knock down of KSP mRNA. The presence of siRNA resulted in the formation of solid lipid nanoparticles when prepared using the thin film and hydration method. LNPs with a relatively modest amount of

  5. Soft computing model for optimized siRNA design by identifying off target possibilities using artificial neural network model.

    PubMed

    Murali, Reena; John, Philips George; Peter S, David

    2015-05-15

    The ability of small interfering RNA (siRNA) to do posttranscriptional gene regulation by knocking down targeted genes is an important research topic in functional genomics, biomedical research and in cancer therapeutics. Many tools had been developed to design exogenous siRNA with high experimental inhibition. Even though considerable amount of work has been done in designing exogenous siRNA, design of effective siRNA sequences is still a challenging work because the target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. In some cases, siRNAs may tolerate mismatches with the target mRNA, but knockdown of genes other than the intended target could make serious consequences. Hence to design siRNAs, two important concepts must be considered: the ability in knocking down target genes and the off target possibility on any nontarget genes. So before doing gene silencing by siRNAs, it is essential to analyze their off target effects in addition to their inhibition efficacy against a particular target. Only a few methods have been developed by considering both efficacy and off target possibility of siRNA against a gene. In this paper we present a new design of neural network model with whole stacking energy (ΔG) that enables to identify the efficacy and off target effect of siRNAs against target genes. The tool lists all siRNAs against a particular target with their inhibition efficacy and number of matches or sequence similarity with other genes in the database. We could achieve an excellent performance of Pearson Correlation Coefficient (R=0. 74) and Area Under Curve (AUC=0.906) when the threshold of whole stacking energy is ≥-34.6 kcal/mol. To the best of the author's knowledge, this is one of the best score while considering the "combined efficacy and off target possibility" of siRNA for silencing a gene. The proposed model

  6. Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs.

    PubMed

    Hübner, Nadja C; Wang, Lily Hui-Ching; Kaulich, Manuel; Descombes, Patrick; Poser, Ina; Nigg, Erich A

    2010-04-01

    The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101-114, 2007). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1-Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of "bypass suppression." In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling.

  7. In Silico, In Vitro, and In Vivo Studies Indicate the Potential Use of Bolaamphiphiles for Therapeutic siRNAs Delivery

    PubMed Central

    Kim, Taejin; Afonin, Kirill A.; Viard, Mathias; Koyfman, Alexey Y; Sparks, Selene; Heldman, Eliahu; Grinberg, Sarina; Linder, Charles; Blumenthal, Robert P; Shapiro, Bruce A

    2013-01-01

    Specific small interfering RNAs (siRNAs) designed to silence different oncogenic pathways can be used for cancer therapy. However, non-modified naked siRNAs have short half-lives in blood serum and encounter difficulties in crossing biological membranes due to their negative charge. These obstacles can be overcome by using siRNAs complexed with bolaamphiphiles, consisting of two positively charged head groups that flank an internal hydrophobic chain. Bolaamphiphiles have relatively low toxicities, long persistence in the blood stream, and most importantly, in aqueous conditions can form poly-cationic micelles thus, becoming amenable to association with siRNAs. Herein, two different bolaamphiphiles with acetylcholine head groups attached to an alkyl chain in two distinct configurations are compared for their abilities to complex with siRNAs and deliver them into cells inducing gene silencing. Our explicit solvent molecular dynamics (MD) simulations showed that bolaamphiphiles associate with siRNAs due to electrostatic, hydrogen bonding, and hydrophobic interactions. These in silico studies are supported by various in vitro and in cell culture experimental techniques as well as by some in vivo studies. Results demonstrate that depending on the application, the extent of siRNA chemical protection, delivery efficiency, and further intracellular release can be varied by simply changing the type of bolaamphiphile used. PMID:23511334

  8. Noninvasive Drug Delivery Using Ultrasound: Targeting Melanoma Using siRNA Against Mutant (V600E) B-Raf

    NASA Astrophysics Data System (ADS)

    Tran, Melissa A.; Gowda, Raghavendra; Park, Eun-Joo; Adair, James; Smith, Nadine; Kester, Mark; Robertson, Gavin P.

    2009-04-01

    Melanoma is the most deadly form of skin cancer. Currently early surgical removal is the best treatment option for melanoma patients with little hope of successful treatment of late stage melanoma. Clearly new treatment options must be explored. Topical administration of drugs provides the advantage of being able to apply large quantities of drug in close proximity to the tumor without the issue of systemic side effects. However, the natural barrier formed by the skin must first be overcome for topical treatment to become a viable option. With this in mind we have sought to use low-frequency ultrasound to transiently permeabilize the stratum corneum and successfully deliver liposomal siRNA to melanoma cells residing at the basement membrane. B-Raf is one of the most frequently activated genes in melanoma, making it an ideal candidate for targeting via siRNA. The novel liposomes used in this study load siRNA, protect if from the outside environment and lead to knockdown of target message. Combining ultrasound with liposomal siRNA we show that siRNA can be delivered into melanoma cells. Additionally, we show that siRNA to mutant B-Raf can effectively inhibit melanoma growth in reconstructs and in mice by 60% and 30% respectively. Therefore, ultrasound with liposomal siRNA is a potentially valuable treatment option for melanoma patients.

  9. Upper Extremity Length Equalization

    PubMed Central

    DeCoster, Thomas A.; Ritterbusch, John; Crawford, Mark

    1992-01-01

    Significant upper extremity length inequality is uncommon but can cause major functional problems. The ability to position and use the hand may be impaired by shortness of any of the long bones of the upper extremity. In many respects upper and lower extremity length problems are similar. They most commonly occur after injury to a growing bone and the treatment modalities utilized in the lower extremity may be applied to the upper extremity. These treatment options include epiphysiodesis, shortening osteotomy, angulatory correction osteotomy and lengthening. This report reviews the literature relative to upper extremity length inequality and equalization and presents an algorithm for evaluation and planning appropriate treatment for patients with this condition. This algorithm is illustrated by two clinical cases of posttraumatic shortness of the radius which were effectively treated. ImagesFigure 1Figure 2Figure 3

  10. Cheminformatics Approach to Gene Silencing: Z Descriptors of Nucleotides and SVM Regression Afford Predictive Models for siRNA Potency.

    PubMed

    Ebalunode, Jerry O; Zheng, Weifan

    2010-12-17

    Short interfering RNA mediated gene silencing technology has been through tremendous development over the past decade, and has found broad applications in both basic biomedical research and pharmaceutical development. Critical to the effective use of this technology is the development of reliable algorithms to predict the potency and selectivity of siRNAs under study. Existing algorithms are mostly built upon sequence information of siRNAs and then employ statistical pattern recognition or machine learning techniques to derive rules or models. However, sequence-based features have limited ability to characterize siRNAs, especially chemically modified ones. In this study, we proposed a cheminformatics approach to describe siRNAs. Principal component scores (z1, z2, z3, z4) have been derived for each of the 5 nucleotides (A, U, G, C, T) from the descriptor matrix computed by the MOE program. Descriptors of a given siRNA sequence are simply the concatenation of the z values of its composing nucleotides. Thus, for each of the 2431 siRNA sequences in the Huesken dataset, 76 descriptors were generated for the 19-NT representation, and 84 descriptors were generated for the 21-NT representation of siRNAs. Support Vector Machine regression (SVMR) was employed to develop predictive models. In all cases, the models achieved Pearson correlation coefficient r and R about 0.84 and 0.65 for the training sets and test sets, respectively. A minimum of 25 % of the whole dataset was needed to obtain predictive models that could accurately predict 75 % of the remaining siRNAs. Thus, for the first time, a cheminformatics approach has been developed to successfully model the structure-potency relationship in siRNA-based gene silencing data, which has laid a solid foundation for quantitative modeling of chemically modified siRNAs.

  11. Gut Microbiota and Extreme Longevity.

    PubMed

    Biagi, Elena; Franceschi, Claudio; Rampelli, Simone; Severgnini, Marco; Ostan, Rita; Turroni, Silvia; Consolandi, Clarissa; Quercia, Sara; Scurti, Maria; Monti, Daniela; Capri, Miriam; Brigidi, Patrizia; Candela, Marco

    2016-06-06

    The study of the extreme limits of human lifespan may allow a better understanding of how human beings can escape, delay, or survive the most frequent age-related causes of morbidity, a peculiarity shown by long-living individuals. Longevity is a complex trait in which genetics, environment, and stochasticity concur to determine the chance to reach 100 or more years of age [1]. Because of its impact on human metabolism and immunology, the gut microbiome has been proposed as a possible determinant of healthy aging [2, 3]. Indeed, the preservation of host-microbes homeostasis can counteract inflammaging [4], intestinal permeability [5], and decline in bone and cognitive health [6, 7]. Aiming at deepening our knowledge on the relationship between the gut microbiota and a long-living host, we provide for the first time the phylogenetic microbiota analysis of semi-supercentenarians, i.e., 105-109 years old, in comparison to adults, elderly, and centenarians, thus reconstructing the longest available human microbiota trajectory along aging. We highlighted the presence of a core microbiota of highly occurring, symbiotic bacterial taxa (mostly belonging to the dominant Ruminococcaceae, Lachnospiraceae, and Bacteroidaceae families), with a cumulative abundance decreasing along with age. Aging is characterized by an increasing abundance of subdominant species, as well as a rearrangement in their co-occurrence network. These features are maintained in longevity and extreme longevity, but peculiarities emerged, especially in semi-supercentenarians, describing changes that, even accommodating opportunistic and allochthonous bacteria, might possibly support health maintenance during aging, such as an enrichment and/or higher prevalence of health-associated groups (e.g., Akkermansia, Bifidobacterium, and Christensenellaceae).

  12. How extreme is extreme hourly precipitation?

    NASA Astrophysics Data System (ADS)

    Papalexiou, Simon Michael; Dialynas, Yannis G.; Pappas, Christoforos

    2016-04-01

    The importance of accurate representation of precipitation at fine time scales (e.g., hourly), directly associated with flash flood events, is crucial in hydrological design and prediction. The upper part of a probability distribution, known as the distribution tail, determines the behavior of extreme events. In general, and loosely speaking, tails can be categorized in two families: the subexponential and the hyperexponential family, with the first generating more intense and more frequent extremes compared to the latter. In past studies, the focus has been mainly on daily precipitation, with the Gamma distribution being the most popular model. Here, we investigate the behaviour of tails of hourly precipitation by comparing the upper part of empirical distributions of thousands of records with three general types of tails corresponding to the Pareto, Lognormal, and Weibull distributions. Specifically, we use thousands of hourly rainfall records from all over the USA. The analysis indicates that heavier-tailed distributions describe better the observed hourly rainfall extremes in comparison to lighter tails. Traditional representations of the marginal distribution of hourly rainfall may significantly deviate from observed behaviours of extremes, with direct implications on hydroclimatic variables modelling and engineering design.

  13. 21st Birthday Drinking: Extremely Extreme

    ERIC Educational Resources Information Center

    Rutledge, Patricia C.; Park, Aesoon; Sher, Kenneth J.

    2008-01-01

    Despite public recognition of the hazards of 21st birthday drinking, there is little empirical information concerning its prevalence, severity, and risk factors. Data from a sample of 2,518 college students suggest that 21st birthday drinking poses an extreme danger: (a) 4 of every 5 participants (83%) reported drinking to celebrate, (b) birthday…

  14. NIR light controlled photorelease of siRNA and its targeted intracellular delivery based on upconversion nanoparticles

    NASA Astrophysics Data System (ADS)

    Yang, Yanmei; Liu, Fang; Liu, Xiaogang; Xing, Bengang

    2012-12-01

    The most notable role of small interfering RNA (siRNA) is in RNA interference (RNAi) and post-transcriptional gene silencing, which leads to a surge of interest in RNAi for both biomedical research and therapeutic applications. However, ``naked'' siRNA cannot cross cellular membranes freely because of highly negative charges which limits its utility for gene therapy. In this work, a system of near-infrared (NIR) light-induced siRNA release from silica coated upconversion nanoparticles (Si-UCNPs) is presented. These Si-UCNPs were functionalized with cationic photocaged linkers through covalent bonding, which could effectively adsorb anionic siRNA through electrostatic attractions and were easily internalized by living cells. Upon NIR light irradiation, the photocaged linker on the Si-UCNPs surface could be cleaved by the upconverted UV light and thus initiated the intracellular release of the siRNA. The in vitro agarose gel electrophoresis and intracellular imaging results indicated that the Si-UCNPs-based gene carrier system allowed effective siRNA delivery and the applications of NIR light instead of direct high energy UV irradiation may greatly guarantee less cell damage.The most notable role of small interfering RNA (siRNA) is in RNA interference (RNAi) and post-transcriptional gene silencing, which leads to a surge of interest in RNAi for both biomedical research and therapeutic applications. However, ``naked'' siRNA cannot cross cellular membranes freely because of highly negative charges which limits its utility for gene therapy. In this work, a system of near-infrared (NIR) light-induced siRNA release from silica coated upconversion nanoparticles (Si-UCNPs) is presented. These Si-UCNPs were functionalized with cationic photocaged linkers through covalent bonding, which could effectively adsorb anionic siRNA through electrostatic attractions and were easily internalized by living cells. Upon NIR light irradiation, the photocaged linker on the Si-UCNPs surface

  15. Li-7 abundances in halo stars: Testing stellar evolution models and the primordial Li-7 abundance

    NASA Technical Reports Server (NTRS)

    Chaboyer, Brian; Demarque, P.

    1994-01-01

    A large number of stellar evolution models with (Fe/H) = -2.3 and -3.3 have been calculated in order to determine the primordial Li-7 abundance and to test current stellar evolution models by a comparison to the extensive database of accurate Li abundances in extremely metal-poor halo stars observed by Thorburn (1994). Standard models with gray atmospheres do a very good job of fitting the observed Li abundances in stars hotter than approximately 5600 K. They predict a primordial. Li-7 abundance of log N(Li) = 2.24 +/- 0.03. Models which include microscopic diffusion predict a downward curvature in the Li-7 destruction isochrones at hot temperatures which is not present in the observations. Thus, the observations clearly rule out models which include uninhibited microscopic diffusion of Li-7 from the surface of the star. Rotational mixing inhibits the microscopic diffusion and the (Fe/H) = -2.28 stellar models which include both diffusion and rotational mixing provide an excellent match to the mean trend in T(sub eff) which is present in the observations. Both the plateau stars and the heavily depleted cool stars are well fit by these models. The rotational mixing leads to considerable Li-7 depletion in these models and the primordial Li-7 abundance inferred from these models is log N(Li) = 3.08 +/- 0.1. However, the (Fe/H) = -3.28 isochrones reveal problems with the combined models. These isochrones predict a trend of decreasing log N(Li) with increasing T(sub eff) which is not present in the observations. Possible causes for this discrepancy are discussed.

  16. The boron abundance of Procyon

    NASA Technical Reports Server (NTRS)

    Lemke, Michael; Lambert, David L.; Edvardsson, Bengt

    1993-01-01

    The B I 2496.8 A resonance line and HST/GHRS echelle spectra are used with model atmospheres and synthetic spectra to derive the B abundance of the F dwarfs Procyon (Alpha Canis Minoris), Theta Ursae Majoris, and Iota Pegasi. The B abundance of Theta UMa and Iota Peg is similar to that derived by Boesgaard and Heacox (1978) from the B II resonance line in spectra of A- and B-type stars. These two dwarfs show normal abundances of Li, Be, and B. Procyon, which is highly depleted in Li and Be, is depleted in B by a factor of at least 3. Comparison of the spectra of Procyon and the halo dwarf HD 140283 shows that the B abundance assigned by Duncan et al. (1992) to three halo dwarfs is not greatly overestimated as a result of contamination of the B I line by an unidentified line.

  17. Ammonia abundances in four comets

    NASA Astrophysics Data System (ADS)

    Wyckoff, S.; Tegler, S. C.; Engel, L.

    1991-02-01

    NH2 emission band strengths were measured in four comets and the NH2 column densities were determined in order to measure the ammonia content of the comets. The mean ammonia/water abundance ratio derived for the four comets is found to be 0.13 + or - 0.06 percent, with no significant variation among the comets. The uniformity of this abundance attests to a remarkable degree of chemical homogeneity over large scales in the comet-forming region of the primordial solar nebula, and contrasts with the CO abundance variations found previously in comets. The N2 and NH3 abundances indicate a condensation temperature in the range 20-160 K, consistent with virtually all comet formation hypotheses.

  18. The boron abundance of Procyon

    NASA Astrophysics Data System (ADS)

    Lemke, Michael; Lambert, David L.; Edvardsson, Bengt

    1993-05-01

    The B I 2496.8 A resonance line and HST/GHRS echelle spectra are used with model atmospheres and synthetic spectra to derive the B abundance of the F dwarfs Procyon (Alpha Canis Minoris), Theta Ursae Majoris, and Iota Pegasi. The B abundance of Theta UMa and Iota Peg is similar to that derived by Boesgaard and Heacox (1978) from the B II resonance line in spectra of A- and B-type stars. These two dwarfs show normal abundances of Li, Be, and B. Procyon, which is highly depleted in Li and Be, is depleted in B by a factor of at least 3. Comparison of the spectra of Procyon and the halo dwarf HD 140283 shows that the B abundance assigned by Duncan et al. (1992) to three halo dwarfs is not greatly overestimated as a result of contamination of the B I line by an unidentified line.

  19. siRNA targeting PLK-1 induces apoptosis of synoviocytes in rheumatoid arthritis

    SciTech Connect

    Wada, Makoto; Kawahito, Yutaka . E-mail: kawahity@koto.kpu-m.ac.jp; Kimura, Shinya; Kohno, Masataka; Ishino, Hidetaka; Kimura, Mizuho; Omoto, Atsushi; Yamamoto, Aihiro; Hamaguchi, Masahide; Tsubouchi, Yasunori; Tokunaga, Daisaku; Hojo, Tatsuya; Ashihara, Eishi; Maekawa, Taira; Yoshikawa, Toshikazu

    2007-06-01

    Polo-like kinase-1 (PLK-1) is a member of the PLK family and participates in the control of cell mitosis. Here, we show that immunoreactive PLK-1 is strongly expressed in synoviocytes and some infiltrative mononuclear cells in synovial tissues from patients with rheumatoid arthritis (RA), while patients with osteoarthritis and injury show little or no expression of PLK-1 in synovial tissues. Western blot analysis shows that PLK is expressed and its expression is enhanced by IL-1{beta} in RA synoviocytes. IL-1{beta} also enhanced the cell growth of RA synoviocytes. Moreover, siRNA targeted against PLK-1 significantly decreases the expression of PLK-1 of RA synoviocytes stimulated by IL-1{beta} and suppresses the proliferation of these synoviocytes through apoptosis. These findings suggest that PLK-1 plays a critical role in the proliferation of RA synoviocytes leading to bone destruction, and siRNA against PLK-1 is potentially useful for the treatment of RA.

  20. Lipidoid-coated iron oxide nanoparticles for efficient DNA and siRNA delivery.

    PubMed

    Jiang, Shan; Eltoukhy, Ahmed A; Love, Kevin T; Langer, Robert; Anderson, Daniel G

    2013-03-13

    The safe, targeted and effective delivery of gene therapeutics remains a significant barrier to their broad clinical application. Here we develop a magnetic nucleic acid delivery system composed of iron oxide nanoparticles and cationic lipid-like materials termed lipidoids. Coated nanoparticles are capable of delivering DNA and siRNA to cells in culture. The mean hydrodynamic size of these nanoparticles was systematically varied and optimized for delivery. While nanoparticles of different sizes showed similar siRNA delivery efficiency, nanoparticles of 50-100 nm displayed optimal DNA delivery activity. The application of an external magnetic field significantly enhanced the efficiency of nucleic acid delivery, with performance exceeding that of the commercially available lipid-based reagent, Lipofectamine 2000. The iron oxide nanoparticle delivery platform developed here offers the potential for magnetically guided targeting, as well as an opportunity to combine gene therapy with MRI imaging and magnetic hyperthermia.

  1. Single-pot enzymatic synthesis of Dicer-substrate siRNAs.

    PubMed

    Guiley, Keelan Z; Pratt, Ashley J; MacRae, Ian J

    2012-03-01

    We describe an inexpensive and efficient method for generating functional pools of Dicer-substrate small interfering RNAs (siRNAs) in a single reaction tube. The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25-27 nt RNA pools during in vitro transcription. The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells. The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.

  2. Nanoparticle-mediated delivery of siRNA for effective lung cancer therapy.

    PubMed

    Kim, Young-Dong; Park, Tae-Eun; Singh, Bijay; Maharjan, Sushila; Choi, Yun-Jaie; Choung, Pill-Hoon; Arote, Rohidas B; Cho, Chong-Su

    2015-01-01

    Lung cancer is one of the most lethal diseases worldwide, and the survival rate is less than 15% even after the treatment. Unfortunately, chemotherapeutic treatments for lung cancer are accompanied by severe side effects, lack of selectivity and multidrug resistance. In order to overcome the limitations of conventional chemotherapy, nanoparticle-mediated RNA interference drugs represent a potential new approach due to selective silencing effect of oncogenes and multidrug resistance related genes. In this review, we provide recent advancements on nanoparticle-mediated siRNA delivery strategies including lipid system, polymeric system and rigid nanoparticles for lung cancer therapies. Importantly, codelivery of siRNA with conventional anticancer drugs and recent theranostic agents that offer great potential for lung cancer therapy is covered.

  3. Inhibition of Hepatitis B virus cccDNA replication by siRNA

    SciTech Connect

    Li Guiqiu; Gu Hongxi . E-mail: hxgu2432@163.com; Li Di; Xu Weizhen

    2007-04-06

    The development of an effective therapy for Hepatitis B virus (HBV) infection is still a challenge. Progress in RNA interference (RNAi) has shed slight on developing a new anti-HBV strategy. Here, we present a series of experiments showing a significant reduction in HBV transcripts and replication intermediates in HepG2.2.15 cells by vector-based siRNA targeted nuclear localization signal (NLS) region. More importantly, we showed that siRNA1 markedly inhibited HBV covalently closed circular DNA (cccDNA) replication. Our results indicated that HBV NLS may serve as a novel RNAi target to combat HBV infection, which can enhance anti-HBV efficacy and overcome the drawbacks of current therapies.

  4. Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model.

    PubMed

    Singer, Oded; Marr, Robert A; Rockenstein, Edward; Crews, Leslie; Coufal, Nicole G; Gage, Fred H; Verma, Inder M; Masliah, Eliezer

    2005-10-01

    In Alzheimer disease, increased beta-secretase (BACE1) activity has been associated with neurodegeneration and accumulation of amyloid precursor protein (APP) products. Thus, inactivation of BACE1 could be important in the treatment of Alzheimer disease. In this study, we found that lowering BACE1 levels using lentiviral vectors expressing siRNAs targeting BACE1 reduced amyloid production and the neurodegenerative and behavioral deficits in APP transgenic mice, a model of Alzheimer disease. Our results suggest that lentiviral vector delivery of BACE1 siRNA can specifically reduce the cleavage of APP and neurodegeneration in vivo and indicate that this approach could have potential therapeutic value for treatment of Alzheimer disease.

  5. Element Abundance Determination in Hot Evolved Stars

    NASA Astrophysics Data System (ADS)

    Werner, Klaus

    The hydrogen-deficiency in extremely hot post-AGB stars of spectral class PG1159 is probably caused by a (very) late helium-shell flash or a AGB final thermal pulse that consumes the hydrogen envelope, exposing the usually-hidden intershell region. Thus, the photospheric element abundances of these stars allow us to draw conclusions about details of nuclear burning and mixing processes in the precursor AGB stars. We compare predicted element abundances to those determined by quantitative spectral analyses performed with advanced non-LTE model atmospheres. A good qualitative and quantitative agreement is found for many species (He, C, N, O, Ne, F, Si, Ar) but discrepancies for others (P, S, Fe) point at shortcomings in stellar evolution models for AGB stars. Almost all of the chemical trace elements in these hot stars can only be identified in the UV spectral range. The Far Ultraviolet Spectroscopic Explorer and the Hubble Space Telescope played a crucial role for this research.

  6. Chlorine Abundances in Cool Stars

    NASA Astrophysics Data System (ADS)

    Maas, Z. G.; Pilachowski, C. A.; Hinkle, K.

    2016-12-01

    Chlorine abundances are reported in 15 evolved giants and 1 M dwarf in the solar neighborhood. The Cl abundance was measured using the vibration-rotation 1-0 P8 line of H35Cl at 3.69851 μm. The high-resolution L-band spectra were observed using the Phoenix infrared spectrometer on the Kitt Peak Mayall 4 m telescope. The average [35Cl/Fe] abundance in stars with -0.72 < [Fe/H] < 0.20 is [35Cl/Fe] = (-0.10 ± 0.15) dex. The mean difference between the [35Cl/Fe] ratios measured in our stars and chemical evolution model values is (0.16 ± 0.15) dex. The [35Cl/Ca] ratio has an offset of ˜0.35 dex above model predictions, suggesting that chemical evolution models are underproducing Cl at the high metallicity range. Abundances of C, N, O, Si, and Ca were also measured in our spectral region and are consistent with F and G dwarfs. The Cl versus O abundances from our sample match Cl abundances measured in planetary nebula and H ii regions. In one star where both H35Cl and H37Cl could be measured, a 35Cl/37Cl isotope ratio of 2.2 ± 0.4 was found, consistent with values found in the Galactic ISM and predicted chemical evolution models.

  7. Tunable Delivery of siRNA from a Biodegradable Scaffold to Promote Angiogenesis In Vivo

    PubMed Central

    Nelson, Christopher E.; Kim, Arnold J.; Adolph, Elizabeth J.; Gupta, Mukesh K.; Yu, Fang; Hocking, Kyle M.; Davidson, Jeffrey M.; Guelcher, Scott A.; Duvall, Craig L.

    2014-01-01

    A system has been engineered for temporally controlled delivery of siRNA from biodegradable tissue regenerative scaffolds. Therapeutic application of this approach to silence prolyl hydroxylase domain 2 promoted expression of pro-angiogenic genes controlled by HIF1α and enhanced scaffold vascularization in vivo. This technology provides a new standard for efficient and controllable gene silencing to modulate host response within regenerative biomaterials. PMID:24338842

  8. Visualization of self-delivering hydrophobically modified siRNA cellular internalization

    PubMed Central

    Ly, Socheata; Navaroli, Deanna M.; Didiot, Marie-Cécile; Cardia, James; Pandarinathan, Lakshmipathi; Alterman, Julia F.; Fogarty, Kevin; Standley, Clive; Lifshitz, Lawrence M.; Bellve, Karl D.; Prot, Matthieu; Echeverria, Dimas; Corvera, Silvia; Khvorova, Anastasia

    2017-01-01

    siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention. PMID:27899655

  9. Low generation polyamine dendrimers bearing flexible tetraethylene glycol as nanocarriers for plasmids and siRNA

    NASA Astrophysics Data System (ADS)

    Sharma, Rishi; Zhang, Issan; Shiao, Tze Chieh; Pavan, Giovanni M.; Maysinger, Dusica; Roy, René

    2016-02-01

    Low G1 generation polyamine dendrimers built around programmable, flexible, and short tetraethyleneglycol branches were readily prepared in a divergent manner using a combination of orthogonal AB3 or AB5 units and highly efficient chemical transformations based on Cu(i) catalyzed alkyne-azide cycloaddition (CUAAC) and thiol-ene click reactions. The constructs showed that the G1 polyamines with only twelve and eighteen amine surface groups can successfully deliver siRNA in human cells, with transfection efficiency comparable to that of Lipofectamine 2000®. Measurements of cell viability following transfection of plasmid DNA and siRNA showed that the dendritic polyamines are less cytotoxic than Lipofectamine 2000® and are thus preferable for biological applications.Low G1 generation polyamine dendrimers built around programmable, flexible, and short tetraethyleneglycol branches were readily prepared in a divergent manner using a combination of orthogonal AB3 or AB5 units and highly efficient chemical transformations based on Cu(i) catalyzed alkyne-azide cycloaddition (CUAAC) and thiol-ene click reactions. The constructs showed that the G1 polyamines with only twelve and eighteen amine surface groups can successfully deliver siRNA in human cells, with transfection efficiency comparable to that of Lipofectamine 2000®. Measurements of cell viability following transfection of plasmid DNA and siRNA showed that the dendritic polyamines are less cytotoxic than Lipofectamine 2000® and are thus preferable for biological applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06757j

  10. Acid-Degradable Cationic Dextran Particles for the Delivery of siRNA Therapeutics

    PubMed Central

    Cohen, Jessica L.; Schubert, Stephanie; Wich, Peter R.; Cui, Lina; Cohen, Joel A.; Mynar, Justin L.; Fréchet, Jean M. J.

    2011-01-01

    We report a new acid-sensitive, biocompatible and biodegradable microparticulate delivery system, spermine modified acetalated-dextran (Spermine-Ac-DEX), which can be used to efficiently encapsulate siRNA. These particles demonstrated efficient gene knockdown in HeLa-luc cells with minimal toxicity. This knockdown was comparable to that obtained using Lipofectamine, a commercially available transfection reagent generally limited to in vitro use due to its high toxicity. PMID:21539393

  11. Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA.

    PubMed

    Thi, Emily P; Mire, Chad E; Ursic-Bedoya, Raul; Geisbert, Joan B; Lee, Amy C H; Agans, Krystle N; Robbins, Marjorie; Deer, Daniel J; Fenton, Karla A; MacLachlan, Ian; Geisbert, Thomas W

    2014-08-20

    Marburg virus (MARV) and the closely related filovirus Ebola virus cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with mortality rates up to 90%. There are no vaccines or drugs approved for human use, and no postexposure treatment has completely protected nonhuman primates against MARV-Angola, the strain associated with the highest rate of mortality in naturally occurring human outbreaks. Studies performed with other MARV strains assessed candidate treatments at times shortly after virus exposure, before signs of disease are detectable. We assessed the efficacy of lipid nanoparticle (LNP) delivery of anti-MARV nucleoprotein (NP)-targeting small interfering RNA (siRNA) at several time points after virus exposure, including after the onset of detectable disease in a uniformly lethal nonhuman primate model of MARV-Angola HF. Twenty-one rhesus monkeys were challenged with a lethal dose of MARV-Angola. Sixteen of these animals were treated with LNP containing anti-MARV NP siRNA beginning at 30 to 45 min, 1 day, 2 days, or 3 days after virus challenge. All 16 macaques that received LNP-encapsulated anti-MARV NP siRNA survived infection, whereas the untreated or mock-treated control subjects succumbed to disease between days 7 and 9 after infection. These results represent the successful demonstration of therapeutic anti-MARV-Angola efficacy in nonhuman primates and highlight the substantial impact of an LNP-delivered siRNA therapeutic as a countermeasure against this highly lethal human disease.

  12. Enhanced Wound Healing Using Topically Administered Nanoparticle Encapsulated siRNA

    DTIC Science & Technology

    2013-11-01

    treatment protocols using our original nanoparticle formulation. Starting next quarter, we will begin to test new nanoparticle formulations. One issue... nanoparticle siRNA showed a significant increase in rate of axonal outgrowth in comparison to controls (Students t- test , pɘ.05). Please note that our...2. Generate usable quantities of quantities of optimized nanoparticles This task has been completed as proposed FY12 Quarter 4 Task 3. Test

  13. CpG-STAT3siRNA for Castration-Resistant Prostate Cancer Therapy

    DTIC Science & Technology

    2015-12-01

    host, likely through activation of host innate immunity. We also verified CpG-STAT3 siRNA efficacy in Ras/Myc oncogene-driven and STAT3-dependent...Kawai T, Akira S (2010) The role of pattern- recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11:373–384 10...Cancer Immunotherapeutics & Tumor Immunology , Beckman Research Institute at City of Hope, Duarte, CA 91010, USA 2Bioinformatics Core Facility, Beckman

  14. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    PubMed

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls.

  15. MicroRNA-directed siRNA biogenesis in Caenorhabditis elegans.

    PubMed

    Corrêa, Régis L; Steiner, Florian A; Berezikov, Eugene; Ketting, René F

    2010-04-08

    RNA interference (RNAi) is a post-transcriptional silencing process, triggered by double-stranded RNA (dsRNA), leading to the destabilization of homologous mRNAs. A distinction has been made between endogenous RNAi-related pathways and the exogenous RNAi pathway, the latter being essential for the experimental use of RNAi. Previous studies have shown that, in Caenorhabditis elegans, a complex containing the enzymes Dicer and the Argonaute RDE-1 process dsRNA. Dicer is responsible for cleaving dsRNA into short interfering RNAs (siRNAs) while RDE-1 acts as the siRNA acceptor. RDE-1 then guides a multi-protein complex to homologous targets to trigger mRNA destabilization. However, endogenous role(s) for RDE-1, if any, have remained unexplored. We here show that RDE-1 functions as a scavenger protein, taking up small RNA molecules from many different sources, including the microRNA (miRNA) pathway. This is in striking contrast to Argonaute proteins functioning directly in the miRNA pathway, ALG-1 and ALG-2: these proteins exclusively bind miRNAs. While playing no significant role in the biogenesis of the main pool of miRNAs, RDE-1 binds endogenous miRNAs and triggers RdRP activity on at least one perfectly matching, endogenous miRNA target. The resulting secondary siRNAs are taken up by a set of Argonaute proteins known to act as siRNA acceptors in exogenous RNAi, resulting in strong mRNA destabilization. Our results show that RDE-1 in an endogenous setting is actively screening the transcriptome using many different small RNAs, including miRNAs, as a guide, with implications for the evolution of transcripts with a potential to be recognized by Dicer.

  16. Cationic dendritic starch as a vehicle for photodynamic therapy and siRNA co-delivery.

    PubMed

    Engelberth, Sarah A; Hempel, Nadine; Bergkvist, Magnus

    2017-03-01

    Cationic enzymatically synthesized glycogen (cESG) is a naturally-derived, nano-scale carbohydrate dendrite that has shown promise as a cellular delivery vehicle owing to its flexibility in chemical modifications, biocompatibility and relative low cost. In the present work, cESG was modified and evaluated as a vehicle for tetraphenylporphinesulfonate (TPPS) in order to improve cellular delivery of this photosensitizer and investigate the feasibility of co-delivery with short interfering ribonucleic acid (siRNA). TPPS was electrostatically condensed with cESG, resulting in a sub-50nm particle with a positive zeta potential of approximately 5mV. When tested in normal ovarian surface epithelial and ovarian clear cell carcinoma cell culture models, encapsulation of TPPS in cESG significantly improved cell death in response to light treatment compared to free drug alone. Dosages as low as 0.16μM TPPS resulted in cellular death upon illumination with a 4.8J/cm(2) light dosage, decreasing viability by 96%. cESG-TPPS was then further evaluated as a co-delivery system with siRNA for potential combination therapy, by charge-based condensation of an siRNA directed at reducing expression of manganese superoxide dismutase (Sod2) as a proof of principle target. Simultaneous delivery of TPPS and siRNA was achieved, reducing Sod2 protein expression to 48%, while maintaining the photodynamic properties of TPPS under light exposure and maintaining low dark toxicity. This study demonstrates the versatility of cESG as a platform for dual delivery of small molecules and oligonucleotides, and the potential for further development of this system in combination therapy applications.

  17. Multifunctional polymeric micelles for delivery of drugs and siRNA

    PubMed Central

    Jhaveri, Aditi M.; Torchilin, Vladimir P.

    2014-01-01

    Polymeric micelles, self-assembling nano-constructs of amphiphilic copolymers with a core-shell structure have been used as versatile carriers for delivery of drugs as well as nucleic acids. They have gained immense popularity owing to a host of favorable properties including their capacity to effectively solubilize a variety of poorly soluble pharmaceutical agents, biocompatibility, longevity, high stability in vitro and in vivo and the ability to accumulate in pathological areas with compromised vasculature. Moreover, additional functions can be imparted to these micelles by engineering their surface with various ligands and cell-penetrating moieties to allow for specific targeting and intracellular accumulation, respectively, to load them with contrast agents to confer imaging capabilities, and incorporating stimuli-sensitive groups that allow drug release in response to small changes in the environment. Recently, there has been an increasing trend toward designing polymeric micelles which integrate a number of the above functions into a single carrier to give rise to “smart,” multifunctional polymeric micelles. Such multifunctional micelles can be envisaged as key to improving the efficacy of current treatments which have seen a steady increase not only in hydrophobic small molecules, but also in biologics including therapeutic genes, antibodies and small interfering RNA (siRNA). The purpose of this review is to highlight recent advances in the development of multifunctional polymeric micelles specifically for delivery of drugs and siRNA. In spite of the tremendous potential of siRNA, its translation into clinics has been a significant challenge because of physiological barriers to its effective delivery and the lack of safe, effective and clinically suitable vehicles. To that end, we also discuss the potential and suitability of multifunctional polymeric micelles, including lipid-based micelles, as promising vehicles for both siRNA and drugs. PMID:24795633

  18. Development of siRNA Technology to Prevent Scar Formation in Tendon Repair

    DTIC Science & Technology

    2012-10-01

    Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes . Faseb...Groups and Antisense Oligonucleotide (ASO) Treatment : Anti-sense RNA technologies: Under normal conditions cells produce small interfering (si) RNAs...into four treatment groups: (1) Control (scrambled siRNA; 300microgram/microliter); and (2) Smad3 ASO, (3) Tgf-beta 1 ASO, or (4) Ctgf (Connective

  19. Development of siRNA Technology to Prevent Scar Formation in Tendon Repair

    DTIC Science & Technology

    2013-12-01

    animal model. This is an established and previously published model in our laboratory. Animal Groups and Antisense Oligonucleotide (ASO) Treatment ...to 8-week-old male mice were obtained from Jackson Laboratories. After surgery, mice were randomly divided into four treatment groups: (1) Control...scrambled siRNA; 300microgram/microliter); and (2) Smad3 ASO, (3) Tgf-beta 1 ASO, or (4) Ctgf (Connective Tissue Growth Factor) ASO. Treatments were

  20. Detailed abundances in EMP dwarfs from SDSS

    NASA Astrophysics Data System (ADS)

    Sbordone, Luca; Caffau, Elisabetta; Bonifacio, Piercarlo

    2012-09-01

    We report on the current status of an ongoing survey to select extremely metal poor (EMP) turn-off (TO) stars from Sloan Digital Sky Survey (SDSS) spectra, and determine their detailed chemical composition through high resolution follow-up. So far, 26 stars have been observed with UVESatVLT and X-SHOOTERatVLT, all but two showing an iron content below [Fe/H]=-3. Among them we detected the current record holder for the lowest total metallicity (SDSS J102915+172927, Z=10-5 Zsolar), four carbon-enhanced extremely metal poor objects (CEMP), as well as subsets with enhanced Ni and Mn. Lithium abundances or upper limits were derived, confirming the previously detected ``meltdown'' of the Spite plateau for metallicities below about [Fe/H]=-2.8. SDSS J102915+172927 in particular shows no detectable Li I 670.8 doublet, leading to an upper limit of A(Li)<1.1, hinting to an even deeper Li depletion in TO stars below [Fe/H]=-4. Spectroscopic follow-up is currently being prosecuted by the recently started ESO large program TOPoS, aiming to observe about 80 more EMP candidates.

  1. Combinatorial approach to determine functional group effects on lipidoid-mediated siRNA delivery.

    PubMed

    Mahon, Kerry P; Love, Kevin T; Whitehead, Kathryn A; Qin, June; Akinc, Akin; Leshchiner, Elizaveta; Leshchiner, Ignaty; Langer, Robert; Anderson, Daniel G

    2010-08-18

    The application of RNA interference (RNAi), either in the clinic or in the laboratory, requires safe and effective delivery methods. Here, we develop a combinatorial approach to synthesize a library of delivery vectors based on two lipid-like substrates with known siRNA delivery capabilities. Members of this library have a mixture of lipid-like tails and feature appendages containing hydroxyl, carbamate, ether, or amine functional groups as well as variations in alkyl chain length and branching. Using a luciferase reporter system in HeLa cells, we studied the relationship between lipid chemical modification and delivery performance in vitro. The impact of the functional group was shown to vary depending on the overall amine content and tail number of the delivery vector. Additionally, in vivo performance was evaluated using a Factor VII knockdown assay. Two library members, each containing ether groups, were found to knock down the target protein at levels comparable to those of the parent delivery vector. These results demonstrate that small chemical changes to the delivery vector impact knockdown efficiency and cell viability both in vitro and in vivo. The work described here identifies new materials for siRNA delivery and provides new insight into the parameters for optimized chemical makeup of lipid-like siRNA delivery materials.

  2. Cell transcytosing poly-arginine coated magnetic nanovector for safe and effective siRNA delivery.

    PubMed

    Veiseh, Omid; Kievit, Forrest M; Mok, Hyejung; Ayesh, Joseph; Clark, Cassra; Fang, Chen; Leung, Matthew; Arami, Hamed; Park, James O; Zhang, Miqin

    2011-08-01

    Lack of safe and effective carriers for delivery of RNA therapeutics remains a barrier to its broad clinical application. We report the development of a cell tanscytosing magnetic nanovector engineered as an siRNA carrier. Iron oxide nanoparticles were modified with poly(ethylene glycol) (PEG), small interfering RNA (siRNA), and a cationic polymer layer. Three nanovector formulations with cationic polymer coatings of poly-arginine (pArg), polylysine (pLys), and polyethylenimine (PEI), respectively, were prepared. The three nanovector formulations where evaluated for safety and ability to promote gene silencing in three types of cancer cells C6/GFP(+), MCF7/GFP(+), and TC2/GFP(+), mimicking human cancers of the brain, breast, and prostate, respectively. Cell viability and fluorescence quantification assays revealed that pArg-coated nanovectors were most effective in promoting gene knockdown and least toxic of the three nanovector formulations tested. Transmission electron microscopy (TEM) imaging of nanovector treated cells further demonstrated that pArg-coated nanovectors enter cells through cell transcytosis, while pLys and PEI coated nanovectors enter cells endocytosis. Our findings suggest that NPs engineered to exploit the cell transcytosis intracellular trafficking pathway may offer a more safe and efficient route for siRNA delivery.

  3. Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis

    PubMed Central

    Li, Shengben; Le, Brandon; Ma, Xuan; Li, Shaofang; You, Chenjiang; Yu, Yu; Zhang, Bailong; Liu, Lin; Gao, Lei; Shi, Ting; Zhao, Yonghui; Mo, Beixin; Cao, Xiaofeng; Chen, Xuemei

    2016-01-01

    Small RNAs are central players in RNA silencing, yet their cytoplasmic compartmentalization and the effects it may have on their activities have not been studied at the genomic scale. Here we report that Arabidopsis microRNAs (miRNAs) and small interfering RNAs (siRNAs) are distinctly partitioned between the endoplasmic reticulum (ER) and cytosol. All miRNAs are associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The MBP association is functionally linked to a deeply conserved and tightly regulated activity of miRNAs – production of phased siRNAs (phasiRNAs) from select target RNAs. The phasiRNA precursor RNAs, thought to be noncoding, are on MBPs and are occupied by ribosomes in a manner that supports miRNA-triggered phasiRNA production, suggesting that ribosomes on the rough ER impact siRNA biogenesis. This study reveals global patterns of cytoplasmic partitioning of small RNAs and expands the known functions of ribosomes and ER. DOI: http://dx.doi.org/10.7554/eLife.22750.001 PMID:27938667

  4. Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers.

    PubMed

    Scholz, Claudia; Wagner, Ernst

    2012-07-20

    Gene therapy offers great opportunities for the treatment of severe diseases including cancer. In recent years the design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of plasmid DNA (pDNA) have brought up a variety of gene delivery vehicles. The more recently emerged gene silencing strategy by the intracellular delivery of small interfering RNA (siRNA) takes benefit from existing expertise in pDNA transfer. Despite common properties however, delivery of siRNA also faces distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action. This review emphasizes the common aspects and main differences between pDNA and siRNA delivery, taking into consideration a wide spectrum of polymer-based, lipidic and peptide carriers. Challenges and opportunities which result from these differences as well as the recent progress made in the optimization of carrier design are presented.

  5. Screening of efficient siRNA carriers in a library of surface-engineered dendrimers

    PubMed Central

    Liu, Hongmei; Chang, Hong; Lv, Jia; Jiang, Cong; Li, Zhenxi; Wang, Fei; Wang, Hui; Wang, Mingming; Liu, Chongyi; Wang, Xinyu; Shao, Naimin; He, Bingwei; Shen, Wanwan; Zhang, Qiang; Cheng, Yiyun

    2016-01-01

    Polymers are widely used as non-viral carriers for siRNA delivery, but concern has also arisen in their limited efficacy and inherent toxicity. Whilst many of previous efforts have been documented towards improving the performance of polymers via chemical modifications, the structure-activity relationships (SAR) of these ligand-modified polymers are not well understood. To address this issue, we systemically prepared a library of surface-engineered dendrimers (>300) as the screening pool to discover efficient siRNA carriers. The modified ligands include alkyls and fluoroalkyls, amino acids, benzene derivatives and heterocyclic compounds. Gene silencing results showed that the lead material shows excellent efficacy even in hard-to-transfect cells such as mesenchymal stem cells. The SAR studies revealed that ligands containing appropriate hydrophobicity, or ligands with both hydrophobic and functional atoms/groups are essential for polymers to achive efficient knockdown efficacy. A second-generation library designed based on the above principles further confirms the proposed design criteria. The results enable the future rational design of potent siRNA carriers. PMID:27121799

  6. Polyspermine imine, a pH responsive polycationic siRNA carrier degradable to endogenous metabolites.

    PubMed

    Du, Zixiu; Xiang, Shengnan; Zang, Yi; Zhou, Yi; Wang, Chuandong; Tang, Hailing; Jin, Tuo; Zhang, Xiaoling

    2014-10-06

    Cationic polymers readily degradable in response to cellular environment are especially favored as easy-formulating materials to pack siRNA into a nanoparticle and to release the cargo in the cytoplasm in time. In addition to the efficiency of cytosomal release, the degradation products should best be free of safety concerns, a typical challenge for cationic polymers. To satisfy the two criteria, we report a new cationic polymer, polyspermine imine, named as PSP-Imine, which is formed by condensing two endogenous molecules, spermine and glyoxal, through conjugated π linkage, -N═C-C═N- (Schiff base reaction), a poly linkage structure sufficiently stable under neutral condition but dissociative under the endosomal pH. Cellular assays under a confocal microscope indicated that the polyplex formed of PSP-Imine readily released the loaded siRNA to the cytoplasm after being engulfed in the target cells and efficiently silenced the target genes in different cell lines and xenograft mouse model of human cervical carcinoma, as compared with nondegradable PEI 25 kDa. Cell viability assays confirmed that PSP-Imine showed no visible cytotoxicity within the concentration being tested. The present study suggests that PSP-Imine is an excellent siRNA condensing material for forming the core of a therapeutically feasible synthetic carrier system.

  7. Polyethyleneimine (PEI) Mediated siRNA Gene Silencing in the Schistosoma mansoni Snail Host, Biomphalaria glabrata

    PubMed Central

    Knight, Matty; Miller, Andre; Liu, Yijia; Scaria, Puthupparampil; Woodle, Martin; Ittiprasert, Wannaporn

    2011-01-01

    An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi) in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI) mediated delivery of long double-stranded (ds) and small interfering (si) RNA. Cellular delivery was evaluated and optimized by using a ‘mock’ fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB) with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx) at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease. PMID:21765961

  8. Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates.

    PubMed

    Dong, Yizhou; Love, Kevin T; Dorkin, J Robert; Sirirungruang, Sasilada; Zhang, Yunlong; Chen, Delai; Bogorad, Roman L; Yin, Hao; Chen, Yi; Vegas, Arturo J; Alabi, Christopher A; Sahay, Gaurav; Olejnik, Karsten T; Wang, Weiheng; Schroeder, Avi; Lytton-Jean, Abigail K R; Siegwart, Daniel J; Akinc, Akin; Barnes, Carmen; Barros, Scott A; Carioto, Mary; Fitzgerald, Kevin; Hettinger, Julia; Kumar, Varun; Novobrantseva, Tatiana I; Qin, June; Querbes, William; Koteliansky, Victor; Langer, Robert; Anderson, Daniel G

    2014-03-18

    siRNA therapeutics have promise for the treatment of a wide range of genetic disorders. Motivated by lipoproteins, we report lipopeptide nanoparticles as potent and selective siRNA carriers with a wide therapeutic index. Lead material cKK-E12 showed potent silencing effects in mice (ED50 ∼ 0.002 mg/kg), rats (ED50 < 0.01 mg/kg), and nonhuman primates (over 95% silencing at 0.3 mg/kg). Apolipoprotein E plays a significant role in the potency of cKK-E12 both in vitro and in vivo. cKK-E12 was highly selective toward liver parenchymal cell in vivo, with orders of magnitude lower doses needed to silence in hepatocytes compared with endothelial cells and immune cells in different organs. Toxicity studies showed that cKK-E12 was well tolerated in rats at a dose of 1 mg/kg (over 100-fold higher than the ED50). To our knowledge, this is the most efficacious and selective nonviral siRNA delivery system for gene silencing in hepatocytes reported to date.

  9. PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery

    PubMed Central

    Zhao, Yi; Wang, Wei; Guo, Shutao; Wang, Yuhua; Miao, Lei; Xiong, Yang; Huang, Leaf

    2016-01-01

    Metformin, a widely implemented anti-diabetic drug, exhibits potent anticancer efficacies. Herein a polymeric construction of Metformin, PolyMetformin (PolyMet) is successfully synthesized through conjugation of linear polyethylenimine (PEI) with dicyandiamide. The delocalization of cationic charges in the biguanide groups of PolyMet reduces the toxicity of PEI both in vitro and in vivo. Furthermore, the polycationic properties of PolyMet permits capture of siRNA into a core-membrane structured lipid-polycation-hyaluronic acid (LPH) nanoparticle for systemic gene delivery. Advances herein permit LPH-PolyMet nanoparticles to facilitate VEGF siRNA delivery for VEGF knockdown in a human lung cancer xenograft, leading to enhanced tumour suppressive efficacy. Even in the absence of RNAi, LPH-PolyMet nanoparticles act similarly to Metformin and induce antitumour efficacy through activation of the AMPK and inhibition of the mTOR. In essence, PolyMet successfully combines the intrinsic anticancer efficacy of Metformin with the capacity to carry siRNA to enhance the therapeutic activity of an anticancer gene therapy. PMID:27264609

  10. Inter-molecular β-sheet structure facilitates lung-targeting siRNA delivery

    PubMed Central

    Zhou, Jihan; Li, Dong; Wen, Hao; Zheng, Shuquan; Su, Cuicui; Yi, Fan; Wang, Jue; Liang, Zicai; Tang, Tao; Zhou, Demin; Zhang, Li-He; Liang, Dehai; Du, Quan

    2016-01-01

    Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-β, which was able to form inter-molecular β-sheet structures. Results showed that K-β peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications. PMID:26955887

  11. RNA Interference Induced by the Cationic Lipid Delivery of siRNA

    NASA Astrophysics Data System (ADS)

    Bouxsein, Nathan

    2005-03-01

    Recent discoveries demonstrate that the introduction of synthetically prepared duplexes of 19-21 bp short interfering RNAs (siRNA) into mammalian cells results in the cleavage of target mRNA leading to post transcriptional gene silencing [1]. Our work focuses on the cationic-lipid (CL) mediated delivery of siRNA into mammalian cell lines in an approach similar to CL based gene delivery [2]. Co-transfection of a target and a non-target reporter plasmid followed by the CL delivery of a sequence specific siRNA allows us to probe the silencing efficiency (SE) of the target plasmid relative to non-specific silencing of both plasmids. We have created a phase diagram for SE as a function of the complex membrane charge density and as a function of the CL:siRNA charge ratio. X-ray diffraction was performed to probe the structure of the complexes at points along the phase diagram. Funding provided by NIH AI-12520, AI-20611 and GM-59288. [1] Elbashir et. al., Nature, 411 494-498 (2001) [2] Ewert et. al., Curr. Med. Chem. 11 133-149 (2004)

  12. Notch1 targeting siRNA delivery nanoparticles for rheumatoid arthritis therapy.

    PubMed

    Kim, Min Ju; Park, Jong-Sung; Lee, So Jin; Jang, Jiyeon; Park, Jin Su; Back, Seung Hyun; Bahn, Gahee; Park, Jae Hyung; Kang, Young Mo; Kim, Sun Hwa; Kwon, Ick Chan; Jo, Dong-Gyu; Kim, Kwangmeyung

    2015-10-28

    Notch pathway plays a pivotal role in synoviocytes involved in progression of rheumatoid arthritis (RA). Herein, we designed the Notch1 targeting siRNA delivery nanoparticles (siRNA-NPs) in order to confirm the anti-inflammatory effect in collagen-induced arthritis (CIA) model. The siRNA-NPs were successfully produced by encapsulating polymerized siRNA (poly-siRNA) into thiolated glycol chitosan (tGC) nanoparticles in aqueous condition. The in vitro Notch1 inhibition of siRNA-NPs in murine macrophage cell (RAW 264.7) was confirmed using confocal microscopy and real time PCR. Fluorescently labeled siRNA-NPs were successfully transfected in RAW 264.7 and modulated the expression of Notch1 in mRNA level. For in vivo study, siRNA-NPs exhibited the higher targeting efficiency in the arthritic joins of CIA mice, confirmed by the near-infrared fluorescence (NIRF) imaging. Furthermore, inhibition of Notch1 with siRNA-NPs resulted in retarded progression of inflammation, bone erosion, and cartilage damage in CIA mice. Novel Notch1 targeting siRNA delivery system of siRNA-NPs showed effective RA treatment by suppressing Notch1 signaling pathway without undesirable severe toxicity. Thus, Notch1 inhibiting siRNA-NPs demonstrated the great potential in RA therapeutics that was hard to be achieved using conventional drugs.

  13. Efficient Inhibition of wear debris-induced inflammation by locally delivered siRNA

    SciTech Connect

    Peng Xiaochun; Tao Kun; Cheng Tao; Zhu Junfeng; Zhang Xianlong

    2008-12-12

    Aseptic loosening is the most common long-term complication of total joint replacement, which is associated with the generation of wear debris. The purpose of this study was to investigate the inhibitory effect of small interfering RNA (siRNA) targeting tumor necrosis factor-{alpha} (TNF-{alpha}) on wear debris-induced inflammation. A local delivery of lentivirus-mediated TNF-{alpha} siRNA into the modified murine air pouch, which was stimulated by polymethylmethacrylate (PMMA) particles, resulted in significant blockage of TNF-{alpha} both in mRNA and protein levels for up to 4 weeks. In addition, significant down-regulation of interleukin-1 (IL-1) and interleukin-6 (IL-6) was observed in TNF-{alpha} siRNA-treated pouches. The safety profile of gene therapy was proven by Bioluminescent assay and quantitative fluorescent flux. Histological analysis revealed less inflammatory responses (thinner pouch membrane and decreased cellular infiltration) in TNF-{alpha} siRNA-treated pouches. These findings suggest that local delivery of TNF-{alpha} siRNA might be an excellent therapeutic candidate to inhibit particle-induced inflammation.

  14. Utility of MicroRNAs and siRNAs in Cervical Carcinogenesis

    PubMed Central

    Díaz-González, Sacnite del Mar; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3′-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer. PMID:25874209

  15. Utility of microRNAs and siRNAs in cervical carcinogenesis.

    PubMed

    Díaz-González, Sacnite del Mar; Deas, Jessica; Benítez-Boijseauneau, Odelia; Gómez-Cerón, Claudia; Bermúdez-Morales, Victor Hugo; Rodríguez-Dorantes, Mauricio; Pérez-Plasencia, Carlos; Peralta-Zaragoza, Oscar

    2015-01-01

    MicroRNAs and siRNAs belong to a family of small noncoding RNAs which bind through partial sequence complementarity to 3'-UTR regions of mRNA from target genes, resulting in the regulation of gene expression. MicroRNAs have become an attractive target for genetic and pharmacological modulation due to the critical function of their target proteins in several signaling pathways, and their expression profiles have been found to be altered in various cancers. A promising technology platform for selective silencing of cell and/or viral gene expression using siRNAs is currently in development. Cervical cancer is the most common cancer in women in the developing world and sexually transmitted infection with HPV is the cause of this malignancy. Therefore, a cascade of abnormal events is induced during cervical carcinogenesis, including the induction of genomic instability, reprogramming of cellular metabolic pathways, deregulation of cell proliferation, inhibition of apoptotic mechanisms, disruption of cell cycle control mechanisms, and alteration of gene expression. Thus, in the present review article, we highlight new research on microRNA expression profiles which may be utilized as biomarkers for cervical cancer. Furthermore, we discuss selective silencing of HPV E6 and E7 with siRNAs which represents a potential gene therapy strategy against cervical cancer.

  16. Liposome-polyethylenimine complexes (DPPC-PEI lipopolyplexes) for therapeutic siRNA delivery in vivo.

    PubMed

    Ewe, Alexander; Panchal, Omkar; Pinnapireddy, Shashank Reddy; Bakowsky, Udo; Przybylski, Susanne; Temme, Achim; Aigner, Achim

    2017-01-01

    Therapeutic applications of RNA interference (RNAi) require efficient siRNA delivery strategies in vivo. Combining lipid-based carriers with polymeric nanoparticles offers the favorable properties of both systems. This is the first study to explore polyethylenimine-based lipopolyplexes comprising a low-molecular weight PEI and the phospholipid DPPC for therapeutic siRNA use. Lipopolyplex structures are analyzed by electron microscopy. Biological efficacies are demonstrated in vitro by cellular uptake, knockdown of the target oncogene survivin, and concomitant cell growth inhibition. Upon systemic administration in tumor-bearing mice, here performed by intraperitoneal (i.p.) injection, radioactive biodistribution assays show lipopolyplex-mediated delivery of intact siRNAs. Absence of blood serum parameter alterations, erythrocyte aggregation or immunostimulation, and the observation of animal well-being and stable body weight confirm biocompatibility. Exploring therapeutic efficacies in a preclinical model, a considerable inhibition of prostate carcinoma xenograft growth is achieved, paralleled by an ~65% survivin knockdown in the tumors. We, thus, demonstrate that PEI-based lipopolyplexes represent an efficient platform for therapeutic use of small RNAs.

  17. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery

    NASA Astrophysics Data System (ADS)

    Lee, Hyukjin; Lytton-Jean, Abigail K. R.; Chen, Yi; Love, Kevin T.; Park, Angela I.; Karagiannis, Emmanouil D.; Sehgal, Alfica; Querbes, William; Zurenko, Christopher S.; Jayaraman, Muthusamy; Peng, Chang G.; Charisse, Klaus; Borodovsky, Anna; Manoharan, Muthiah; Donahoe, Jessica S.; Truelove, Jessica; Nahrendorf, Matthias; Langer, Robert; Anderson, Daniel G.

    2012-06-01

    Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ~ 24.2 min) than the parent siRNA (t1/2 ~ 6 min).

  18. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery.

    PubMed

    Lee, Hyukjin; Lytton-Jean, Abigail K R; Chen, Yi; Love, Kevin T; Park, Angela I; Karagiannis, Emmanouil D; Sehgal, Alfica; Querbes, William; Zurenko, Christopher S; Jayaraman, Muthusamy; Peng, Chang G; Charisse, Klaus; Borodovsky, Anna; Manoharan, Muthiah; Donahoe, Jessica S; Truelove, Jessica; Nahrendorf, Matthias; Langer, Robert; Anderson, Daniel G

    2012-06-03

    Nanoparticles are used for delivering therapeutics into cells. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell-specific internalization, excretion, toxicity and efficacy. A variety of materials have been explored for delivering small interfering RNAs (siRNAs)--a therapeutic agent that suppresses the expression of targeted genes. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, a lack of tissue specificity and potential toxicity. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer-targeting ligands (such as peptides and folate) on the nanoparticle surface can be controlled precisely. We show that at least three folate molecules per nanoparticle are required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t(1/2) ≈ 24.2 min) than the parent siRNA (t(1/2) ≈ 6 min).

  19. The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center.

    PubMed

    Pontes, Olga; Li, Carey Fei; Costa Nunes, Pedro; Haag, Jeremy; Ream, Thomas; Vitins, Alexa; Jacobsen, Steven E; Pikaard, Craig S

    2006-07-14

    In Arabidopsis thaliana, small interfering RNAs (siRNAs) direct cytosine methylation at endogenous DNA repeats in a pathway involving two forms of nuclear RNA polymerase IV (Pol IVa and Pol IVb), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler DRD1, and the de novo cytosine methyltransferase DRM2. We show that RDR2, DCL3, AGO4, and NRPD1b (the largest subunit of Pol IVb) colocalize with siRNAs within the nucleolus. By contrast, Pol IVa and DRD1 are external to the nucleolus and colocalize with endogenous repeat loci. Mutation-induced loss of pathway proteins causes downstream proteins to mislocalize, revealing their order of action. Pol IVa acts first, and its localization is RNA dependent, suggesting an RNA template. We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers.

  20. Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita.

    PubMed

    Leachman, Sancy A; Hickerson, Robyn P; Hull, Peter R; Smith, Frances J D; Milstone, Leonard M; Lane, E Birgitte; Bale, Sherri J; Roop, Dennis R; McLean, W H Irwin; Kaspar, Roger L

    2008-09-01

    The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant-negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real-time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this "first-in-skin" siRNA may herald a paradigm shift in the treatment of dominant-negative genetic disorders.

  1. Inter-molecular β-sheet structure facilitates lung-targeting siRNA delivery

    NASA Astrophysics Data System (ADS)

    Zhou, Jihan; Li, Dong; Wen, Hao; Zheng, Shuquan; Su, Cuicui; Yi, Fan; Wang, Jue; Liang, Zicai; Tang, Tao; Zhou, Demin; Zhang, Li-He; Liang, Dehai; Du, Quan

    2016-03-01

    Size-dependent passive targeting based on the characteristics of tissues is a basic mechanism of drug delivery. While the nanometer-sized particles are efficiently captured by the liver and spleen, the micron-sized particles are most likely entrapped within the lung owing to its unique capillary structure and physiological features. To exploit this property in lung-targeting siRNA delivery, we designed and studied a multi-domain peptide named K-β, which was able to form inter-molecular β-sheet structures. Results showed that K-β peptides and siRNAs formed stable complex particles of 60 nm when mixed together. A critical property of such particles was that, after being intravenously injected into mice, they further associated into loose and micron-sized aggregates, and thus effectively entrapped within the capillaries of the lung, leading to a passive accumulation and gene-silencing. The large size aggregates can dissociate or break down by the shear stress generated by blood flow, alleviating the pulmonary embolism. Besides the lung, siRNA enrichment and targeted gene silencing were also observed in the liver. This drug delivery strategy, together with the low toxicity, biodegradability, and programmability of peptide carriers, show great potentials in vivo applications.

  2. Bivariate extreme value distributions

    NASA Technical Reports Server (NTRS)

    Elshamy, M.

    1992-01-01

    In certain engineering applications, such as those occurring in the analyses of ascent structural loads for the Space Transportation System (STS), some of the load variables have a lower bound of zero. Thus, the need for practical models of bivariate extreme value probability distribution functions with lower limits was identified. We discuss the Gumbel models and present practical forms of bivariate extreme probability distributions of Weibull and Frechet types with two parameters. Bivariate extreme value probability distribution functions can be expressed in terms of the marginal extremel distributions and a 'dependence' function subject to certain analytical conditions. Properties of such bivariate extreme distributions, sums and differences of paired extremals, as well as the corresponding forms of conditional distributions, are discussed. Practical estimation techniques are also given.

  3. Ternary complexes of folate-PEG-appended dendrimer (G4)/α-cyclodextrin conjugate, siRNA and low-molecular-weight polysaccharide sacran as a novel tumor-selective siRNA delivery system.

    PubMed

    Ohyama, Ayumu; Higashi, Taishi; Motoyama, Keiichi; Arima, Hidetoshi

    2017-02-14

    We previously developed a tumor-selective siRNA carrier by preparing polyamidoamine dendrimer (generation 4, G4) conjugates with α-cyclodextrin and folate-polyethylene glycol (Fol-PαC (G4)). In the present study, we developed ternary complexes of Fol-PαC (G4)/siRNA with low-molecular-weight-sacrans to achieve more effective siRNA transfer activity. Among the different molecular-weight sacrans, i.e. sacran 100, 1000 and 10,000 (MW 44,889Da, 943,692Da and 1,488,281Da, respectively), sacran 100 significantly increased the cellular uptake and the RNAi effects of Fol-PαC (G4)/siRNA binary complex with negligible cytotoxicity in KB cells (folate receptor-α positive cells). In addition, the ζ-potential and particle size of Fol-PαC (G4)/siRNA complex were decreased by the ternary complexation with sacran 100. Importantly, the in vivo RNAi effect of the ternary complex after the intravenous administration to tumor-bearing BALB/c mice was significantly higher than that of the binary complex. In conclusion, Fol-PαC (G4)/siRNA/sacran 100 ternary complex has a potential as a novel tumor-selective siRNA delivery system.

  4. Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy

    NASA Astrophysics Data System (ADS)

    Dumitru, Andra C.; Herruzo, Elena T.; Rausell, Estrella; Ceña, Valentin; Garcia, Ricardo

    2015-11-01

    We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding events.

  5. Non-Covalently Functionalized of Single-Walled Carbon Nanotubes by DSPE-PEG-PEI for SiRNA Delivery.

    PubMed

    Siu, King Sun; Zhang, Yujuan; Zheng, Xiufen; Koropatnick, James; Min, Wei-Ping

    2016-01-01

    The expression of a gene can be specifically downregulated by small interfering RNA (SiRNA). Modified carbon nanotubes (CNT) can be used to protect SiRNA and facilitate its entry into cells. Regardless of that, simple and efficient functionalization of CNT is lacking. Effective SiRNA delivery can be carried out using non-covalently functionalized CNT, where non-covalent (versus covalent) functionalization is simpler and more expeditious. Non-covalently functionalized single walled carbon nanotubes (SWCNT) that include a lipopolymer are described here. Polyethylenimine (PEI) conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) was generated and the products used to disperse CNT to form DSPE-PEG-PEI/CNT (DGI/C), an agent capable of facilitating SiRNA delivery to cells in vitro and organs and cells in vivo.

  6. Unbinding forces and energies between a siRNA molecule and a dendrimer measured by force spectroscopy.

    PubMed

    Dumitru, Andra C; Herruzo, Elena T; Rausell, Estrella; Ceña, Valentin; Garcia, Ricardo

    2015-12-21

    We have measured the intermolecular forces between small interference RNA (siRNA) and polyamidoamine dendrimers at the single molecular level. A single molecule force spectroscopy approach has been developed to measure the unbinding forces and energies between a siRNA molecule and polyamidoamine dendrimers deposited on a mica surface in a buffer solution. We report three types of unbinding events which are characterized by forces and free unbinding energies, respectively, of 28 pN, 0.709 eV; 38 pN, 0.722 eV; and 50 pN, 0.724 eV. These events reflect different possible electrostatic interactions between the positive charges of one or two dendrimers and the negatively charged phosphate groups of a single siRNA. We have evidence of a high binding affinity of siRNA towards polyamidoamine dendrimers that leads to a 45% probability of measuring specific unbinding events.

  7. Effects of chitosan nanoparticle-mediated BRAF siRNA interference on invasion and metastasis of gastric cancer cells.

    PubMed

    Huo, Jian

    2016-08-01

    To observe the changes in invasion capacity of gastric cancer BGC823 cells after being treated with chitosan-encapsulated BRAF siRNA nanoparticles, and to evaluate the effects of the nanoparticle-mediated BRAF siRNA interference on cell invasion and metastasis, BRAF siRNA was encapsulated with chitosan into nanoparticles sized 350 nm to treat gastric cancer cells. Silencing of BRAF was detected by Western blot and PCR, and cell invasion was observed by the Transwell assay. The nanoparticles significantly downregulated BRAF expression in BGC823 cells (P < 0.01) and inhibited their invasion (P < 0.001). Chitosan nanoparticle-mediated BRAF siRNA interference evidently reduced the invasion capacity of gastric cancers.

  8. Recent In Vivo Evidences of Particle-Based Delivery of Small-Interfering RNA (siRNA) into Solid Tumors

    PubMed Central

    2014-01-01

    Small-interfering RNA (siRNA) is both a powerful tool in research and a promising therapeutic platform to modulate expression of disease-related genes. Malignant tumors are attractive disease targets for nucleic acid-based therapies. siRNA directed against oncogenes, and genes driving metastases or angiogenesis have been evaluated in animal models and in some cases, in humans. The outcomes of these studies indicate that drug delivery is a significant limiting factor. This review provides perspectives on in vivo validated nanoparticle-based siRNA delivery systems. Results of recent advances in liposomes and polymeric and inorganic formulations illustrate the need for mutually optimized attributes for performance in systemic circulation, tumor interstitial space, plasma membrane, and endosomes. Physiochemical properties conducive to efficient siRNA delivery are summarized and directions for future research are discussed. PMID:25221632

  9. [Dose-effect research using nanopatch to deliver siRNA in the inhibition of HPV gene expression].

    PubMed

    Xiong, Zhuo; Dong, Xiaojing; Sun, Peiwen; Zhang, Ying

    2013-12-01

    Hela is the cell line of adenocarcinoma of the uterine cervix, and human papillomavirus (HPV) 18 shows positive. We delivered siRNA with target specifically to HPV18 E7 mRNA into nude mice Hela tumor xenografts by nanopatch to inhibit the HPV gene expression, and further to study the superiority, the best action time and concentration of siRNA of using nanopatch to transfer siRNA in vivo. We designed siRNA that target specifically to HPV18 E7 mRNA (siE7) and checked the effect of siE7 in vitro. Tumor xenografts were transfected with siE7 and GenEscort III by nanopatch. Expression of HPV18 E7 mRNA and protein were detected 0 hours, 24 hours, 48 hours, 72 hours after transfection with PT-PCR and Western blot, and the best action time was analyzed using nanopatch to thansfect siRNA in vivo. We transfected GenEscort III and siE7 of Different concentration into tumor xenografts respectively by nanopatch and intraperitoneal injection. Expression of HPV18 E7 mRNA and protein was detected 72 hours after transfection by PT-PCR and Western blot, to analyze the best action concentration of siRNA and the superiority of using nanopatch to thansfect siRNA in vivo. The results proved that SiE7was efficient to inhibit expression of HPV18 E7 mRNA and to advance Hela apoptosis in vitro. SiE7 transfected by nanopatch into xenografts could inhibit effectively expression of HPV18 E7 mRNA and protein. The best action time and concentration of siRNA of using nanopatch to thansfect siRNA in vivo are 72 hour post-transfection and 2 micromol/L siE7. To compare intraperitoneal injection in delivering siRNA in vivo, the effect of nanopatch is very predominant. It can be well concluded that Nanopatch can effectively transfer siRNA in vivo, which can effectively inhibit the HPV gene expression.

  10. cRGD grafted liposomes containing inorganic nano-precipitate complexed siRNA for intracellular delivery in cancer cells.

    PubMed

    Khatri, Nirav; Baradia, Dipesh; Vhora, Imran; Rathi, Mohan; Misra, Ambikanandan

    2014-05-28

    Development of effective vector for intracellular delivery of siRNA has always been a challenge due to its hydrophilicity, net negative surface charge and sensitivity against nucleases in biological milieu. The present investigation was aimed to develop a novel non-viral liposomal carrier for siRNA delivery. Nano-precipitate of calcium phosphate was entrapped in liposomes composed of a neutral lipid (DPPC), a fusogenic lipid (DOPE), a PEGylated lipid (DSPE-mPEG2000) and cholesterol. siRNA was made permeable through liposomal bilayer and complexed to calcium phosphate precipitates inside the liposomes. siRNA entrapped liposomes were further grafted with cRGD to achieve targeting potential against cancer cells. More than 80% of siRNA was entrapped inside the liposomes having average particle size below 150nm. Cryo-transmission electron microscopy revealed the intra-liposomal calcium phosphate precipitation and unilamellar morphology of prepared liposomes. The viability of A549 lung cancer cells was significantly higher after treatment with siRNA entrapped liposomes as compared to Lipofectamine2000 complexed siRNA. Fluorescent intensity in lung carcinoma cells was significantly higher after exposure to fluorescent siRNA entrapped liposomes than with Lipofectamine2000, which were confirmed by both confocal microscopy and flow cytometry. Live imaging by confocal microscopy ascertained the targeting efficacy of cRGD grafted liposomes compared to naked siRNA and non-grafted liposomes. Developed liposomal formulation showed effective protection of siRNA against serum nucleases along with less haemolytic potential and excellent stability against electrolyte induced flocculation. At 5nM concentration gene expression of target protein was reduced up to 24.1±3.4% while Lipofectamine2000 reduced expression level up to 26.35±1.55%. In vivo toxicity in mice suggested admirable safety profile for developed lipid based delivery vector. These results advocate that prepared

  11. Bach1 siRNA attenuates bleomycin-induced pulmonary fibrosis by modulating oxidative stress in mice

    PubMed Central

    Liu, Yuan; Zheng, Yi

    2017-01-01

    Oxidative stress plays an essential role in inflammation and fibrosis. Bach1 is an important transcriptional repressor that acts by modulating oxidative stress and represents a potential target in the treatment of pulmonary fibrosis (PF). In this study, we knocked down Bach1 using adenovirus-mediated small interfering RNA (siRNA) to determine whether the use of Bach1 siRNA is an effective therapeutic strategy in mice with bleomycin (BLM)-induced PF. Mouse lung fibroblasts (MLFs) were incubated with transforming growth factor (TGF)-β1 (5 ng/ml) and subsequently infected with recombined adenovirus-like Bach1 siRNA1 and Bach1 siRNA2, while an empty adenovirus vector was used as the negative control. The selected Bach1 siRNA with higher interference efficiency was used for the animal experiments. A mouse model of BLM-induced PF was established, and Bach1 siRNA (1×109 PFU) was administered to the mice via the tail vein. The results revealed that the Bach1 mRNA and protein levels were significantly downregulated by Bach1 siRNA. Furthermore, the MLFs infected with Bach1 siRNA exhibited increased mRNA and protein expression levels of heme oxygenase-1 and glutathione peroxidase 1, but decreased levels of TGF-β1 and interleukin-6 in the cell supernatants compared with the cells exposed to TGF-β1 alone. Bach1 knockdown by siRNA also enhanced the expression of antioxidant factors, but suppressed that of fibrosis-related cytokines in mice compared with the BLM group. Finally, the inflammatory infiltration of alveolar and interstitial cells and the destruction of lung structure were significantly attenuated in the mide administered Bach1 siRNA compared with those in the BLM group. On the whole, our findings demonstrate that Bach1 siRNA exerts protective effects against BLM-induced PF in mice. Our data may provide the basis for the development of novel targeted therapeutic strategies for PF. PMID:27959382

  12. Thermal spectrum of uranus: implications for large helium abundance.

    PubMed

    Orton, G S

    1986-02-21

    An analysis of the infrared spectrum of Uranus' disk between 7 micrometers and 3 millimeters suggests a volume mixing ratio for helium in the atmosphere of 40 +/- 20 percent, more than for the sun, Jupiter, or Saturn. Alternative explanations require even more extreme assumptions regarding gas abundances or aerosol vertical distribution and spectral properties. The most serious difficulty with a model containing large amounts of helium is devising a credible evolutionary or chemical model explaining the absence or segregation of so much hydrogen.

  13. Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

    SciTech Connect

    Li, Xiaojie; Zhang, Ling; Shao, Yueting; Liang, Zuowen; Shao, Chen; Wang, Bo; Guo, Baofeng; Li, Na; Zhao, Xuejian; Li, Yang; Xu, Deqi

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Neu3 is as one of the sialidases and regulates cell surface functions. Black-Right-Pointing-Pointer A Neu3-specific siRNA inhibited prostrate cancer cell invasion and migration. Black-Right-Pointing-Pointer The Neu3-specific siRNA inhibited prostate cancer metastasis in mice. Black-Right-Pointing-Pointer Targeting Neu3 may have utility for gene-based therapy of human cancer metastasis. -- Abstract: Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed

  14. Intracellular delivery of peptides and siRNAs using microbubble enhanced focused ultrasound

    NASA Astrophysics Data System (ADS)

    Kinoshita, Manabu; Hynynen, Kullervo

    2006-05-01

    Bioactive substances such as peptides and nucleic acid based agents have attracted great attention for the next generation drug for various diseases. However, the greatest challenge for using these bioactive substances is the development of their delivery system, especially the method for delivering these substances through the cell membrane. With the advancement of ultrasound and ultrasound contrast agent technology, it has become possible to transiently change the permeability of the cell membrane. Moreover, using a focused ultrasound transducer, it is possible to narrow and focus the ultrasound energy within a small target, avoiding damage to the surrounding tissue. In this research we have searched the possibility of delivering the Bak BH3 peptide, the death domain of the Bc1-2 family of proteins, or the short interfering RNA (siRNA) targeting the enhanced green fluorescent protein (EGFP) using microbubble-enhanced focused ultrasound in an in vitro setting. Using a 1.696 MHz focused ultrasound and a microbubble ultrasound contrast agent OPTISON®, we first tested the stability of BH3 peptide under microbubble-enhanced focused ultrasound exposure and proved that the peptide is stable under these circumstances. Next, we have tested the cell-killing effect of the intracellularly delivered Bak BH3 peptide in HeLa and BJAB cell line and observed a statistically enhanced cell death in BJAB cells but not in HeLa cells, leading to the conclusion that intracellularly delivered BH3 peptide by microbubble-enhanced ultrasound can exert its cell killing effect in some cells. We also investigated if we can silence the EGFP expression in the cell by delivering siRNA targeting the EGFP in both transient and stable EGFP expression cell line. Using a 1.653 MHz focused ultrasound and OPTISON®, in both cases, intracellularly delivered siRNA by microbubble-enhanced ultrasound was able to knock down the EGFP expression, which demonstrates the feasibility of using this novel method

  15. Combination siRNA therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro.

    PubMed

    McDonagh, Phillip; Sheehy, Paul A; Norris, Jacqueline M

    2015-03-23

    Virulent biotypes of feline coronavirus (FCoV), commonly referred to as feline infectious peritonitis virus (FIPV), can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. We previously reported the successful in vitro inhibition of FIPV replication by synthetic siRNA mediated RNA interference (RNAi) in an immortalised cell line (McDonagh et al., 2011). A major challenge facing the development of any antiviral strategy is that of resistance, a problem which is particularly acute for RNAi based therapeutics due to the exquisite sequence specificity of the targeting mechanism. The development of resistance during treatment can be minimised using combination therapy to raise the genetic barrier or using highly potent compounds which result in a more rapid and pronounced reduction in the viral replication rate, thereby reducing the formation of mutant, and potentially resistant viruses. This study investigated the efficacy of combination siRNA therapy and its ability to delay or prevent viral escape. Virus serially passaged through cells treated with a single or dual siRNAs rapidly acquired resistance, with mutations identified in the siRNA target sites. Combination therapy with three siRNA prevented viral escape over the course of five passages. To identify more potent silencing molecules we also compared the efficacy, in terms of potency and duration of action, of canonical versus Dicer-substrate siRNAs for two previously identified effective viral motifs. Dicer-substrate siRNAs showed equivalent or better potency than canonical siRNAs for the target sites investigated, and may be a more appropriate molecule for in vivo use. Combined, these data inform the potential therapeutic application of antiviral RNAi against FIPV.

  16. Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production.

    PubMed

    Kandasamy, Suresh K; Fukunaga, Ryuya

    2016-12-06

    The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

  17. Preparation and in vitro evaluation of vaginal formulations including siRNA and paclitaxel-loaded SLNs for cervical cancer.

    PubMed

    Büyükköroğlu, Gülay; Şenel, Behiye; Başaran, Ebru; Yenilmez, Evrim; Yazan, Yasemin

    2016-12-01

    Cervical cancer is one of the most life threatening types of cancer among women and is generally resistant to chemotherapy. The objective of this study was to prepare a vaginal suppository containing a chemotherapeutic agent and a genetic material that can be applied locally for cervical cancer. Paclitaxel was selected as the chemotherapeutic agent and siRNA which inhibits BCL-2 oncogene was selected as the genetic material. Bcl-2 siRNA, paclitaxel and paclitaxel/Bcl-2 siRNA combination were incorporated into solid lipid nanoparticles (SLNs) and were dispersed separately in vaginal suppositories prepared with PEG 6000. Physicochemical properties of SLNs, their cytotoxicities on HeLa cell lines and also the effect of SLNs on the total protein amount of the cells were examined followed by the investigation of release rates of the active materials from the SLNs prepared. Average diameters of all SLNs prepared were below 180nm with a positive zeta potential value between +22.20 and +48.16mV at the pH range of 4.2 and 7.4. The release of Bcl-2 siRNA from SLNs incorporated Bcl-2 siRNA and the release of paclitaxel (PTX) from PTX incorporated SLNs were completed within 12h and 36h. SLNs incorporating Bcl-2 siRNA and paclitaxel/Bcl-2 siRNA were found to be more toxic when compared to paclitaxel incorporated SLN and placebo SLN. The disintegration of the vaginal suppositories as well as the release of the SLNs was completed within 2 h. This study indicates that vaginal suppository containing SLNs can bring the advantages of the simultaneous delivery of paclitaxel and siRNA via vaginal route with no help from professionals.

  18. Chitosan-based nanoparticles for survivin targeted siRNA delivery in breast tumor therapy and preventing its metastasis

    PubMed Central

    Sun, Ping; Huang, Wei; Jin, Mingji; Wang, Qiming; Fan, Bo; Kang, Lin; Gao, Zhonggao

    2016-01-01

    Nanoparticle-mediated small interfering RNA (siRNA) delivery is a promising therapeutic strategy in various cancers. However, it is difficult to deliver degradative siRNA to tumor tissue, and thus a safe and efficient vector for siRNA delivery is essential for cancer therapy. In this study, poly(ethylene glycol)-modified chitosan (PEG-CS) was synthesized successfully for delivering nucleic acid drug. We deemed that PEGylated CS could improve its solubility by forming a stable siRNA loaded in nanoparticles, and enhancing transfection efficiency of siRNA-loaded CS nanoparticles in cancer cell line. The research results showed that siRNA loaded in PEGylated CS (PEG-CS/siRNA) nanoparticles with smaller particle size had superior structural stability in the physical environment compared to CS nanoparticles. The data of in vitro antitumor activity revealed that 4T1 tumor cell growth was significantly inhibited and cellular uptake of PEG-CS/siRNA nanoparticles in 4T1 cells was dramatically enhanced compared to naked siRNA groups. The results from flow cytometry and confocal laser scanning microscopy showed that PEG-CS/siRNA nanoparticles were more easily taken up than naked siRNA. Importantly, PEG-CS/siRNA nanoparticles significantly reduced the growth of xenograft tumors of 4T1 cells in vivo. It has been demonstrated that the PEG-CS is a safe and efficient vector for siRNA delivery, and it can effectively reduce tumor growth and prevent metastasis. PMID:27729789

  19. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors.

    PubMed

    Ganesh, Shanthi; Iyer, Arun K; Morrissey, David V; Amiji, Mansoor M

    2013-04-01

    Anticancer therapeutics employing RNA interference mechanism holds promising potentials for sequence-specific silencing of target genes. However targeted delivery of siRNAs to tumor tissues and cells and more importantly, their intracellular release at sites of interest still remains a major challenge that needs to be addressed before this technique could become a clinically viable option. In the current study, we have engineered and screened a series of CD44 targeting hyaluronic acid (HA) based self-assembling nanosystems for targeted siRNA delivery. The HA polymer was functionalized with lipids of varying carbon chain lengths/nitrogen content, as well as polyamines for assessing siRNA encapsulation. From the screens, several HA-derivatives were identified that could stably encapsulate/complex siRNAs and form self-assembled nanosystems, as determined by gel retardation assays and dynamic light scattering. Many HA derivatives could transfect siRNAs into cancer cells overexpressing CD44 receptors. Interestingly, blocking the CD44 receptors on the cells using free excess soluble HA prior to incubation of cy3-labeled-siRNA loaded HA nano-assemblies resulted in >90% inhibition of the receptor mediated uptake, confirming target specificity. In addition, SSB/PLK1 siRNA encapsulated in HA-PEI/PEG nanosystems demonstrated dose dependent and target specific gene knockdown in both sensitive and resistant A549 lung cancer cells overexpressing CD44 receptors. More importantly, these siRNA encapsulated nanosystems demonstrated tumor selective uptake and target specific gene knock down in vivo in solid tumors as well as in metastatic tumors. The HA based nanosystems thus portend to be promising siRNA delivery vectors for systemic targeting of CD44 overexpressing cancers including tumor initiating (stem-) cells and metastatic lesions.

  20. Chitosan-based nanoparticles for survivin targeted siRNA delivery in breast tumor therapy and preventing its metastasis.

    PubMed

    Sun, Ping; Huang, Wei; Jin, Mingji; Wang, Qiming; Fan, Bo; Kang, Lin; Gao, Zhonggao

    Nanoparticle-mediated small interfering RNA (siRNA) delivery is a promising therapeutic strategy in various cancers. However, it is difficult to deliver degradative siRNA to tumor tissue, and thus a safe and efficient vector for siRNA delivery is essential for cancer therapy. In this study, poly(ethylene glycol)-modified chitosan (PEG-CS) was synthesized successfully for delivering nucleic acid drug. We deemed that PEGylated CS could improve its solubility by forming a stable siRNA loaded in nanoparticles, and enhancing transfection efficiency of siRNA-loaded CS nanoparticles in cancer cell line. The research results showed that siRNA loaded in PEGylated CS (PEG-CS/siRNA) nanoparticles with smaller particle size had superior structural stability in the physical environment compared to CS nanoparticles. The data of in vitro antitumor activity revealed that 4T1 tumor cell growth was significantly inhibited and cellular uptake of PEG-CS/siRNA nanoparticles in 4T1 cells was dramatically enhanced compared to naked siRNA groups. The results from flow cytometry and confocal laser scanning microscopy showed that PEG-CS/siRNA nanoparticles were more easily taken up than naked siRNA. Importantly, PEG-CS/siRNA nanoparticles significantly reduced the growth of xenograft tumors of 4T1 cells in vivo. It has been demonstrated that the PEG-CS is a safe and efficient vector for siRNA delivery, and it can effectively reduce tumor growth and prevent metastasis.

  1. Bio-inspired materials in drug delivery: Exploring the role of pulmonary surfactant in siRNA inhalation therapy.

    PubMed

    De Backer, Lynn; Cerrada, Alejandro; Pérez-Gil, Jesús; De Smedt, Stefaan C; Raemdonck, Koen

    2015-12-28

    Many pathologies of the respiratory tract are inadequately treated with existing small molecule-based therapies. The emergence of RNA interference (RNAi) enables the post-transcriptional silencing of key molecular disease factors that cannot readily be targeted with conventional small molecule drugs. Pulmonary administration of RNAi effectors, such as small interfering RNA (siRNA), allows direct delivery into the lung tissue, hence reducing systemic exposure. Unfortunately, the clinical translation of RNAi is severely hampered by inefficient delivery of siRNA therapeutics towards the cytoplasm of the target cells. In order to have a better control of the siRNA delivery process, both extra- and intracellular, siRNAs are typically formulated in nanosized delivery vehicles (nanoparticles, NPs). In the lower airways, which are the targeted sites of action for multiple pulmonary disorders, these siRNA-loaded NPs will encounter the pulmonary surfactant (PS) layer, covering the entire alveolar surface. The interaction between the instilled siRNA-loaded NPs and the PS at this nano-bio interface results in the adsorption of PS components onto the surface of the NPs. The formation of this so-called biomolecular corona conceals the original NP surface and will therefore profoundly determine the biological efficacy of the NP. Though this interplay has initially been regarded as a barrier towards efficient siRNA delivery to the respiratory target cell, recent reports have illustrated that the interaction with PS might also be beneficial for local pulmonary siRNA delivery.

  2. Spray-dried powders enhance vaginal siRNA delivery by potentially modulating the mucus molecular sieve structure

    PubMed Central

    Wu, Na; Zhang, Xinxin; Li, Feifei; Zhang, Tao; Gan, Yong; Li, Juan

    2015-01-01

    Vaginal small interfering RNA (siRNA) delivery provides a promising strategy for the prevention and treatment of vaginal diseases. However, the densely cross-linked mucus layer on the vaginal wall severely restricts nanoparticle-mediated siRNA delivery to the vaginal epithelium. In order to overcome this barrier and enhance vaginal mucus penetration, we prepared spray-dried powders containing siRNA-loaded nanoparticles. Powders with Pluronic F127 (F127), hydroxypropyl methyl cellulose (HPMC), and mannitol as carriers were obtained using an ultrasound-assisted spray-drying technique. Highly dispersed dry powders with diameters of 5–15 μm were produced. These powders showed effective siRNA protection and sustained release. The mucus-penetrating properties of the powders differed depending on their compositions. They exhibited different potential of opening mesh size of molecular sieve in simulated vaginal mucus system. A powder formulation with 0.6% F127 and 0.1% HPMC produced the maximum increase in the pore size of the model gel used to simulate vaginal mucus by rapidly extracting water from the gel and interacting with the gel; the resulting modulation of the molecular sieve effect achieved a 17.8-fold improvement of siRNA delivery in vaginal tract and effective siRNA delivery to the epithelium. This study suggests that powder formulations with optimized compositions have the potential to alter the steric barrier posed by mucus and hold promise for effective vaginal siRNA delivery. PMID:26347257

  3. Nose-to-brain delivery of BACE1 siRNA loaded in solid lipid nanoparticles for Alzheimer's therapy.

    PubMed

    Rassu, Giovanna; Soddu, Elena; Posadino, Anna Maria; Pintus, Gianfranco; Sarmento, Bruno; Giunchedi, Paolo; Gavini, Elisabetta

    2017-04-01

    We designed a delivery system to obtain an efficient and optimal nose-to-brain transport of BACE1 siRNA, potentially useful in the treatment of Alzheimer's disease. We selected a cell-penetrating peptide, the short peptide derived from rabies virus glycoprotein known as RVG-9R, to increase the transcellular pathway in neuronal cells. The optimal molar ratio between RVG-9R and BACE1 siRNA was elucidated. The complex between the two was then encapsulated. We propose chitosan-coated and uncoated solid lipid nanoparticles (SLNs) as a nasal delivery system capable of exploiting both olfactory and trigeminal nerve pathways. The coating process had an effect on the zeta potential, obtaining positively-charged nanoparticles, and on siRNA protection. The positive charge of the coating formulation ensured mucoadhesiveness to the particles and also prolonged residence time in the nasal cavity. We studied the cellular transport of siRNA released from the SLNs using Caco-2 as a model of epithelial-like phenotypes. We found that siRNA permeates the monolayer to a greater extent when released from any of the studied formulations than from bare siRNA, and primarily from chitosan-coated SLNs.

  4. Small-sized, stable lipid nanoparticle for the efficient delivery of siRNA to human immune cell lines

    PubMed Central

    Nakamura, Takashi; Kuroi, Moeka; Fujiwara, Yuki; Warashina, Shota; Sato, Yusuke; Harashima, Hideyoshi

    2016-01-01

    Gene silencing by small interfering RNA (siRNA) is useful for analyzing the functions of human immune cells. However, the transfection of siRNA to human immune cells is difficult. Here, we used a multifunctional envelope-type nanodevice (MEND) containing YSK12-C4 (YSK12-MEND) to efficiently introduce siRNA to human immune cell lines, Jurkat, THP-1, KG-1 and NK92. The YSK12-MEND was transfected to human immune cell lines at a siRNA dose range of 1–30 nM, resulting that maximum gene silencing efficiencies at the mRNA level in Jurkat, THP-1, KG-1 and NK92 were 96%, 96%, 91% and 75%, respectively. The corresponding values for Lipofectamine RNAiMAX (RNAiMAX) were 37%, 56%, 43% and 19%, respectively. The process associated with cellular uptake played a role in effective gene silencing effect of the YSK12-MEND. The small size and high non-aggregability of the YSK12-MEND were advantageous for the cellular internalization of siRNA to immune cell lines. In the case of RNAiMAX, a drastic increase in particles size was observed in the medium used, which inhibited cellular uptake. The YSK12-MEND reported in herein appears to be appropriate for delivering siRNA to human immune cells, and the small particle size and non-aggregability are essential properties. PMID:27892533

  5. Spray-dried powders enhance vaginal siRNA delivery by potentially modulating the mucus molecular sieve structure.

    PubMed

    Wu, Na; Zhang, Xinxin; Li, Feifei; Zhang, Tao; Gan, Yong; Li, Juan

    2015-01-01

    Vaginal small interfering RNA (siRNA) delivery provides a promising strategy for the prevention and treatment of vaginal diseases. However, the densely cross-linked mucus layer on the vaginal wall severely restricts nanoparticle-mediated siRNA delivery to the vaginal epithelium. In order to overcome this barrier and enhance vaginal mucus penetration, we prepared spray-dried powders containing siRNA-loaded nanoparticles. Powders with Pluronic F127 (F127), hydroxypropyl methyl cellulose (HPMC), and mannitol as carriers were obtained using an ultrasound-assisted spray-drying technique. Highly dispersed dry powders with diameters of 5-15 μm were produced. These powders showed effective siRNA protection and sustained release. The mucus-penetrating properties of the powders differed depending on their compositions. They exhibited different potential of opening mesh size of molecular sieve in simulated vaginal mucus system. A powder formulation with 0.6% F127 and 0.1% HPMC produced the maximum increase in the pore size of the model gel used to simulate vaginal mucus by rapidly extracting water from the gel and interacting with the gel; the resulting modulation of the molecular sieve effect achieved a 17.8-fold improvement of siRNA delivery in vaginal tract and effective siRNA delivery to the epithelium. This study suggests that powder formulations with optimized compositions have the potential to alter the steric barrier posed by mucus and hold promise for effective vaginal siRNA delivery.

  6. Non-targeting siRNA induces NPGPx expression to cooperate with exoribonuclease XRN2 for releasing the stress.

    PubMed

    Wei, Pei-Chi; Lo, Wen-Ting; Su, Mei-I; Shew, Jin-Yuh; Lee, Wen-Hwa

    2012-01-01

    Short interfering RNAs (siRNAs) target specific mRNAs for their degradation mediated by RNA-induced silencing complex (RISC). Persistent activation of siRNA-RISC frequently leads to non-targeting toxicity. However, how cells mediate this stress remains elusive. In this communication, we found that the presence of non-targeting siRNA selectively induced the expression of an endoplasmic reticulum (ER)-resident protein, non-selenocysteine containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), but not other ER-stress proteins including GRP78, Calnexin and XBP1. Cells suffering from constant non-targeting siRNA stress grew slower and prolonged G1 phase, while NPGPx-depleted cells accumulated mature non-targeting siRNA and underwent apoptosis. Upon the stress, NPGPx covalently bound to exoribonuclease XRN2, facilitating XRN2 to remove accumulated non-targeting siRNA. These results suggest that NPGPx serves as a novel responder to non-targeting siRNA-induced stress in facilitating XRN2 to release the non-targeting siRNA accumulation.

  7. Influence of SiRNA targeting survivin on chemosensitivity of H460/cDDP lung cancer cells.

    PubMed

    Yang, H; Fu, J-H; Hu, Y; Huang, W-Z; Zheng, B; Wang, G; Zhang, X; Wen, J

    2008-01-01

    We investigated the difference in survivin expression between a multidrug-resistant lung cancer cell line (H460/cDDP) and its parental counterpart (H460) and the influence of siRNA targeting survivin on the chemosensitivity of H460/cDDP. SiRNA targeting survivin was transfected into H460/cDDP cells using a liposome approach. Survivin mRNA and protein expression were significantly higher in H460/cDDP than H460 cells. The median inhibitory concentrations (IC(50)s) for cisplatin and paclitaxol in vitro against H460/cDDP cells were significantly lower in cells treated with survivin-specific siRNA than in control cells. Apoptosis and cleaved caspase-3 expression were analysed using annexin V and Western blotting, respectively, and showed a significant increase in apoptosis after treatment with the chemotherapeutic agents plus specific siRNA. Specific siRNA sensitized H460/cDDP cells to both cisplatin and paclitaxol. Thus, survivin appears to participate in the multidrug resistance mechanism of H460/cDDP cells and siRNA targeting survivin has the potential to increase the sensitivity of drug-resistant cancer cells to anticancer drugs.

  8. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments

    PubMed Central

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-01-01

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments. PMID:27278626

  9. Small RNA transcriptomes of mangroves evolve adaptively in extreme environments.

    PubMed

    Wen, Ming; Lin, Xingqin; Xie, Munan; Wang, Yushuai; Shen, Xu; Liufu, Zhongqi; Wu, Chung-I; Shi, Suhua; Tang, Tian

    2016-06-09

    MicroRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) are key players in plant stress responses. Here, we present the sRNA transcriptomes of mangroves Bruguiera gymnorrhiza and Kandelia candel. Comparative computational analyses and target predictions revealed that mangroves exhibit distinct sRNA regulatory networks that differ from those of glycophytes. A total of 32 known and three novel miRNA families were identified. Conserved and mangrove-specific miRNA targets were predicted; the latter were widely involved in stress responses. The known miRNAs showed differential expression between the mangroves and glycophytes, reminiscent of the adaptive stress-responsive changes in Arabidopsis. B. gymnorrhiza possessed highly abundant but less conserved TAS3 trans-acting siRNAs (tasiRNAs) in addition to tasiR-ARFs, with expanded potential targets. Our results indicate that the evolutionary alteration of sRNA expression levels and the rewiring of sRNA-regulatory networks are important mechanisms underlying stress adaptation. We also identified sRNAs that are involved in salt and/or drought tolerance and nutrient homeostasis as possible contributors to mangrove success in stressful environments.

  10. Extremal surface barriers

    NASA Astrophysics Data System (ADS)

    Engelhardt, Netta; Wall, Aron C.

    2014-03-01

    We present a generic condition for Lorentzian manifolds to have a barrier that limits the reach of boundary-anchored extremal surfaces of arbitrary dimension. We show that any surface with nonpositive extrinsic curvature is a barrier, in the sense that extremal surfaces cannot be continuously deformed past it. Furthermore, the outermost barrier surface has nonnegative extrinsic curvature. Under certain conditions, we show that the existence of trapped surfaces implies a barrier, and conversely. In the context of AdS/CFT, these barriers imply that it is impossible to reconstruct the entire bulk using extremal surfaces. We comment on the implications for the firewall controversy.

  11. Solar and stellar photospheric abundances

    NASA Astrophysics Data System (ADS)

    Allende Prieto, Carlos

    2016-12-01

    The determination of photospheric abundances in late-type stars from spectroscopic observations is a well-established field, built on solid theoretical foundations. Improving those foundations to refine the accuracy of the inferred abundances has proven challenging, but progress has been made. In parallel, developments on instrumentation, chiefly regarding multi-object spectroscopy, have been spectacular, and a number of projects are collecting large numbers of observations for stars across the Milky Way and nearby galaxies, promising important advances in our understanding of galaxy formation and evolution. After providing a brief description of the basic physics and input data involved in the analysis of stellar spectra, a review is made of the analysis steps, and the available tools to cope with large observational efforts. The paper closes with a quick overview of relevant ongoing and planned spectroscopic surveys, and highlights of recent research on photospheric abundances.

  12. Robust Abundance Estimation in Animal Abundance Surveys with Imperfect Detection

    EPA Science Inventory

    Surveys of animal abundance are central to the conservation and management of living natural resources. However, detection uncertainty complicates the sampling process of many species. One sampling method employed to deal with this problem is depletion (or removal) surveys in whi...

  13. The solar abundance of beryllium

    NASA Technical Reports Server (NTRS)

    Ross, J. E.; Aller, L. H.

    1974-01-01

    The solar abundance of beryllium is deduced from high-resolution Kitt Peak observations of the 3130.43- and 3131.08-A lines of Be II interpreted by the method of spectrum synthesis. The results are in good agreement with those previously obtained by Grevesse (1968) and by Hauge and Engvold (1968) and indicate that in the photospheric layers, beryllium is depleted below the chondritic value by a factor of about two. It is found that the beryllium abundance is equal to logN(Be)/N(H) + 12 = 1.08 plus or minus 0.05.

  14. Extreme environments and exobiology.

    PubMed

    Friedmann, E I

    1993-01-01

    Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.

  15. Survival of extreme opinions

    NASA Astrophysics Data System (ADS)

    Hsu, Jiann-wien; Huang, Ding-wei

    2009-12-01

    We study the survival of extreme opinions in various processes of consensus formation. All the opinions are treated equally and subjected to the same rules of changing. We investigate three typical models to reach a consensus in each case: (A) personal influence, (B) influence from surroundings, and (C) influence to surroundings. Starting with uniformly distributed random opinions, our calculated results show that the extreme opinions can survive in both models (A) and (B), but not in model (C). We obtain a conclusion that both personal influence and passive adaptation to the environment are not sufficient enough to eradicate all the extreme opinions. Only the active persuasion to change the surroundings eliminates the extreme opinions completely.

  16. Extreme environments and exobiology

    NASA Technical Reports Server (NTRS)

    Friedmann, E. I.

    1993-01-01

    Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.

  17. USACE Extreme Sea levels

    DTIC Science & Technology

    2014-03-14

    into Extreme Water Level Characterization 9 September 2013 Attendees: Heidi Moritz, Kate White, Jonathan Simm, Robert Nicholls, Peter Hawkes...adaptation. Robert Nicholls raised the question of how well do we feel that we understand the present extreme climate? We should start with this area...the peer-review and acceptance process for a journal paper. Robert suggested that most of the papers which are needed for an analysis today may be

  18. In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes

    PubMed Central

    ElHefnawi, Mahmoud; Kim, TaeKyu; Kamar, Mona A.; Min, Saehong; Hassan, Nafisa M.; El-Ahwany, Eman; Kim, Heeyoung; Zada, Suher; Amer, Marwa; Windisch, Marc P.

    2016-01-01

    RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide

  19. Dexamethasone-conjugated polyethylenimine/MIF siRNA complex regulation of particulate matter-induced airway inflammation.

    PubMed

    Choi, Moonhwan; Lee, Minhyung; Rhim, Taiyoun

    2013-10-01

    Inhalation of airborne particulate matter (PM), such as silicon dioxide (SiO2) and titanium dioxide (TiO2), induces acute lung inflammation. siRNA therapy has been proposed as a method to repair acute lung inflammation. To determine whether DEXA-PEI/MIF siRNA contributes to SiO2-induced acute lung inflammation repair, we administered Dexa-PEI/MIF siRNA in SiO2-treated Beas-2b cells and instilled DEXA-PEI-MIF siRNA intratracheally in mice with SiO2-induced acute lung inflammation. Using genetic (MIF mRNA RT-PCR), histological (H&E and PAS) and immunohistochemical (MIF and Muc5ac) analyses, we estimated the acute lung inflammation in Beas-2b cells and BALB/c mice. Cells and mice treated with SiO2 particles demonstrated pulmonary inflammation. DEXA-PEI/MIF siRNA restricted the extent of the pulmonary inflammation reaction to SiO2 in cells and mice. In case of SiO2-treated Beas-2b cells, only DEXA-PEI treatment failed to effectively regulate MIF mRNA release. At the same time, only DEXA-PEI treatment adjusted the amount of MIF mRNA to some extent in SiO2-treated BALB/c mice. siRNA treatment did not markedly control MIF mRNA release in mice. We also observed that the amount of MIF mRNA was decreased in cells and mice treated with DEXA-PEI/MIF siRNA. The increase of MIF mRNA markedly increased Muc5ac; in contrast, the decrease of MIF mRNA using DEXA-PEI/MIF siRNA effectively lowered Muc5ac in SiO2-treated cells and mice. These results suggest that DEXA-PEI plays a role in delivering siRNA to the nucleus as a carrier and limits the extent of acute lung inflammation. MIF siRNA also contributed to the reparative lung response in SiO2-induced pulmonary inflammation.

  20. THE SOLAR FLARE IRON ABUNDANCE

    SciTech Connect

    Phillips, K. J. H.; Dennis, B. R. E-mail: Brian.R.Dennis@nasa.gov

    2012-03-20

    The abundance of iron is measured from emission line complexes at 6.65 keV (Fe line) and 8 keV (Fe/Ni line) in RHESSI X-ray spectra during solar flares. Spectra during long-duration flares with steady declines were selected, with an isothermal assumption and improved data analysis methods over previous work. Two spectral fitting models give comparable results, viz., an iron abundance that is lower than previous coronal values but higher than photospheric values. In the preferred method, the estimated Fe abundance is A(Fe) = 7.91 {+-} 0.10 (on a logarithmic scale, with A(H) = 12) or 2.6 {+-} 0.6 times the photospheric Fe abundance. Our estimate is based on a detailed analysis of 1898 spectra taken during 20 flares. No variation from flare to flare is indicated. This argues for a fractionation mechanism similar to quiet-Sun plasma. The new value of A(Fe) has important implications for radiation loss curves, which are estimated.

  1. A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects

    PubMed Central

    Bramsen, Jesper B.; Pakula, Malgorzata M.; Hansen, Thomas B.; Bus, Claus; Langkjær, Niels; Odadzic, Dalibor; Smicius, Romualdas; Wengel, Suzy L.; Chattopadhyaya, Jyoti; Engels, Joachim W.; Herdewijn, Piet; Wengel, Jesper; Kjems, Jørgen

    2010-01-01

    Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2–8 of either siRNA strand counting from the 5′-end) and complementary sequences in the 3′UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA–target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low. PMID:20453030

  2. Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

    NASA Astrophysics Data System (ADS)

    Xiao, Yuling; Jaskula-Sztul, Renata; Javadi, Alireza; Xu, Wenjin; Eide, Jacob; Dammalapati, Ajitha; Kunnimalaiyaan, Muthusamy; Chen, Herbert; Gong, Shaoqin

    2012-10-01

    A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The

  3. Actinide abundances in ordinary chondrites

    USGS Publications Warehouse

    Hagee, B.; Bernatowicz, T.J.; Podosek, F.A.; Johnson, M.L.; Burnett, D.S.; Tatsumoto, M.

    1990-01-01

    Measurements of 244Pu fission Xe, U, Th, and light REE (LREE) abundances, along with modal petrographic determinations of phosphate abundances, were carried out on equilibrated ordinary chondrites in order to define better the solar system Pu abundance and to determine the degree of variation of actinide and LREE abundances. Our data permit comparison of the directly measured Pu/ U ratio with that determined indirectly as (Pu/Nd) ?? (Nd/U) assuming that Pu behaves chemically as a LREE. Except for Guaren??a, and perhaps H chondrites in general, Pu concentrations are similar to that determined previously for St. Se??verin, although less precise because of higher trapped Xe contents. Trapped 130Xe 136Xe ratios appear to vary from meteorite to meteorite, but, relative to AVCC, all are similar in the sense of having less of the interstellar heavy Xe found in carbonaceous chondrite acid residues. The Pu/U and Pu/Nd ratios are consistent with previous data for St. Se??verin, but both tend to be slightly higher than those inferred from previous data on Angra dos Reis. Although significant variations exist, the distribution of our Th/U ratios, along with other precise isotope dilution data for ordinary chondrites, is rather symmetric about the CI chondrite value; however, actinide/(LREE) ratios are systematically lower than the CI value. Variations in actinide or LREE absolute and relative abundances are interpreted as reflecting differences in the proportions and/or compositions of more primitive components (chondrules and CAI materials?) incorporated into different regions of the ordinary chondrite parent bodies. The observed variations of Th/U, Nd/U, or Ce/U suggest that measurements of Pu/U on any single equilibrated ordinary chondrite specimen, such as St. Se??verin, should statistically be within ??20-30% of the average solar system value, although it is also clear that anomalous samples exist. ?? 1990.

  4. Electronics for Extreme Environments

    NASA Technical Reports Server (NTRS)

    Patel, J. U.; Cressler, J.; Li, Y.; Niu, G.

    2001-01-01

    Most of the NASA missions involve extreme environments comprising radiation and low or high temperatures. Current practice of providing friendly ambient operating environment to electronics costs considerable power and mass (for shielding). Immediate missions such as the Europa orbiter and lander and Mars landers require the electronics to perform reliably in extreme conditions during the most critical part of the mission. Some other missions planned in the future also involve substantial surface activity in terms of measurements, sample collection, penetration through ice and crust and the analysis of samples. Thus it is extremely critical to develop electronics that could reliably operate under extreme space environments. Silicon On Insulator (SOI) technology is an extremely attractive candidate for NASA's future low power and high speed electronic systems because it offers increased transconductance, decreased sub-threshold slope, reduced short channel effects, elimination of kink effect, enhanced low field mobility, and immunity from radiation induced latch-up. A common belief that semiconductor devices function better at low temperatures is generally true for bulk devices but it does not hold true for deep sub-micron SOI CMOS devices with microscopic device features of 0.25 micrometers and smaller. Various temperature sensitive device parameters and device characteristics have recently been reported in the literature. Behavior of state of the art technology devices under such conditions needs to be evaluated in order to determine possible modifications in the device design for better performance and survivability under extreme environments. Here, we present a unique approach of developing electronics for extreme environments to benefit future NASA missions as described above. This will also benefit other long transit/life time missions such as the solar sail and planetary outposts in which electronics is out open in the unshielded space at the ambient space

  5. Extreme Programming: Maestro Style

    NASA Technical Reports Server (NTRS)

    Norris, Jeffrey; Fox, Jason; Rabe, Kenneth; Shu, I-Hsiang; Powell, Mark

    2009-01-01

    "Extreme Programming: Maestro Style" is the name of a computer programming methodology that has evolved as a custom version of a methodology, called extreme programming that has been practiced in the software industry since the late 1990s. The name of this version reflects its origin in the work of the Maestro team at NASA's Jet Propulsion Laboratory that develops software for Mars exploration missions. Extreme programming is oriented toward agile development of software resting on values of simplicity, communication, testing, and aggressiveness. Extreme programming involves use of methods of rapidly building and disseminating institutional knowledge among members of a computer-programming team to give all the members a shared view that matches the view of the customers for whom the software system is to be developed. Extreme programming includes frequent planning by programmers in collaboration with customers, continually examining and rewriting code in striving for the simplest workable software designs, a system metaphor (basically, an abstraction of the system that provides easy-to-remember software-naming conventions and insight into the architecture of the system), programmers working in pairs, adherence to a set of coding standards, collaboration of customers and programmers, frequent verbal communication, frequent releases of software in small increments of development, repeated testing of the developmental software by both programmers and customers, and continuous interaction between the team and the customers. The environment in which the Maestro team works requires the team to quickly adapt to changing needs of its customers. In addition, the team cannot afford to accept unnecessary development risk. Extreme programming enables the Maestro team to remain agile and provide high-quality software and service to its customers. However, several factors in the Maestro environment have made it necessary to modify some of the conventional extreme

  6. Immunostimulatory motifs enhance antiviral siRNAs targeting highly pathogenic avian influenza H5N1.

    PubMed

    Stewart, Cameron R; Karpala, Adam J; Lowther, Sue; Lowenthal, John W; Bean, Andrew G

    2011-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi)-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN) production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (si)RNAs in chicken cells (i) mode of synthesis and (ii) nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5'-UGUGU-3' is a key determinant in inducing high levels of expression of IFN-α, -β, -λ and interleukin 1-β in chicken cells. Positioning of this 5'-UGUGU-3' motif at the 5'-end of the sense strand of siRNAs, but not the 3'-end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257) tagged with 5'-UGUGU-3' induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics.

  7. Polyphosphoester nanoparticles as biodegradable platform for delivery of multiple drugs and siRNA

    PubMed Central

    Elzeny, Hadeel; Zhang, Fuwu; Ali, Esraa N; Fathi, Heba A; Zhang, Shiyi; Li, Richen; El-Mokhtar, Mohamed A; Hamad, Mostafa A; Wooley, Karen L; Elsabahy, Mahmoud

    2017-01-01

    Delivery of multiple therapeutics and/or diagnostic agents to diseased tissues is challenging and necessitates the development of multifunctional platforms. Among the various strategies for design of multifunctional nanocarriers, biodegradable polyphosphoester (PPE) polymers have been recently synthesized via a rapid and simple synthetic strategy. In addition, the chemical structure of the polymer could be tuned to form nanoparticles with varying surface chemistries and charges, which have shown exceptional safety and biocompatibility as compared to several commercial agents. The purpose of this study was to exploit a mixture of PPE nanoparticles of cationic and neutral surface charges for multiple delivery of anticancer drugs (ie, sorafenib and paclitaxel) and nucleic acids (ie, siRNA). Cationic PPE polymers could efficiently complex siRNA, and the stability of the nanoparticles could be maintained in physiological solutions and upon freeze-drying and were able to deliver siRNA in vivo when injected intravenously in mice. Commercially available cationic polyethylenimine polymer had LD50 of ca. 61.7 mg/kg in mice, whereas no animal died after injection of the cationic PPE polymer at a dose of >130 mg/kg. Neutral PPE nanoparticles were able to encapsulate two hydrophobic drugs, namely, sorafenib and paclitaxel, which are commonly used for the treatment of hepatocellular carcinoma. Mixing the neutral and cationic PPE nanoparticles did not result in any precipitation, and the size characteristics of both types of nanoparticles were maintained. Hence, PPE polymers might have potential for the delivery of multiple drugs and diagnostic agents to diseased tissues via simple synthesis of the individual polymers and assembly into nanoparticles that can host several drugs while being mixed in the same administration set, which is of importance for industrial and clinical development. PMID:28260861

  8. Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses

    PubMed Central

    Charbonnel, Cyril; Elvira-Matelot, Emilie; Bochnakian, Aurore; Comella, Pascale; Mallory, Allison C.; Lepère, Gersende; Sáez-Vásquez, Julio; Vaucheret, Hervé

    2015-01-01

    Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant–virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses. PMID:26696443

  9. Efficient silencing of EGFP reporter gene with siRNA delivered by asymmetrical N4,N9-diacyl spermines.

    PubMed

    Metwally, Abdelkader A; Reelfs, Olivier; Pourzand, Charareh; Blagbrough, Ian S

    2012-07-02

    It is important to obtain structure-activity relationship (SAR) data across cationic lipids for the self-assembly and nonviral intracellular delivery of siRNA. The aims of this work are to carry out a SAR study on the efficiency of asymmetrical N(4),N(9)-diacyl spermines in siRNA delivery and EGFP reporter gene silencing, with comparisons to selected mixtures composed of symmetrical N(4),N(9)-diacyl spermines. Another important aim of these studies is to quantify the changes in cell viability, assayed with alamarBlue, as a function of lipid structure. Therefore, we have designed, synthesized, purified, and assayed novel cationic lipids that are asymmetrical lipopolyamines based on spermine. Flow cytometry and fluorescence microscopy in an EGFP stably transfected HeLa cell line, measuring both delivery of fluorescently tagged siRNAs and silencing the EGFP signal, allowed quantitation of the differences between asymmetrical cationic lipids, mixtures of their symmetrical counterparts, and comparison with commercial nonviral delivery agents. Intracellular delivery of siRNA and gene silencing by siRNA differ with different hydrophobic domains. In these asymmetrical N(4),N(9)-diacyl spermines, lipids that enhance siRNA uptake do not necessarily enhance siRNA-induced inhibition of gene expression: C18 and longer saturated chains promote uptake, while more unsaturated C18 chains promote gene silencing. These properties are efficiently demonstrated in a new nontoxic cationic lipid siRNA vector, N(4)-linoleoyl-N(9)-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), which is also shown to be comparable with or superior to TransIT-TKO and Lipofectamine 2000.

  10. Functionalized, biodegradable hydrogels for control over sustained and localized siRNA delivery to incorporated and surrounding cells.

    PubMed

    Nguyen, Khanh; Dang, Phuong Ngoc; Alsberg, Eben

    2013-01-01

    Currently, the most severe limitation to applying RNA interference technology is delivery, including localizing the molecules to a specific site of interest to target a specific cell population and sustaining the presentation of these molecules for a controlled period of time. In this study, we engineered a functionalized, biodegradable system created by covalent incorporation of cationic linear polyethyleneimine (LPEI) into photocrosslinked dextran (DEX) hydrogels through a biodegradable ester linkage. The key innovation of this system is that control over the sustained release of short interference RNA (siRNA) was achieved, as LPEI could electrostatically interact with siRNA to maintain siRNA within the hydrogels and degradation of the covalent ester linkages between the LPEI and the hydrogels led to tunable release of LPEI/siRNA complexes over time. The covalent conjugation of LPEI did not affect the swelling or degradation properties of the hydrogels, and the addition of siRNA and LPEI had minimal effect on their mechanical properties. These hydrogels exhibited low cytotoxicity against human embryonic kidney 293 cells (HEK293). The release profiles could be tailored by varying DEX (8 and 12% w/w) and LPEI (0, 5, 10 μg/100 μl gel) concentrations with nearly 100% cumulative release achieved at day 9 (8% w/w gel) and day 17 (12% w/w gel). The released siRNA exhibited high bioactivity with cells surrounding and inside the hydrogels over an extended time period. This controllable and sustained siRNA delivery hydrogel system that permits tailored siRNA release profiles may be valuable to guide cell fate for regenerative medicine and other therapeutic applications such as cancer treatment.

  11. A laminar flow electroporation system for efficient DNA and siRNA delivery.

    PubMed

    Wei, Zewen; Zhao, Deyao; Li, Xueming; Wu, Mengxi; Wang, Wei; Huang, Huang; Wang, Xiaoxia; Du, Quan; Liang, Zicai; Li, Zhihong

    2011-08-01

    By introducing a hydrodynamic mechanism into a microfluidics-based electroporation system, we developed a novel laminar flow electroporation system with high performance. The laminar buffer flow implemented in the system separated the cell suspension flow from the electrodes, thereby excluding many unfavorable effects due to electrode reaction during electroporation, such as hydrolysis, bubble formation, pH change, and heating. Compared to conventional microfluidic electroporation systems, these improvements significantly enhanced transfection efficiency and cell viability. Furthermore, successful electrotransfection of plasmid DNA and, more importantly, synthetic siRNA, was demonstrated in several hard-to-transfect cell types using this system.

  12. Element abundances at high redshift

    NASA Technical Reports Server (NTRS)

    Meyer, David M.; Welty, D. E.; York, D. G.

    1989-01-01

    Abundances of Si(+), S(+), Cr(+), Mn(+), Fe(_), and Zn(+) are considered for two absorption-line systems in the spectrum of the QSO PKS 0528 - 250. Zinc and sulfur are underabundant, relative to H, by a factor of 10 compared to their solar and Galactic interstellar abundances. The silicon-, chromium-, iron-, and nickel-to-hydrogen ratios are less than the solar values and comparable to the local interstellar ratios. A straightforward interpretation is that nucleosynthesis in these high-redshift systems has led to only about one-tenth as much heavy production as in the gas clouds around the sun, and that the amount of the observed underabundances attributable to grain depletion is small. The dust-to-gas ratio in these clouds is less than 8 percent of the Galactic value.

  13. Coronal abundances and their variation

    NASA Technical Reports Server (NTRS)

    Saba, Julia L. R.

    1994-01-01

    This contract supports the investigation of elemental abundances in the solar corona, principally through analysis of high-resolution software X-ray spectra from the Flat Crystal Spectrometer on NASA's Solar Maximum Mission. The goals of the study are a characterization of the mean values of relative abundances of elements accessible in the FCS data, and information on the extent and circumstances of their variability. This report is a summation of the data analysis and reporting activities which occurred since the last report, submitted two months early, in April 1994, to facilitate evaluation of the first year's progress for contract renewal. Hence this report covers the period 15 April 1994 - 15 December 1994. A list of publications resulting from this research is included.

  14. HMGB1 siRNA can reduce damage to retinal cells induced by high glucose in vitro and in vivo

    PubMed Central

    Jiang, Shuang; Chen, Xiaolong

    2017-01-01

    Background Diabetic retinopathy (DR), one of the most common complications of late-phase diabetes, is associated with many risk factors, among which continuous low-grade inflammation is one of the principal ones. As such, lowering inflammation levels and maintain the viability of human retinal endothelial cells (HRECs) are critical for DR therapy. HMGB1 is a well-known proinflammatory cytokine. However, whether HMGB1 small interfering RNA (siRNA) can protect retina cells under a high-glucose environment from morphological changes and functional abnormalities remain undetermined. We aimed to investigate the effect of HMGB1 siRNA on retinal cells in DR. Materials and methods A total of 80 adult Wistar rats were randomly divided into four groups (n=20 each): normal control, diabetes mellitus (DM), scrambled (Scr) siRNA, and HMGB1 siRNA. Rats in the DM, Scr siRNA, and siRNA groups were established by intraperitoneal injection of streptozotocin. At 16 weeks after injection, rats in the siRNA and Scr-siRNA groups were intravitreally injected with 2 μL HMGB1 siRNA and 2 μL Scr-siRNA, while rats in the control and DM groups were intravitreally injected with the same dose of sterile saline. At 1 week after injections, we performed the following experiments. Immunohistochemical staining and real-time quantitative polymerase chain reaction were performed to test HMGB1 protein and messenger RNA expression in retinas. We performed TUNEL assays to detect retinal cell apoptosis and electroretinography to detect retinal function. In HRECs treated with high glucose, proliferation, morphology, apoptosis, super-oxide dismutase (SOD), and reactive oxygen species production were detected. Western blot was applied to determine the expressions of HMGB1 and its related protein and apoptosis protein. Results Intravitreal injection of HMGB1 siRNA reduced protein and messenger RNA expression of HMGB1 (both P<0.05). Intravitreal injection of HMGB1 siRNA reduced apoptosis of retinal cells (P

  15. Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs

    PubMed Central

    2011-01-01

    Background Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection. Results Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved

  16. The CALIFA survey: Oxygen abundances

    NASA Astrophysics Data System (ADS)

    Sánchez, S. F.; Aff001

    We present here the last results we obtained on the spatial resolved analysis of the ionized gas of disk-dominated galaxies based on CALIFA data. CALIFA is an ongoing IFS survey of galaxies in the Local Univese (0.005 < z < 0.03) that has already obtained spectroscopic information up to ~2.5r e with a spatial resolution better than ~1 kpc for a total number of an statiscal sample of galaxies of different morphological types, covering the CM-diagram up to Mr<-18 mag. With nearly 2000 spectra obtained for each galaxy, CALIFA offer one of the best IFU data to study the starformation histories and chemical enrichment of galaxies. In this article we focus on the main results based on the analysis of the oxygen abundances based on the study of ionized gas in H ii regions and individual spaxels, and their relations with the global properties of galaxies. In summary we have found that: (1) the -Z relation does not present a secondary relation with the star-formation rate, when the abundance is measured at the effective radius; (2) the oxygen abundance present a strong correlation with the stellar surface density (Σ-Z relation); (3) the oxygen abundance profiles present three well defined regimes, (a) an overall negative radial gradient, between 0.5-2 r e , with a characteristic slope of α O/H ~-0.1 dex/r e , (b) an universal flatenning beyond >2r e and (c) an inner drop at <0.5r e which presence depends on the mass. All these results indicates that disk-galaxies present an overall inside-out growth, although with clear deviations from this simple scenario.

  17. The solar abundance of thulium

    NASA Technical Reports Server (NTRS)

    Ross, J. E.; Aller, L. H.

    1974-01-01

    Consideration of one relatively unblended line of the solar spectrum, namely, the 3131.258-A line of Tm II, which yields a thulium abundance of 0.80 plus or minus 0.10 with the Corliss and Bozman (1962) f-value. The uncertainty of this figure is discussed in conjunction with the contradictory findings of some other investigators. The need for further detailed study of the lanthanides by the method of spectrum synthesis is pointed out.

  18. Adventure and Extreme Sports.

    PubMed

    Gomez, Andrew Thomas; Rao, Ashwin

    2016-03-01

    Adventure and extreme sports often involve unpredictable and inhospitable environments, high velocities, and stunts. These activities vary widely and include sports like BASE jumping, snowboarding, kayaking, and surfing. Increasing interest and participation in adventure and extreme sports warrants understanding by clinicians to facilitate prevention, identification, and treatment of injuries unique to each sport. This article covers alpine skiing and snowboarding, skateboarding, surfing, bungee jumping, BASE jumping, and whitewater sports with emphasis on epidemiology, demographics, general injury mechanisms, specific injuries, chronic injuries, fatality data, and prevention. Overall, most injuries are related to overuse, trauma, and environmental or microbial exposure.

  19. Occult fractures of extremities.

    PubMed

    Ahn, Joong Mo; El-Khoury, Georges Y

    2007-05-01

    Recent advances in cross-sectional imaging, particularly in CT and MR imaging, have given these modalities a prominent role in the diagnosis of fractures of the extremities. This article describes the clinical application and imaging features of cross-sectional imaging (CT and MR imaging) in the evaluation of patients who have occult fractures of the extremities. Although CT or MR imaging is not typically required for evaluation of acute fractures, these modalities could be helpful in the evaluation of the occult osseous injuries in which radiographic findings are equivocal or inconclusive.

  20. Chlorine Abundances in Martian Meteorites

    NASA Technical Reports Server (NTRS)

    Bogard, D.D.; Garrison, D.H.; Park, J.

    2009-01-01

    Chlorine measurements made in martian surface rocks by robotic spacecraft typically give Chlorine (Cl) abundances of approximately 0.1-0.8%. In contrast, Cl abundances in martian meteorites appear lower, although data is limited, and martian nakhlites were also subjected to Cl contamination by Mars surface brines. Chlorine abundances reported by one lab for whole rock (WR) samples of Shergotty, ALH77005, and EET79001 range 108-14 ppm, whereas Cl in nakhlites range 73-1900 ppm. Measurements of Cl in various martian weathering phases of nakhlites varied 0.04-4.7% and reveal significant concentration of Cl by martian brines Martian meteorites contain much lower Chlorine than those measured in martian surface rocks and give further confirmation that Cl in these surface rocks was introduced by brines and weathering. It has been argued that Cl is twice as effective as water in lowering the melting point and promoting melting at shallower martian depths, and that significant Cl in the shergottite source region would negate any need for significant water. However, this conclusion was based on experiments that utilized Cl concentrations more analogous to martian surface rocks than to shergottite meteorites, and may not be applicable to shergottites.

  1. Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia

    PubMed Central

    Carradec, Quentin; Götz, Ulrike; Arnaiz, Olivier; Pouch, Juliette; Simon, Martin; Meyer, Eric; Marker, Simone

    2015-01-01

    In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3′-to-5′ and 5′-to-3′ transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism. PMID:25593325

  2. Targeted exosome-mediated delivery of opioid receptor Mu siRNA for the treatment of morphine relapse

    PubMed Central

    Liu, Yuchen; Li, Dameng; Liu, Zhengya; Zhou, Yu; Chu, Danping; Li, Xihan; Jiang, Xiaohong; Hou, Dongxia; Chen, Xi; Chen, Yuda; Yang, Zhanzhao; Jin, Ling; Jiang, Waner; Tian, Chenfei; Zhou, Geyu; Zen, Ke; Zhang, Junfeng; Zhang, Yujing; Li, Jing; Zhang, Chen-Yu

    2015-01-01

    Cell-derived exosomes have been demonstrated to be efficient carriers of small RNAs to neighbouring or distant cells, highlighting the preponderance of exosomes as carriers for gene therapy over other artificial delivery tools. In the present study, we employed modified exosomes expressing the neuron-specific rabies viral glycoprotein (RVG) peptide on the membrane surface to deliver opioid receptor mu (MOR) siRNA into the brain to treat morphine addiction. We found that MOR siRNA could be efficiently packaged into RVG exosomes and was associated with argonaute 2 (AGO2) in exosomes. These exosomes efficiently and specifically delivered MOR siRNA into Neuro2A cells and the mouse brain. Functionally, siRNA-loaded RVG exosomes significantly reduced MOR mRNA and protein levels. Surprisingly, MOR siRNA delivered by the RVG exosomes strongly inhibited morphine relapse via the down-regulation of MOR expression levels. In conclusion, our results demonstrate that targeted RVG exosomes can efficiently transfer siRNA to the central nervous system and mediate the treatment of morphine relapse by down-regulating MOR expression levels. Our study provides a brand new strategy to treat drug relapse and diseases of the central nervous system. PMID:26633001

  3. Nek2 siRNA therapy using a portal venous port-catheter system for liver metastasis in pancreatic cancer.

    PubMed

    Kokuryo, Toshio; Hibino, Shigeru; Suzuki, Kazushi; Watanabe, Katsutaka; Yokoyama, Yukihiro; Nagino, Masato; Senga, Takeshi; Hamaguchi, Michinari

    2016-09-01

    Nek2 (NIMA-related kinase 2) is a serine-threonine kinase and human homolog of the mitotic regulator NIMA of Aspergillus nidulan. We reported the efficiency of Nek2 siRNA in several cancer xenograft models using cholangiocarcinoma, breast cancer and colorectal cancer. Pancreatic cancer is difficult to treat due to its rapid progression and resistance to chemotherapy. Novel treatments are urgently required to improve survival in pancreatic cancer, and siRNA are a promising therapeutic option. However, finding an in vivo drug delivery system of siRNA remains a major problem for clinical application. In this study, the overexpression of Nek2 was identified in pancreatic cancer cell lines. Nek2 siRNA inhibited tumor growth in a subcutaneous xenograft mouse model of pancreatic cancer, prolonged the survival time in an intraperitoneal xenograft mouse model and efficiently prevented the progression of liver metastasis using a portal venous port-catheter system. Taken together, Nek2 is an effective therapeutic target in pancreatic cancer. An adequate delivery system is considered important in treating advanced pancreatic cancer, such as peritoneal dissemination and liver metastasis. Further investigations are required on the safety and side effects of the portal venous port-catheter system. We hope that Nek2 siRNA will be a novel therapeutic strategy for pancreatic cancer with liver metastasis and peritoneal dissemination.

  4. Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia.

    PubMed

    Carradec, Quentin; Götz, Ulrike; Arnaiz, Olivier; Pouch, Juliette; Simon, Martin; Meyer, Eric; Marker, Simone

    2015-02-18

    In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3'-to-5' and 5'-to-3' transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism.

  5. The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects

    PubMed Central

    Kamola, Piotr J.; Nakano, Yuko; Takahashi, Tomoko; Wilson, Paul A.; Ui-Tei, Kumiko

    2015-01-01

    RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA ‘specificity’ design rules. PMID:26657993

  6. Intranasal siRNA administration reveals IGF2 deficiency contributes to impaired cognition in Fragile X syndrome mice

    PubMed Central

    Pardo, Marta; Cheng, Yuyan; Velmeshev, Dmitry; Magistri, Marco; Martinez, Ana; Faghihi, Mohammad A.; Jope, Richard S.; Beurel, Eleonore

    2017-01-01

    Molecular mechanisms underlying learning and memory remain imprecisely understood, and restorative interventions are lacking. We report that intranasal administration of siRNAs can be used to identify targets important in cognitive processes and to improve genetically impaired learning and memory. In mice modeling the intellectual deficiency of Fragile X syndrome, intranasally administered siRNA targeting glycogen synthase kinase-3β (GSK3β), histone deacetylase-1 (HDAC1), HDAC2, or HDAC3 diminished cognitive impairments. In WT mice, intranasally administered brain-derived neurotrophic factor (BDNF) siRNA or HDAC4 siRNA impaired learning and memory, which was partially due to reduced insulin-like growth factor-2 (IGF2) levels because the BDNF siRNA– or HDAC4 siRNA–induced cognitive impairments were ameliorated by intranasal IGF2 administration. In Fmr1–/– mice, hippocampal IGF2 was deficient, and learning and memory impairments were ameliorated by IGF2 intranasal administration. Therefore intranasal siRNA administration is an effective means to identify mechanisms regulating cognition and to modulate therapeutic targets. PMID:28352664

  7. Balancing polymer hydrophobicity for ligand presentation and siRNA delivery in dual function CXCR4 inhibiting polyplexes.

    PubMed

    Wang, Y; Li, J; Chen, Y; Oupický, D

    2015-07-01

    In the present study, a series of copolymers (PAMD-Ch) was synthesized by grafting polymeric Plerixafor/AMD3100 (PAMD) with different amounts of cholesterol and the effect of cholesterol modification on siRNA delivery was investigated. PAMD-Ch/siRNA polyplexes exhibited improved colloidal and enzymatic stability when compared with PAMD/siRNA polyplexes containing no cholesterol. PAMD-Ch with low (17 wt%) and medium (25 wt%) cholesterol content exhibited CXCR4 antagonism comparable to unmodified PAMD. Cholesterol modification increased cell uptake of siRNA polyplexes and significantly decreased sensitivity of siRNA transfection to the presence of serum. When used to deliver anticancer siRNA against polo-like kinase 1 (PLK1), polyplexes based on PAMD-Ch with 17 wt% cholesterol exhibited the highest cancer cell killing activity both in serum-free and serum-containing conditions. Overall, the results of this study validate cholesterol modified PAMD as dual-function delivery vectors suitable for efficient delivery of anticancer siRNA and simultaneous CXCR4 inhibition for combined anticancer therapies.

  8. Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome.

    PubMed

    Zhao, Yun-Chun; Zhang, Li; Feng, Shi-Sen; Hong, Lu; Zheng, Hai-Li; Chen, Li-Li; Zheng, Xiao-Ling; Ye, Yi-Qing; Zhao, Meng-Dan; Wang, Wen-Xi; Zheng, Cai-Hong

    A novel cationic cholesterol derivative-based small interfering RNA (siRNA) interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-chems) liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100-200 nm and zeta potentials of 40-50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells.

  9. Novel polyacrylate-based cationic nanoparticles for survivin siRNA delivery combined with mitoxantrone for treatment of breast cancer.

    PubMed

    Arami, Sanam; Mahdavi, Majid; Rashidi, Mohammad Reza; Fathi, Marziyeh; Hejazi, Mohammad-Saeid; Samadi, Nasser

    2016-11-01

    As a gene delivery method in breast cancer therapy, knocking down the undesired genes in the cancerous cells would be promising. Inhibitors of Apoptosis Protein (IAP) family genes are some of the genes whose responsibility is inhibition of apoptosis in cells. Silencing these genes seems to be helpful directing the tumor cells to death. siRNA sequence designed against survivin anti-apoptotic gene can play this role if carried to the cytoplasm. Here we prepared a positive charged biocompatible nano-sized particle made up of a Fe3O4 core covered respectively by polyacrylate (PA) and polyethyleneimine (PEI) layer, which could successfully deliver the siRNA into the MCF-7 cells. The particle structure was checked and having less than 50 nm diameter in size, positive charge and, safety towards MCF-7 cells besides being able to form nanoplexes with the siRNA strand helps it entering into the biologic assays part. The siRNA delivery evaluated via flowcytometry. Apoptosis induction was determined by DAPI staining. The efficiency of survivin gene knockdown was evaluated in mRNA and protein levels using Real time PCR and western blotting methods. Overall, the Fe3O4-PA-PEI nanoparticles can deliver siRNA effectively into the cytoplasm of the MCF-7 breast cancer cells and induce apoptosis.

  10. Intramyocardial Injection of siRNAs Can Efficiently Establish Myocardial Tissue-Specific Renalase Knockdown Mouse Model

    PubMed Central

    Liu, Ju; Zhang, Hui

    2016-01-01

    Ischaemia/reperfusion (I/R) injury will cause additional death of cardiomyocytes in ischaemic heart disease. Recent studies revealed that renalase was involved in the I/R injury. So, the myocardial tissue-specific knockdown mouse models were needed for the investigations of renalase. To establish the mouse models, intramyocardial injection of siRNAs targeting renalase was performed in mice. The wild distribution and high transfection efficiency of the siRNAs were approved. And the renalase expression was efficiently suppressed in myocardial tissue. Compared with the high cost, time consumption, and genetic compensation risk of the Cre/loxP technology, RNA interference (RNAi) technology is much cheaper and less time-consuming. Among the RNAi technologies, injection of siRNAs is safer than virus. And considering the properties of the I/R injury mouse models, the efficiency and durability of injection with siRNAs are acceptable for the studies. Altogether, intramyocardial injection of siRNAs targeting renalase is an economical, safe, and efficient method to establish myocardial tissue-specific renalase knockdown mouse models. PMID:27868059

  11. Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome

    PubMed Central

    Zhao, Yun-Chun; Zhang, Li; Feng, Shi-Sen; Hong, Lu; Zheng, Hai-Li; Chen, Li-Li; Zheng, Xiao-Ling; Ye, Yi-Qing; Zhao, Meng-Dan; Wang, Wen-Xi; Zheng, Cai-Hong

    2016-01-01

    A novel cationic cholesterol derivative-based small interfering RNA (siRNA) interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-chems) liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100–200 nm and zeta potentials of 40–50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells. PMID:27799771

  12. Balancing polymer hydrophobicity for ligand presentation and siRNA delivery in dual function CXCR4 inhibiting polyplexes

    PubMed Central

    Wang, Y.; Li, J.; Chen, Y.; Oupický, D.

    2015-01-01

    In the present study, a series of copolymers (PAMD-Ch) was synthesized by grafting polymeric Plerixafor/AMD3100 (PAMD) with different amounts of cholesterol and the effect of cholesterol modification on siRNA delivery was investigated. PAMD-Ch/siRNA polyplexes exhibited improved colloidal and enzymatic stability when compared with PAMD/siRNA polyplexes containing no cholesterol. PAMD-Ch with low (17 wt%) and medium (25 wt%) cholesterol content exhibited CXCR4 antagonism comparable to unmodified PAMD. Cholesterol modification increased cell uptake of siRNA polyplexes and significantly decreased sensitivity of siRNA transfection to the presence of serum. When used to deliver anticancer siRNA against polo-like kinase 1 (PLK1), polyplexes based on PAMD-Ch with 17 wt% cholesterol exhibited the highest cancer cell killing activity both in serum-free and serum-containing conditions. Overall, the results of this study validate cholesterol modified PAMD as dual-function delivery vectors suitable for efficient delivery of anticancer siRNA and simultaneous CXCR4 inhibition for combined anticancer therapies. PMID:26146552

  13. siRNA delivery targeting to the lung via agglutination-induced accumulation and clearance of cationic tetraamino fullerene

    NASA Astrophysics Data System (ADS)

    Minami, Kosuke; Okamoto, Koji; Doi, Kent; Harano, Koji; Noiri, Eisei; Nakamura, Eiichi

    2014-05-01

    The efficient treatment of lung diseases requires lung-selective delivery of agents to the lung. However, lung-selective delivery is difficult because the accumulation of micrometer-sized carriers in the lung often induces inflammation and embolization-related toxicity. Here we demonstrate a lung-selective delivery system of small interfering RNA (siRNA) by controlling the size of carrier vehicle in blood vessels. The carrier is made of tetra(piperazino)fullerene epoxide (TPFE), a water-soluble cationic tetraamino fullerene. TPFE and siRNA form sub-micrometer-sized complexes in buffered solution and these complexes agglutinate further with plasma proteins in the bloodstream to form micrometer-sized particles. The agglutinate rapidly clogs the lung capillaries, releases the siRNA into lung cells to silence expression of target genes, and is then cleared rapidly from the lung after siRNA delivery. We applied our delivery system to an animal model of sepsis, indicating the potential of TPFE-based siRNA delivery for clinical applications.

  14. An extreme Population II dwarf without lithium

    SciTech Connect

    Hobbs, L.M.; Thorburn, J.A.; Welty, D.E. Chicago, University, IL )

    1991-06-01

    G186 - 26 is an apparently normal, Population II dwarf with Fe/H = {minus} 2.9 and Te = 6220 K. A high-dispersion spectrogram of this extreme halo star recorded at the Li I 6707 line shows no detectable surface lithium, at an abundance upper limit N(Li) = 12 + log (Li/H) not greater than 1.23. In comparison with the uniform lithium abundance N(Li) of about 2.17 found previously in 11 other halo dwarfs with Fe/H less than about {minus}2.6, the minimum deficiency of surface lithium in G 186 {minus} 26 therefore exceeds a factor of 8. 19 refs.

  15. A single dose of EGLN1 siRNA yields increased erythropoiesis in nonhuman primates.

    PubMed

    Abrams, Marc T; Koser, Martin; Burchard, Julja; Strapps, Walter; Mehmet, Huseyin; Gindy, Marian; Zaller, Dennis; Sepp-Lorenzino, Laura; Stickens, Dominique

    2014-12-01

    Decreased production of erythropoietin (EPO) causes anemia in patients with chronic kidney disease, and recombinant human EPO is used to treat renal failure associated anemia. The liver, the main EPO-producing organ in utero, maintains the capacity to produce EPO in the adult but in insufficient quantities to restore hemoglobin levels to normal in patients with impaired renal function. Inhibition of prolyl-4-hydroxylase domain (PHD) proteins is known to cause an increase in EPO production through its effects on hypoxia inducible factor. Here, we utilized small interfering RNA (siRNA) targeting EGLN1, the gene encoding the PHD2 protein, to investigate the phenotypic consequences in nonhuman primates. A single, well-tolerated intravenous dose of an optimized EGLN1 siRNA encapsulated in a lipid nanoparticle formulation caused robust mRNA silencing in the liver, leading to increases in serum EPO and hemoglobin. The siRNA-induced erythropoiesis was dose-dependent and was sustained for at least 2 months. These data point to the potential for an RNA interference-based, liver-targeted therapeutic approach for the treatment of anemia.

  16. Inhalation Properties and Stability of Nebulized Naked siRNA Solution for Pulmonary Therapy.

    PubMed

    Tahara, Kohei; Hashimoto, Wakana; Takeuchi, Hirofumi

    2016-01-01

    The use of naked unmodified small interfering RNA (N-siRNA) without vector has previously been investigated as a pulmonary therapy. However, little is known regarding stabilities and aerodynamic particle sizes of N-siRNA-containing droplets; nebulizers have not yet been optimized for N-siRNA solutions. Thus, in this study, we investigated the feasibility of inhaled N-siRNA solutions for pulmonary therapy using nebulization. Various nebulizers and N-siRNA concentrations were assessed in terms of siRNA integrity after nebulization, and inhalation properties including aerodynamic particle size were examined. In comparison with ultrasonic-, air-jet-, and vibrating-mesh nebulizers, N-siRNA integrity was not affected by nebulization. Thus, in further experiments, performances of N-siRNA aerosols with different nebulizers and N-siRNA concentrations were evaluated and screened using an aerodynamic particle sizer (APS) which employed the time-of-flight principle or a cascade impactor. Mean mass aerodynamic diameters of N-siRNA-containing droplets from vibrating-mesh nebulizers tended to decrease with increasing N-siRNA concentrations, reflecting the influence of N-siRNA solutions on surface tension, as indicated by contact angles. These data indicate the utility of APS instruments for investigating the nebulized characteristics of expensive drugs including siRNAs and may facilitate the development of N-siRNA inhalation formulations.

  17. Gene Silencing of SOCS3 by siRNA Intranasal Delivery Inhibits Asthma Phenotype in Mice

    PubMed Central

    Mazzeo, Carla; Gámez, Cristina; Rodriguez Marco, Ainara; de Zulueta, Ana; Sanz, Veronica; Bilbao, Izaskun; Ruiz-Cabello, Jesús; Zubeldia, Jose M.; del Pozo, Victoria

    2014-01-01

    Suppresors of cytokine signaling (SOCS) proteins regulate cytokine responses and control immune balance. Several studies have confirmed that SOCS3 is increased in asthmatic patients, and SOCS3 expression is correlated with disease severity. The objective of this study was to evaluate if delivering of SOCS3 short interfering RNA (siRNA) intranasally in lungs could be a good therapeutic approach in an asthma chronic mouse model. Our results showed that intranasal treatment with SOCS3-siRNA led to an improvement in the eosinophil count and the normalization of hyperresponsiveness to methacholine. Concomitantly, this treatment resulted in an improvement in mucus secretion, a reduction in lung collagen, which are prominent features of airway remodeling. The mechanism implies JAK/STAT and RhoA/Rho-kinase signaling pathway, because we found a decreasing in STAT3 phosphorylation status and down regulation of RhoA/Rho-kinase protein expression. These results might lead to a new therapy for the treatment of chronic asthma. PMID:24637581

  18. Dry Powder Formulation of Plasmid DNA and siRNA for Inhalation.

    PubMed

    Chow, Michael Y T; Lam, Jenny K W

    2015-01-01

    Nucleic acid therapeutics has huge potential for the treatment of a wide range of diseases including respiratory diseases. Plasmid DNA (pDNA) and small interfering RNA (siRNA) are the two most widely investigated nucleic acids for therapeutic development. However, efficient and safe delivery of nucleic acids is still a major hurdle in translating nucleic acid therapy into clinical practice. For the treatment of respiratory diseases, administration via inhalation is the most direct and effective way to deliver therapeutic nucleic acids to the lungs. Although liquid aerosol formulation is investigated in most of the studies, it is not desirable in terms of maintaining the stability of nucleic acid especially during long-term storage. This problem could be circumvented by formulating the therapeutic nucleic acids into dry powder for inhalation, and should be considered as the future direction of developing inhalable nucleic acids. In this review, the three major particle engineering methods investigated for the preparation of inhalable pDNA and siRNA formulations, including spray drying (SD), spray freeze drying (SFD) and supercritical fluid (SFC) drying, are discussed and compared. Moreover, common assessment methods and the challenges of evaluating the biological activities of inhalable nucleic acid powders are also reviewed.

  19. Efficient Gene Silencing in Metastatic Tumor by siRNA Formulated in Surface-modified Nanoparticles

    PubMed Central

    Li, Shyh-Dar; Chono, Sumio; Huang, Leaf

    2009-01-01

    We have developed a nanoparticle (NP) formulation for systemically delivering siRNA into metastatic tumors. The NP, composed of nucleic acids, a polycationic peptide and cationic liposome, was prepared in a self-assembling process. The NP was then modified by PEG-lipid containing a targeting ligand, anisamide, and thus was decorated for targeting sigma receptor expressing B16F10 tumor. The activity of the targeted NP was compared with the naked NP (no PEGylation) and non-targeted NP (no ligand). The delivery efficiency of the targeted NP was 4-fold higher than the non-targeted NP and could be competed by excess free ligand. Luciferase siRNA was used to evaluate the gene silencing activity in the B16F10 cells, which were stably transduced with a luciferase gene, in a lung metastasis model. The gene silencing activity of the targeted NP was significantly higher than the other formulations and lasted for 4 days. While confocal microscopy showed the naked NP provided no tissue selectivity and non-targeted NP was ineffective for tumor uptake, the targeted NP effectively penetrated the lung metastasis, but not the liver. It resulted in 70-80% gene silencing in the metastasis model after a single i.v. injection (150 μg siRNA/kg). This effective formulation also showed very little immunotoxicity. PMID:18083264

  20. A synthetic lethal siRNA screen identifying genes mediating sensitivity to a PARP inhibitor.

    PubMed

    Turner, Nicholas C; Lord, Christopher J; Iorns, Elizabeth; Brough, Rachel; Swift, Sally; Elliott, Richard; Rayter, Sydonia; Tutt, Andrew N; Ashworth, Alan

    2008-05-07

    Inhibitors of poly (ADP-ribose)-polymerase-1 (PARP) are highly lethal to cells with deficiencies in BRCA1, BRCA2 or other components of the homologous recombination pathway. This has led to PARP inhibitors entering clinical trials as a potential therapy for cancer in carriers of BRCA1 and BRCA2 mutations. To discover new determinants of sensitivity to these drugs, we performed a PARP-inhibitor synthetic lethal short interfering RNA (siRNA) screen. We identified a number of kinases whose silencing strongly sensitised to PARP inhibitor, including cyclin-dependent kinase 5 (CDK5), MAPK12, PLK3, PNKP, STK22c and STK36. How CDK5 silencing mediates sensitivity was investigated. Previously, CDK5 has been suggested to be active only in a neuronal context, but here we show that CDK5 is required in non-neuronal cells for the DNA-damage response and, in particular, intra-S and G(2)/M cell-cycle checkpoints. These results highlight the potential of synthetic lethal siRNA screens with chemical inhibitors to define new determinants of sensitivity and potential therapeutic targets.

  1. Ultradeformable cationic liposomes for delivery of small interfering RNA (siRNA) into human primary melanocytes.

    PubMed

    Geusens, B; Lambert, J; De Smedt, S C; Buyens, K; Sanders, N N; Van Gele, M

    2009-02-10

    The aim of this work was to develop a system that can deliver siRNA into cells present in the human epidermis. More specifically, we wanted to block the expression of a specific Myosin Va exon F containing isoform that is physiologically involved in melanosome transport in human melanocytes. Therefore, we prepared and investigated the capacity of ultradeformable cationic liposomes (UCLs) to deliver siRNA in hard-to-transfect human primary melanocytes. UCLs were formulated from different w:w ratios (6:1, 8:1 and 10:1) of the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and the edge activator sodium cholate. Subsequently, UCL/siRNA complexes were prepared and their particle size, surface charge, deformability, cytotoxicity, transfection efficiency and long-term stability were tested. The best results were obtained with UCLs composed of a DOTAP/NaChol ratio of 6:1 (w:w) which are promising for future in vivo experiments.

  2. Going beyond the liver: progress and challenges of targeted delivery of siRNA therapeutics.

    PubMed

    Lorenzer, Cornelia; Dirin, Mehrdad; Winkler, Anna-Maria; Baumann, Volker; Winkler, Johannes

    2015-04-10

    Therapeutic gene silencing promises significant progress in pharmacotherapy, including considerable expansion of the druggable target space and the possibility for treating orphan diseases. Technological hurdles have complicated the efficient use of therapeutic oligonucleotides, and siRNA agents suffer particularly from insufficient pharmacokinetic properties and poor cellular uptake. Intense development and evolution of delivery systems have resulted in efficient uptake predominantly in liver tissue, in which practically all nanoparticulate and liposomal delivery systems show the highest accumulation. The most efficacious strategies include liposomes and bioconjugations with N-acetylgalactosamine. Both are in early clinical evaluation stages for treatment of liver-associated diseases. Approaches for achieving knockdown in other tissues and tumors have been proven to be more complicated. Selective targeting to tumors may be enabled through careful modulation of physical properties, such as particle size, or by taking advantage of specific targeting ligands. Significant barriers stand between sufficient accumulation in other organs, including endothelial barriers, cellular membranes, and the endosome. The brain, which is shielded by the blood-brain barrier, is of particular interest to facilitate efficient oligonucleotide therapy of neurological diseases. Transcytosis of the blood-brain barrier through receptor-specific docking is investigated to increase accumulation in the central nervous system. In this review, the current clinical status of siRNA therapeutics is summarized, as well as innovative and promising preclinical concepts employing tissue- and tumor-targeted ligands. The requirements and the respective advantages and drawbacks of bioconjugates and ligand-decorated lipid or polymeric particles are discussed.

  3. Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates.

    PubMed

    Thi, Emily P; Mire, Chad E; Lee, Amy C H; Geisbert, Joan B; Zhou, Joy Z; Agans, Krystle N; Snead, Nicholas M; Deer, Daniel J; Barnard, Trisha R; Fenton, Karla A; MacLachlan, Ian; Geisbert, Thomas W

    2015-05-21

    The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.

  4. siRNA screen identifies QPCT as a druggable target for Huntington’s disease

    PubMed Central

    Jimenez-Sanchez, Maria; Lam, Wun; Tarditi, Alessia; Menzies, Fiona; Dami, Teresa Ed; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P.; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P.; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O’Kane, Cahir J.; Caricasole, Andrea; Rubinsztein, David C.

    2015-01-01

    Huntington’s disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified novel modifiers of mutant HTT toxicity by performing a large-scale “druggable genome” siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen, and which also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone alpha B-crystallin and reduced the aggregation of diverse proteins. We generated novel QPCT inhibitors using in silico methods followed by in vitro screens, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a novel HD druggable target affecting mutant huntingtin aggregation, and provide proof-of-principle for a discovery pipeline from druggable genome screen to drug development. PMID:25848931

  5. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

    PubMed

    Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

    2015-05-30

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies.

  6. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome

    PubMed Central

    Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

    2015-01-01

    Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. DOI: http://dx.doi.org/10.7554/eLife.06602.001 PMID:26026149

  7. siRNA screen identifies QPCT as a druggable target for Huntington's disease.

    PubMed

    Jimenez-Sanchez, Maria; Lam, Wun; Hannus, Michael; Sönnichsen, Birte; Imarisio, Sara; Fleming, Angeleen; Tarditi, Alessia; Menzies, Fiona; Ed Dami, Teresa; Xu, Catherine; Gonzalez-Couto, Eduardo; Lazzeroni, Giulia; Heitz, Freddy; Diamanti, Daniela; Massai, Luisa; Satagopam, Venkata P; Marconi, Guido; Caramelli, Chiara; Nencini, Arianna; Andreini, Matteo; Sardone, Gian Luca; Caradonna, Nicola P; Porcari, Valentina; Scali, Carla; Schneider, Reinhard; Pollio, Giuseppe; O'Kane, Cahir J; Caricasole, Andrea; Rubinsztein, David C

    2015-05-01

    Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development.

  8. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity

    NASA Astrophysics Data System (ADS)

    Whitehead, Kathryn A.; Dorkin, J. Robert; Vegas, Arturo J.; Chang, Philip H.; Veiseh, Omid; Matthews, Jonathan; Fenton, Owen S.; Zhang, Yunlong; Olejnik, Karsten T.; Yesilyurt, Volkan; Chen, Delai; Barros, Scott; Klebanov, Boris; Novobrantseva, Tatiana; Langer, Robert; Anderson, Daniel G.

    2014-06-01

    One of the most significant challenges in the development of clinically viable delivery systems for RNA interference therapeutics is to understand how molecular structures influence delivery efficacy. Here, we have synthesized 1,400 degradable lipidoids and evaluate their transfection ability and structure-function activity. We show that lipidoid nanoparticles mediate potent gene knockdown in hepatocytes and immune cell populations on IV administration to mice (siRNA EC50 values as low as 0.01 mg kg-1). We identify four necessary and sufficient structural and pKa criteria that robustly predict the ability of nanoparticles to mediate greater than 95% protein silencing in vivo. Because these efficacy criteria can be dictated through chemical design, this discovery could eliminate our dependence on time-consuming and expensive cell culture assays and animal testing. Herein, we identify promising degradable lipidoids and describe new design criteria that reliably predict in vivo siRNA delivery efficacy without any prior biological testing.

  9. Major degradable polycations as carriers for DNA and siRNA.

    PubMed

    Islam, Mohammad Ariful; Park, Tae-Eun; Singh, Bijay; Maharjan, Sushila; Firdous, Jannatul; Cho, Myung-Haing; Kang, Sang-Kee; Yun, Cheol-Heui; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-10

    Non-viral gene delivery systems are one of the most potential alternatives to viral vectors because of their less immunogenicity, less toxicity and easy productivity in spite of their low capacity of gene transfection using DNA or silencing using siRNA compared to that of viral vectors. Among non-viral systems, the polycationic derivatives are the most popular gene carriers since they can effectively condense nucleic acids to transfer into the cells, especially the polyethylenimine (PEI) which has been used as a golden standard polymer owing to its high buffering ability for endosomal escape of gene to be expressed. However, PEI has severe problems for its toxicity due to the high positive charge density and non-degradability although the toxicity of PEI depends on its molecular weight (MW) and structure. Therefore, a considerable attention has been paid on synthesis of degradable PEI derivatives using low MW one because low MW PEI is much less toxic than high MW PEI. Other degradable polycationic gene carriers such as polyamidoamines (PAA) and cyclodextrin (CD)-based polycations are also in a significant interest because of their high transfection efficiency with low toxicity. This review in detail explains the recent developments on these three major degradable polycations as promising carriers for deoxyribonucleic acid (DNA) and small interfering RNA (siRNA).

  10. Tumor acidity-sensitive linkage-bridged block copolymer for therapeutic siRNA delivery.

    PubMed

    Xu, Cong-Fei; Zhang, Hou-Bing; Sun, Chun-Yang; Liu, Yang; Shen, Song; Yang, Xian-Zhu; Zhu, Yan-Hua; Wang, Jun

    2016-05-01

    The design of ideal nanoparticle delivery systems should be capable of meeting the requirements of several stages of drug delivery, including prolonged circulation, enhanced accumulation and penetration in the tumor, facilitated cellular internalization and rapid release of the active drug in the tumor cells. However, among the current design strategies, meeting the requirements of one stage often conflicts with the other. Herein, a tumor pH-labile linkage-bridged block copolymer of poly(ethylene glycol) with poly(lacide-co-glycolide) (PEG-Dlinkm-PLGA) was used for siRNA delivery to fulfill all aforementioned requirements of these delivery stages. The obtained siRNA-encapsulating PEG-Dlinkm-PLGA nanoparticle gained efficiently prolonged circulation in the blood and preferential accumulation in tumor sites via the PEGylation. Furthermore, the PEG surface layer was detached in response to the tumor acidic microenvironment to facilitate cellular uptake, and the siRNA was rapidly released within tumor cells due to the hydrophobic PLGA layer. Hence, PEG-Dlinkm-PLGA nanoparticles met the requirements of several stages of drug delivery, and resulted in the enhanced therapeutic effect of the nanoparticular delivery systems.

  11. siRNA mediated gene silencing in Fusarium sp. HKF15 for overproduction of bikaverin.

    PubMed

    Deshmukh, Radhika; Purohit, Hemant J

    2014-04-01

    Fusarium sp. HKF15 is an isolate from effluent treatment plant which produces bikaverin. Bikaverin is a polyketide having antitumor and antibiotic potential. Acetyl coenzyme A is a common precursor for bikaverin as well as carotenoids and gibberellins. A polyketide synthase gene bik1 is responsible for bikaverin production whereas, hydroxymethyl glutaryl coenzyme A reductase (hmgR) and farnesyl pyrophosphate synthase (fpps) are carotenoid and gibberellin pathway genes. Aim of this study was assessing siRNA mediated gene silencing for bikaverin overproduction with down-regulation of carotenoid and gibberellin pathway. HKF15 protoplasts derived from glucose grown culture were treated with 200pmolml(-1)hmgR and fpps siRNAs separately. Along with down-regulation of target genes, there was 2.4-fold increase in bik1 gene expression. The silencing was effective till 48h with a 41% increase in bikaverin production. The study proposes a strategy for manipulation of physiology towards desired secondary metabolite overproduction.

  12. Targeted tumor-penetrating siRNA nanocomplexes for credentialing the ovarian cancer target ID4

    PubMed Central

    Ren, Yin; Cheung, Hiu Wing; von Maltzhan, Geoffrey; Agrawal, Amit; Cowley, Glenn S.; Weir, Barbara A.; Boehm, Jesse S.; Tamayo, Pablo; Karst, Alison M.; Liu, Joyce F.; Hirsch, Michelle S.; Mesirov, Jill P.; Drapkin, Ronny; Root, David E.; Lo, Justin; Fogal, Valentina; Ruoslahti, Erkki; Hahn, William C.; Bhatia, Sangeeta N.

    2013-01-01

    The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance, because many of these targets are not amenable to manipulation by small molecules or antibodies. RNAi provides a direct way to study putative cancer targets; however, specific delivery of therapeutics to the tumor parenchyma remains an intractable problem. We describe a platform for the discovery and initial validation of cancer targets, composed of a systematic effort to identify amplified and essential genes in human cancer cell lines and tumors partnered with a novel modular delivery technology. We developed a tumor-penetrating nanocomplex (TPN) comprised of siRNA complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enabled the specific delivery of siRNA deep into the tumor parenchyma. We employed TPN in vivo to evaluate inhibitor of DNA binding 4 (ID4) as a novel oncogene. Treatment of ovarian tumor-bearing mice with ID4-specific TPN suppressed growth of established tumors and significantly improved survival. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers, but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets. PMID:22896676

  13. siRNA repositioning for guide strand selection by human Dicer complexes.

    PubMed

    Noland, Cameron L; Ma, Enbo; Doudna, Jennifer A

    2011-07-08

    The human ribonuclease Dicer and its double-stranded RNA (dsRNA)-binding protein (dsRBP) partners TRBP and PACT play important roles in the biogenesis of regulatory RNAs. Following dicing, one dsRNA product strand is preferentially assembled into an RNA-induced silencing complex (RISC). The mechanism of strand selection in humans and the possible role of Dicer in this process remain unclear. Here we demonstrate that dsRNAs undergo significant repositioning within Dicer complexes following dicing. This repositioning enables directional binding of RNA duplexes, thereby biasing their orientation for guide strand selection according to the thermodynamic properties of the helix. Our findings indicate that Dicer is itself capable of sensing siRNA thermodynamic asymmetry regardless of the dsRBP to which it is bound. These results support a model in which Dicer employs two distinct RNA-binding sites-one for dsRNA processing and the other for sensing of siRNA thermodynamic asymmetry-during RISC loading in humans.

  14. Elemental Abundances from Very Low Abundance HII Regions

    NASA Astrophysics Data System (ADS)

    Skillman, Evan D.; Terlevich, Roberto J.; Terlevich, Elena

    1992-12-01

    In 1987 we initiated a program to mitigate the deficiency of known low metallicity galaxies. Following our discoveries of very low abundance H II regions in nearby dwarf galaxies (Skillman et al. 1988, 1989a,b), we used the IDS on the INT to to collect spectra of dwarf galaxies in the Second Byurakan Survey (SBS) of UV excess galaxies. Our survey of over 40 SBS galaxies was completed in January 1990 and we have identified roughly one dozen very low metallicity H II galaxies. Now, with a significant sample of these galaxies, several observational programs are possible; foremost of these is the measurement of the primordial helium abundance (eg., Pagel et al. 1992). We report here on observations from March 1991 and 1992 using the ISIS spectrograph on the WHT to obtain very high quality spectra of 8 of these newly discovered metal-poor galaxies. The ISIS double spectrograph allows simultaneous observations of the blue (3600 - 5100 Angstroms) and red (6300 - 6800 Angstroms). Thus, He, N, O, Ne and S abundances can be derived with relatively small observational uncertainties. We compare our new observations with those in the literature. Our preliminary analysis indicates a slightly larger scatter in He/H at low O/H than had been seen previously. The small scatter may have been due simply to the paucity of observations at low metallicity. References: Pagel, B.E.J., Simonson, E.A., Terlevich, R.J., & Edmunds, M.G. 1992, MNRAS, 255, 325 Skillman, E.D., Kennicutt, R.C., & Hodge, P.W. 1989a, ApJ, 347, 875 Skillman, E.D., Melnick, J., Terlevich, R., & Moles, M. 1988, A&A, 196, 31 Skillman, E.D., Terlevich, R., & Melnick, J. 1989b, MNRAS, 240, 563

  15. The Diffuse Extreme Ultraviolet Background

    NASA Technical Reports Server (NTRS)

    Vallerga, John; Slavin, Jonathan

    1996-01-01

    Observations of the diffuse EUV background towards 138 different directions using the spectrometers aboard the Extreme Ultraviolet Explorer satellite (EUVE) have been combined into a spectrum from 150A to 730A and represent an effective exposure of 18 million seconds. There is no significant evidence of any non-local line flux in the resultant spectrum such as that from a hot coronal plasma. These results are inconsistent with the Wisconsin C and B broad-band surveys assuming the source is a logT = 5.8 - 6.1 hot plasma in ionization equilibrium with solar abundances, confirming the previous result of Jelinksy, Vallerga and Edelstein) (hereafter Paper 1) using an observation along the ecliptic with the same instrument. To make these results consistent with the previous broad-band surveys, the plasma responsible for the emission must either be depleted in Fe by a factor of approximately 6, be behind an absorbing slab of neutral H with a column of 2 x 10(exp 19)/sq cm, or not be in collisional ionization equilibrium (CIE). One such non-CIE model (Breitswerdt and Schmutzier) that explains the soft x-ray results is also inconsistent with this EUV data.

  16. Role of generation, architecture, pH and ionic strength on successful siRNA delivery and transfection by hybrid PPV-PAMAM dendrimers.

    PubMed

    Pavan, G M; Monteagudo, S; Guerra, J; Carrión, B; Ocaña, V; Rodríguez-Lopez, J; Danani, A; Pérez-Martínez, F C; Ceña, V

    2012-01-01

    Small interfering RNA (siRNA) constitutes an excellent way of knocking down genes. However, it requires the use of delivery systems to reach the target cells, especially to neuronal cells. Dendrimers are one of the most widely used synthetic nanocarriers for siRNA delivery. However, due to the complexity of the dendrimer-siRNA interactions, when a new dendritic carrier is designed it is difficult to predict its efficiency to bind and to deliver siRNA. At the same time it is not easy to understand the origin of eventual limited functionalities. We have modeled the interactions between two dendrimers (TDG-G1 and TDG-G2) and siRNA using molecular dynamics (MD) simulation. The results were compared to experimental physico-chemical parameters such as siRNA complexation, complex stability, size, and zeta potentials and biological effects such as down-regulation of a specific RNA expression in cortical neurons in culture. Data indicate that the combination of rigid core and flexible branches guarantees strong siRNA binding, which is important to have a good transfection profile. However, the successful nanocarrier for siRNA delivery (TDG-G1) is identified not only by a high affinity for siRNA, but by a favorable equilibrium between a strong binding and the ability to release siRNA to exert its biological action. The conditions under which the dendriplex is formed are also relevant for transfection efficiency and biological activity.

  17. Protection of shrimp against white spot syndrome virus (WSSV) with β-1,3-D-glucan-encapsulated vp28-siRNA particles.

    PubMed

    Zhu, Fei; Zhang, Xiaobo

    2012-02-01

    White spot syndrome virus (WSSV) is a major shrimp viral pathogen responsible for large economic losses to shrimp aquaculture all over the world. The RNAi mediated by siRNA contributes a new strategy to control this viral disease. However, the efficient approach to deliver the siRNA into shrimp remains to be addressed. In this investigation, an antiviral vp28-siRNA was encapsulated in β-1,3-D-glucan, and then the β-1,3-D-glucan-encapsulated vp28-siRNA particles (GeRPs) were delivered into Marsupenaeus japonicus shrimp. The results showed that the vp28-siRNA in GeRPs could be released in hemocytes of shrimp. It was found that the GeRPs containing the vp28-siRNA inhibited the replication of WSSV in vivo, which presented a better antiviral activity than the non-encapsulated vp28-siRNA. Further evidence indicated that the mortality of WSSV-infected shrimp was significantly delayed by the GeRPs containing vp28-siRNA. Therefore, our study presented that the glucan-encapsulated siRNA might represent a novel potential therapeutic or preventive approach to control the shrimp disease.

  18. Stimuli-responsive hybrid nanocarriers developed by controllable integration of hyperbranched PEI with mesoporous silica nanoparticles for sustained intracellular siRNA delivery

    PubMed Central

    Prabhakar, Neeraj; Zhang, Jixi; Desai, Diti; Casals, Eudald; Gulin-Sarfraz, Tina; Näreoja, Tuomas; Westermarck, Jukka; Rosenholm, Jessica M

    2016-01-01

    Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with the challenge being to deliver it in a sustained manner. The combination of mesoporous silica nanoparticles (MSNs) and polycations in the confined pore space allows for incorporation and controlled release of therapeutic siRNA payloads. We hereby constructed MSNs with expanded mesopores and pore-surface-hyperbranched poly(ethyleneimine) (PEI) tethered with redox-cleavable linkers that could carry a high payload of siRNA (120 mg·g−1). The developed nanocarriers were efficiently taken up by cancer cells and were subsequently able to escape to the cytoplasm from the endosomes, most likely owing to the integrated PEI. Triggered by the intracellular redox conditions, the siRNA was sustainably released inside the cells over a period of several days. Functionality of siRNAs was demonstrated by using cell-killing siRNA as cargo. Despite not being the aim of the developed system, in vitro experiments using cell-killing siRNAs showed that the efficacy of siRNA transfection was comparable to the commercial in vitro transfection agent Lipofectamine. Consequently, the developed MSN-based delivery system offers a potential approach to hybrid nanocarriers for more efficient and long-term siRNA delivery and, in a longer perspective, in vivo gene silencing for RNA interference (RNAi) therapy. PMID:27994460

  19. Stimuli-responsive hybrid nanocarriers developed by controllable integration of hyperbranched PEI with mesoporous silica nanoparticles for sustained intracellular siRNA delivery.

    PubMed

    Prabhakar, Neeraj; Zhang, Jixi; Desai, Diti; Casals, Eudald; Gulin-Sarfraz, Tina; Näreoja, Tuomas; Westermarck, Jukka; Rosenholm, Jessica M

    Small interfering RNA (siRNA) is a highly potent drug in gene-based therapy with the challenge being to deliver it in a sustained manner. The combination of mesoporous silica nanoparticles (MSNs) and polycations in the confined pore space allows for incorporation and controlled release of therapeutic siRNA payloads. We hereby constructed MSNs with expanded mesopores and pore-surface-hyperbranched poly(ethyleneimine) (PEI) tethered with redox-cleavable linkers that could carry a high payload of siRNA (120 mg·g(-1)). The developed nanocarriers were efficiently taken up by cancer cells and were subsequently able to escape to the cytoplasm from the endosomes, most likely owing to the integrated PEI. Triggered by the intracellular redox conditions, the siRNA was sustainably released inside the cells over a period of several days. Functionality of siRNAs was demonstrated by using cell-killing siRNA as cargo. Despite not being the aim of the developed system, in vitro experiments using cell-killing siRNAs showed that the efficacy of siRNA transfection was comparable to the commercial in vitro transfection agent Lipofectamine. Consequently, the developed MSN-based delivery system offers a potential approach to hybrid nanocarriers for more efficient and long-term siRNA delivery and, in a longer perspective, in vivo gene silencing for RNA interference (RNAi) therapy.

  20. Late Embryogenesis Abundant (LEA) proteins in legumes.

    PubMed

    Battaglia, Marina; Covarrubias, Alejandra A

    2013-01-01

    Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions.

  1. Late Embryogenesis Abundant (LEA) proteins in legumes

    PubMed Central

    Battaglia, Marina; Covarrubias, Alejandra A.

    2013-01-01

    Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions. PMID:23805145

  2. Hydrological extremes and security

    NASA Astrophysics Data System (ADS)

    Kundzewicz, Z. W.; Matczak, P.

    2015-04-01

    Economic losses caused by hydrological extremes - floods and droughts - have been on the rise. Hydrological extremes jeopardize human security and impact on societal livelihood and welfare. Security can be generally understood as freedom from threat and the ability of societies to maintain their independent identity and their functional integrity against forces of change. Several dimensions of security are reviewed in the context of hydrological extremes. The traditional interpretation of security, focused on the state military capabilities, has been replaced by a wider understanding, including economic, societal and environmental aspects that get increasing attention. Floods and droughts pose a burden and serious challenges to the state that is responsible for sustaining economic development, and societal and environmental security. The latter can be regarded as the maintenance of ecosystem services, on which a society depends. An important part of it is water security, which can be defined as the availability of an adequate quantity and quality of water for health, livelihoods, ecosystems and production, coupled with an acceptable level of water-related risks to people, environments and economies. Security concerns arise because, over large areas, hydrological extremes - floods and droughts - are becoming more frequent and more severe. In terms of dealing with water-related risks, climate change can increase uncertainties, which makes the state's task to deliver security more difficult and more expensive. However, changes in population size and development, and level of protection, drive exposure to hydrological hazards.

  3. Astron extreme lightweighting

    NASA Astrophysics Data System (ADS)

    Tromp, Niels; Drost, Marco; Pragt, Johan

    2004-09-01

    Producing extreme light weighted structures by combining a new design concept with the most recent production machines and production software tools. Weight reductions of up to 50% compared to the traditional techniques are feasible with the same stiffness performance. Suitable for standard materials like aluminium and steel, for single construction parts out of mono material and with a single production process. Astronomical instruments for space applications and ground-based applications require more and more extreme light and extreme stiff structures. The traditional technique like 3-axis or multisided machining of metal parts seems limited and not suitable for the next generation instruments. New materials with new production technologies are used more and more with all their specialties and restrictions. ASTRON developed a new structural design of traditional materials with heritage optimized for production with the most recent milling machines. The structural shapes are closely linked to the extremes of 5-axis simultaneous milling. The design and production process is patented and now free for publication.

  4. Silencing by blasting: combination of laser pulse induced stress waves and magnetophoresis for siRNA delivery

    NASA Astrophysics Data System (ADS)

    Babincová, M.; Babincová, N.; Durdík, S.; Bergemann, C.; Sourivong, P.

    2016-06-01

    A new method is developed for efficient delivery of short interference RNA into cells using combination of magnetophoresis for pre-concentration of siRNA-magnetic nanoparticle complex on the surface of cells with subsequent nanosecond laser pulse generating stress waves in transfection chamber, which is able to permeabilize cell membrane for the facilitated delivery of siRNA into the cell interior. As has been shown using siRNA inducing cell apoptosis, combination of these two physical factors increased the efficiency of three different human carcinoma cells transfection to 93%, 89%, and 84%, for HeLa (cervical carcinoma), MCF-7 (breast carcinoma), and UCI-107 (ovarian carcinoma) cells, respectively. This new physical method of siRNA delivery may have therefore far reaching applications in biotechnology and functional genomics.

  5. Enhanced apoptosis of ovarian cancer cells via nanocarrier-mediated codelivery of siRNA and doxorubicin

    PubMed Central

    Zou, Seyin; Cao, Nuo; Cheng, Du; Zheng, Rongqin; Wang, Jin; Zhu, Kangshun; Shuai, Xintao

    2012-01-01

    A folate conjugated ternary copolymer, FA–PEG–PEI–PCL, of poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and poly(ɛ-caprolactone) (PCL) was synthesized. The copolymer self-assembled into cationic micelles capable of co-delivering siRNA and the anticancer drug doxorubicin (DOX). This dual functional nanocarrier demonstrated low cytotoxicity and high performance in drug/siRNA delivery. Upon the codelivery of siRNA, targeting the Bcl-2 gene, and DOX, using the folate-targeted nanocarrier, DOX-induced apoptosis in the skov-3 cells overexpressing folate receptor was significantly enhanced through a mechanism of downregulating the antiapoptotic protein Bcl-2, while simultaneously upregulating the proapoptotic protein Bax. This work suggested that the combination of Bcl-2 siRNA and DOX therapies is feasible, based on our dual functional nanocarrier, which set up a good basis for a future in vivo test. PMID:22888237

  6. Selective nuclear localization of siRNA by metallic versus semiconducting single wall carbon nanotubes in keratinocytes

    PubMed Central

    Huzil, John Torin; Saliaj, Evi; Ivanova, Marina V; Gharagozloo, Marjan; Loureiro, Maria Jimena; Lamprecht, Constanze; Korinek, Andreas; Chen, Ding Wen; Foldvari, Marianna

    2015-01-01

    Background: The potential use of carbon nanotubes (CNTs) in gene therapy as delivery systems for nucleic acids has been recently recognized. Here, we describe that metallic versus semiconducting single-wall CNTs can produce significant differences in transfection rate and cellular distribution of siRNA in murine PAM212 keratinocytes. Results/Methodology: The results of cell interaction studies, coupled with supportive computational simulations and ultrastructural studies revealed that the use of metallic single wall CNTs resulted in siRNA delivery into both the cytoplasm and nucleus of keratinocytes, whereas semiconducting CNTs resulted in delivery only to the cytoplasm. Conclusion: Using enriched fractions of metallic or semiconducting CNTs for siRNA complex preparation may provide specific subcellular targeting advantages. PMID:28031892

  7. Biocompatible and colloidally stabilized mPEG-PE/calcium phosphate hybrid nanoparticles loaded with siRNAs targeting tumors.

    PubMed

    Gao, Pei; Zhang, Xiangyu; Wang, Hongzhi; Zhang, Qinghong; Li, He; Li, Yaogang; Duan, Yourong

    2016-01-19

    Calcium phosphate nanoparticles are safe and effective delivery vehicles for small interfering RNA (siRNA), as a result of their excellent biocompatibility. In this work, mPEG-PE (polyethylene glycol-L-α-phosphatidylethanolamine) was synthesized and used to prepare nanoparticles composed of mPEG-PE and calcium phosphate for siRNA delivery. Calcium phosphate and mPEG-PE formed the stable hybrid nanoparticles through self-assembly resulting from electrostatic interaction in water. The average size of the hybrid nanoparticles was approximately 53.2 nm with a negative charge of approximately -16.7 mV, which was confirmed by dynamic light scattering (DLS) measurements. The nanoparticles exhibited excellent stability in serum and could protect siRNA from ribonuclease (RNase) degradation. The cellular internalization of siRNA-loaded nanoparticles was evaluated in SMMC-7721 cells using a laser scanning confocal microscope (CLSM) and flow cytometry. The hybrid nanoparticles could efficiently deliver siRNA to cells compared with free siRNA. Moreover, the in vivo distribution of Cy5-siRNA-loaded hybrid nanoparticles was observed after being injected into tumor-bearing nude mice. The nanoparticles concentrated in the tumor regions through an enhanced permeability and retention (EPR) effect based on the fluorescence intensities of tissue distribution. A safety evaluation of the nanoparticles was performed both in vitro and in vivo demonstrating that the hybrid nanoparticle delivery system had almost no toxicity. These results indicated that the mPEG-PE/CaP hybrid nanoparticles could be a stable, safe and promising siRNA nanocarrier for anticancer therapy.

  8. Biocompatible and colloidally stabilized mPEG-PE/calcium phosphate hybrid nanoparticles loaded with siRNAs targeting tumors

    PubMed Central

    Gao, Pei; Zhang, Xiangyu; Wang, Hongzhi; Zhang, Qinghong

    2016-01-01

    Calcium phosphate nanoparticles are safe and effective delivery vehicles for small interfering RNA (siRNA), as a result of their excellent biocompatibility. In this work, mPEG-PE (polyethylene glycol-L-α-phosphatidylethanolamine) was synthesized and used to prepare nanoparticles composed of mPEG-PE and calcium phosphate for siRNA delivery. Calcium phosphate and mPEG-PE formed the stable hybrid nanoparticles through self-assembly resulting from electrostatic interaction in water. The average size of the hybrid nanoparticles was approximately 53.2 nm with a negative charge of approximately −16.7 mV, which was confirmed by dynamic light scattering (DLS) measurements. The nanoparticles exhibited excellent stability in serum and could protect siRNA from ribonuclease (RNase) degradation. The cellular internalization of siRNA-loaded nanoparticles was evaluated in SMMC-7721 cells using a laser scanning confocal microscope (CLSM) and flow cytometry. The hybrid nanoparticles could efficiently deliver siRNA to cells compared with free siRNA. Moreover, the in vivo distribution of Cy5-siRNA-loaded hybrid nanoparticles was observed after being injected into tumor-bearing nude mice. The nanoparticles concentrated in the tumor regions through an enhanced permeability and retention (EPR) effect based on the fluorescence intensities of tissue distribution. A safety evaluation of the nanoparticles was performed both in vitro and in vivo demonstrating that the hybrid nanoparticle delivery system had almost no toxicity. These results indicated that the mPEG-PE/CaP hybrid nanoparticles could be a stable, safe and promising siRNA nanocarrier for anticancer therapy. PMID:26625203

  9. Galactosylated Liposomes for Targeted Co-Delivery of Doxorubicin/Vimentin siRNA to Hepatocellular Carcinoma

    PubMed Central

    Oh, Hea Ry; Jo, Hyun-Young; Park, James S.; Kim, Dong-Eun; Cho, Je-Yoel; Kim, Pyung-Hwan; Kim, Keun-Sik

    2016-01-01

    The combination of therapeutic nucleic acids and chemotherapeutic drugs has shown great promise for cancer therapy. In this study, asialoglycoprotein receptors (ASGPR) targeting-ligand-based liposomes were tested to determine whether they can co-deliver vimentin siRNA and doxorubicin to hepatocellular carcinoma (HCC) selectively. To achieve this goal, we developed an ASGPR receptor targeted co-delivery system called gal-doxorubicin/vimentin siRNA liposome (Gal-DOX/siRNA-L). The Gal-DOX/siRNA-L was created via electrostatic interaction of galactose linked-cationic liposomal doxorubicin (Gal-DOX-L) on vimentin siRNA. Previous studies have shown that Gal-DOX/siRNA-L inhibited tumor growth by combined effect of DOX and vimentin siRNA than single delivery of either DOX or vimentin siRNA. These Gal-DOX/siRNA-Ls showed stronger affinity to human hepatocellular carcinoma cells (Huh7) than other cells (lung epithelial carcinoma, A549). These liposomes also have demonstrated that novel hepatic drug/gene delivery systems composed of cationic lipid (DMKE: O,O’-dimyristyl-N-lysyl glutamate), cholesterol, galactosylated ceramide, POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), and PEG2000-DSPE (distearoyl phosphatidyl ethanolamine) at 2:1:1:1:0.2 (moral ratios) can be used as an effective drug/gene carrier specifically targeting the liver in vivo. These results suggest that Gal-DOX-siRNA-L could effectively target tumor cells, enhance transfection efficacy and subsequently achieve the co-delivery of DOX and siRNA, demonstrating great potential for synergistic anti-tumor therapy. PMID:28335269

  10. Stability, Intracellular Delivery, and Release of siRNA from Chitosan Nanoparticles Using Different Cross-Linkers

    PubMed Central

    Abdul Ghafoor Raja, Maria; Katas, Haliza; Jing Wen, Thum

    2015-01-01

    Chitosan (CS) nanoparticles have been extensively studied for siRNA delivery; however, their stability and efficacy are highly dependent on the types of cross-linker used. To address this issue, three common cross-linkers; tripolyphosphate (TPP), dextran sulphate (DS) and poly-D-glutamic acid (PGA) were used to prepare siRNA loaded CS-TPP/DS/PGA nanoparticles by ionic gelation method. The resulting nanoparticles were compared with regard to their physicochemical properties including particle size, zeta potential, morphology, binding and encapsulation efficiencies. Among all the formulations prepared with different cross linkers, CS-TPP-siRNA had the smallest particle size (ranged from 127 ± 9.7 to 455 ± 12.9 nm) with zeta potential ranged from +25.1 ± 1.5 to +39.4 ± 0.5 mV, and high entrapment (>95%) and binding efficiencies. Similarly, CS-TPP nanoparticles showed better siRNA protection during storage at 4˚C and as determined by serum protection assay. TEM micrographs revealed the assorted morphology of CS-TPP-siRNA nanoparticles in contrast to irregular morphology displayed by CS-DS-siRNA and CS-PGA-siRNA nanoparticles. All siRNA loaded CS-TPP/DS/PGA nanoparticles showed initial burst release followed by sustained release of siRNA. Moreover, all the formulations showed low and concentration-dependent cytotoxicity with human colorectal cancer cells (DLD-1), in vitro. The cellular uptake studies with CS-TPP-siRNA nanoparticles showed successful delivery of siRNA within cytoplasm of DLD-1 cells. The results demonstrate that ionically cross-linked CS-TPP nanoparticles are biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting. PMID:26068222

  11. Abundance measurements in stellar environments

    SciTech Connect

    Leone, F.

    2014-05-09

    Most of what we know about stars, and systems of stars, is derived from the analysis of their electromagnetic radiation. This lesson is an attempt to describe to Physicists, without any Astrophysical background, the framework to understand the present status of abundance determination in stellar environments and its limit. These notes are dedicated to the recently passed, November 21, 2013, Prof. Dimitri Mihalas who spent his life confuting the 19th century positivist philosopher Auguste Comte who stated that we shall not at all be able to determine the chemical composition of stars.

  12. Abundance measurements in stellar environments

    NASA Astrophysics Data System (ADS)

    Leone, F.

    2014-05-01

    Most of what we know about stars, and systems of stars, is derived from the analysis of their electromagnetic radiation. This lesson is an attempt to describe to Physicists, without any Astrophysical background, the framework to understand the present status of abundance determination in stellar environments and its limit. These notes are dedicated to the recently passed, November 21, 2013, Prof. Dimitri Mihalas who spent his life confuting the 19th century positivist philosopher Auguste Comte who stated that we shall not at all be able to determine the chemical composition of stars.

  13. The solar abundance of Oxygen

    NASA Astrophysics Data System (ADS)

    Grevesse, N.

    2009-07-01

    With Martin Asplund (Max Planck Institute of Astrophysics, Garching) and Jacques Sauval (Observatoire Royal de Belgique, Brussels) I recently published detailed reviews on the solar chemical composition ({Asplund et al. 2005}, {Grevesse et al. 2007}). A new one, with Pat Scott (Stockholm University) as additional co-author, will appear in Annual Review of Astronomy and Astrophysics ({Asplund et al. 2009}). Here we briefly analyze recent works on the solar abundance of Oxygen and recommend a value of 8.70 in the usual astronomical scale.

  14. Targeted in vivo delivery of EGFR siRNA inhibits ovarian cancer growth and enhances drug sensitivity

    PubMed Central

    Satpathy, Minati; Mezencev, Roman; Wang, Lijuan; McDonald, John F.

    2016-01-01

    A functionalized nanohydrogel siRNA delivery system and a mouse model of serous ovarian cancer were used to test predictions from previous cell line studies that knockdown of EGFR (epidermal growth factor receptor) may be of clinical significance in the treatment of epithelial tumors especially with respect to the enhancement of platinum based therapies. Our results support these predictions and suggest that targeted delivery of EGFR siRNA may be an effective strategy for the treatment of ovarian and other epithelial tumors associated with elevated levels of EGFR and especially those demonstrating resistance to platinum-based therapies. PMID:27819259

  15. Cystathionine-gamma-lyase gene silencing with siRNA in monocytes/macrophages protects mice against acute pancreatitis.

    PubMed

    Badiei, A; Chambers, S T; Gaddam, R R; Fraser, R; Bhatia, M

    2016-01-01

    Hydrogen sulphide (H2S) is an endogenous inflammatory mediator produced by cystathionine-γ-lyase (CSE) in monocytes/macrophages. To determine the role of H2S and macrophages in inflammation, we used small interference RNA (siRNA) to target the CSE gene and investigated its effect in a mouse model of acute pancreatitis. Acute pancreatitis is characterised by increased levels of plasma amylase, myeloperoxidase (MPO) activity and pro-inflammatory cytokines and chemokines in the pancreas and lung. SiRNA treatment attenuated inflammation in the pancreas and lungs of mice following caerulein-induced acute pancreatitis. MPO activity increased in caerulein-induced acute pancreatitis (16.21 ± 3.571 SD fold increase over control) and treatment with siRNA significantly reduced this (mean 3.555 ± 2.522 SD fold increase over control) (p < 0.0001). Similarly, lung MPO activity increased following treatment with caerulein (3.56 ± 0.941 SD fold increase over control) while siRNA treatment significantly reduced MPO activity (0.8243 ± 0.4353 SD fold increase over control) (p < 0.0001). Caerulein treatment increased plasma amylase activity (7094 ± 207 U/l) and this significantly decreased following siRNA administration (5895 ± 115 U/l) (p < 0.0001). Cytokine and chemokine levels in caerulein-induced acute pancreatitis reduced following treatment with siRNA. For example, siRNA treatment significantly decreased pancreatic and lung monocyte chemoattractant protein (MCP)-1 (169.8 ± 59.75 SD; 90.01 ± 46.97 SD pg/ml, respectively) compared to caerulein-treated mice (324.7 ± 103.9 SD; 222.8 ± 85.37 SD pg/ml, pancreas and lun,g respectively) (p < 0.0001). These findings show a crucial pro-inflammatory role for H2S synthesised by CSE in macrophages in acute pancreatitis and suggest CSE gene silencing with siRNA as a potential therapeutic approach for this condition.

  16. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    PubMed

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

  17. In Vitro and In Vivo Tumor-Targeting siRNA Delivery Using Folate-PEG-appended Dendrimer (G4)/α-Cyclodextrin Conjugates.

    PubMed

    Ohyama, Ayumu; Higashi, Taishi; Motoyama, Keiichi; Arima, Hidetoshi

    2016-03-16

    We previously reported that folate-polyethylene glycol (PEG)-appended dendrimer (generation 3)/α-cyclodextrin conjugate (Fol-PαC (G3)) shows folate receptor-α (FR-α)-overexpressing tumor cell-selective in vitro siRNA transfer activity. However, Fol-PαC (G3)/siRNA complex did not induce a significant in vivo RNAi effect after intravenous administration to tumor-bearing mice, possibly resulting from immediate dissociation of the complex in blood. Herein, to develop the novel siRNA carrier having high blood circulating ability, high in vivo siRNA transfer activity, and high safety profile, we newly prepared Fol-PαCs with higher generation (G4) and evaluated their potential as tumor-targeting siRNA carriers in vitro and in vivo. Fol-PαC (G4, average degree of substitution of α-cyclodextrin (DSC) 2.9, average degree of substitution of folate-PEG (DSF) 2)/siRNA complex had the prominent RNAi effect through adequate physicochemical properties, FR-α-mediated endocytosis, efficient endosomal escape, and siRNA delivery to cytoplasm with negligible cytotoxicity. Importantly, Fol-PαC (G4, DSC2.9, DSF2) improved the serum stability, blood circulating ability, and in vivo RNAi effects of siRNA, compared to Fol-PαC (G3). Furthermore, Fol-PαC (G4, DSC2.9, DSF2) complex with siRNA against Polo-like kinase 1 (siPLK1) suppressed the tumor growth compared to control siRNA complex. These results suggest that Fol-PαC (G4, DSC2.9, DSF2) has the potential as a novel tumor-targeting siRNA carrier in vitro and in vivo.

  18. Materials in extreme environments.

    SciTech Connect

    Hemley, R. J.; Crabtree, G. W.; Buchanan, M. V.; Materials Science Division; Geophysical Lab.; ORNL

    2009-11-01

    Nature is rich with examples of phenomena and environments we might consider extreme, at least from our familiar experience on Earth's surface: large fluxes of radiation and particles from the Sun, explosive asteroid collisions in space, volcanic eruptions that originate deep underground, extraordinary pressures and temperatures in the interiors of planets and stars, and electromagnetic discharges that occur, say, in sunspots and pulsars. We often intentionally create similar extreme environments - for example, in high-powered lasers, high-temperature turbines, internal-combustion engines, and industrial chemical plants. The response of materials to the broad range of such environments signals the materials underlying structure and dynamics, provides insight into new phenomena, exposes failure modes that limit technological possibility, and presents novel routes for making new materials.

  19. Extremal quantum cloning machines

    SciTech Connect

    Chiribella, G.; D'Ariano, G. M.; Perinotti, P.; Cerf, N.J.

    2005-10-15

    We investigate the problem of cloning a set of states that is invariant under the action of an irreducible group representation. We then characterize the cloners that are extremal in the convex set of group covariant cloning machines, among which one can restrict the search for optimal cloners. For a set of states that is invariant under the discrete Weyl-Heisenberg group, we show that all extremal cloners can be unitarily realized using the so-called double-Bell states, whence providing a general proof of the popular ansatz used in the literature for finding optimal cloners in a variety of settings. Our result can also be generalized to continuous-variable optimal cloning in infinite dimensions, where the covariance group is the customary Weyl-Heisenberg group of displacement000.

  20. Lower extremity venous reflux

    PubMed Central

    Baliyan, Vinit; Tajmir, Shahein; Ganguli, Suvranu; Prabhakar, Anand M.

    2016-01-01

    Venous incompetence in the lower extremity is a common clinical problem. Basic understanding of venous anatomy, pathophysiologic mechanisms of venous reflux is essential for choosing the appropriate treatment strategy. The complex interplay of venous pressure, abdominal pressure, venous valvular function and gravitational force determine the venous incompetence. This review is intended to provide a succinct review of the pathophysiology of venous incompetence and the current role of imaging in its management. PMID:28123974

  1. Religious Extremism in Pakistan

    DTIC Science & Technology

    2014-12-01

    Face (July 2008): 32. 21 Ahmed Rashid , Pakistan on the Brink: The Future of America, Pakistan, and Afghanistan (New York: Viking, 2012). 22 Brian J...promoting extremism. Commentators such as Jessica Stern, Alan Richards, Hussain Haqqani, Ahmed Rashid , and Ali Riaz are a few of the scholars who...www.jstor.org/stable/3183558; See also Ahmed Rashid , Descent Into Chaos: The United States and the Failure of Nation Building in Pakistan, Afghanistan, and

  2. Extreme geomagnetically induced currents

    NASA Astrophysics Data System (ADS)

    Kataoka, Ryuho; Ngwira, Chigomezyo

    2016-12-01

    We propose an emergency alert framework for geomagnetically induced currents (GICs), based on the empirically extreme values and theoretical upper limits of the solar wind parameters and of d B/d t, the time derivative of magnetic field variations at ground. We expect this framework to be useful for preparing against extreme events. Our analysis is based on a review of various papers, including those presented during Extreme Space Weather Workshops held in Japan in 2011, 2012, 2013, and 2014. Large-amplitude d B/d t values are the major cause of hazards associated with three different types of GICs: (1) slow d B/d t with ring current evolution (RC-type), (2) fast d B/d t associated with auroral electrojet activity (AE-type), and (3) transient d B/d t of sudden commencements (SC-type). We set "caution," "warning," and "emergency" alert levels during the main phase of superstorms with the peak Dst index of less than -300 nT (once per 10 years), -600 nT (once per 60 years), or -900 nT (once per 100 years), respectively. The extreme d B/d t values of the AE-type GICs are 2000, 4000, and 6000 nT/min at caution, warning, and emergency levels, respectively. For the SC-type GICs, a "transient alert" is also proposed for d B/d t values of 40 nT/s at low latitudes and 110 nT/s at high latitudes, especially when the solar energetic particle flux is unusually high.

  3. On extreme geomagnetic storms

    NASA Astrophysics Data System (ADS)

    Cid, Consuelo; Palacios, Judith; Saiz, Elena; Guerrero, Antonio; Cerrato, Yolanda

    2014-10-01

    Extreme geomagnetic storms are considered as one of the major natural hazards for technology-dependent society. Geomagnetic field disturbances can disrupt the operation of critical infrastructures relying on space-based assets, and can also result in terrestrial effects, such as the Quebec electrical disruption in 1989. Forecasting potential hazards is a matter of high priority, but considering large flares as the only criterion for early-warning systems has demonstrated to release a large amount of false alarms and misses. Moreover, the quantification of the severity of the geomagnetic disturbance at the terrestrial surface using indices as Dst cannot be considered as the best approach to give account of the damage in utilities. High temporal resolution local indices come out as a possible solution to this issue, as disturbances recorded at the terrestrial surface differ largely both in latitude and longitude. The recovery phase of extreme storms presents also some peculiar features which make it different from other less intense storms. This paper goes through all these issues related to extreme storms by analysing a few events, highlighting the March 1989 storm, related to the Quebec blackout, and the October 2003 event, when several transformers burnt out in South Africa.

  4. Chemical Compositions and Abundance Anomalies in Stellar Coronae ADP 99

    NASA Technical Reports Server (NTRS)

    Drake, Jeremy; Oliversen, Ronald J. (Technical Monitor)

    2003-01-01

    Progress has been made using both EUVE (Extreme Ultraviolet Explorer) and ASCA (Advanced Satellite for Cosmology and Astrophysics) data and a new postdoctoral scientist has now been hired. Stars studied to date include YY Gem (dMe binary), xi Boo A (intermediate activity G8 V), xi UMa (more active G quadruple system) HR1099 (K1 IV + G5 V) RS CVn-like, AU Mic (dMe). In addition to a paper that concentrated on abundancies in HR1099, a paper was recently submitted on the coronal abundances of AR(tilde)Lac that revealed an interesting pattern of overabundances of very low FIP elements (Al and Ca) compared to the low FIP elements Si, Mg and Fe. Two papers are nearing completion on methods of analysis and on the abundances in the corona of AU(tilde)Mic. Additionally, two invited conference proceedings papers are being published on this work. The main conclusion of the study to date is that our existing ideas of coronal abundance anomalies need complete revision. The solar-like FIP effect is replaced by a pattern than appears to enhance high FIP elements rather than low FIP elements in very active stars. The archival studies we are undertaking now are revealing some key details of these patterns, and are beginning to map out the anomalies as a function of spectral type, a key goal of this study.

  5. A Molecular Umbrella Approach to the Intracellular Delivery of siRNA

    PubMed Central

    Cline, Lauren L.; Janout, Vaclav; Fisher, Michael; Juliano, Rudolph L.; Regen, Steven L.

    2011-01-01

    A series of diwalled and tetrawalled molecular umbrellas have been synthesized using cholic acid, spermidine, and lysine as starting material. Coupling of these molecular umbrellas to an octaarginine peptide afforded agents that were capable of promoting the transport of small interfering RNA (siRNA) to HeLa cells, as judged by the knockdown of enhanced green fluorescent protein (eGFP) expression. The efficiency of this knockdown was found to increase with an increasing number of facially amphiphilic walls present, and also when a cleavable disulfide linker was replaced with a non-cleavable, maleimido moiety. The knockdown efficiency that was observed for one tetrawalled molecular umbrella-octaargine conjugate was comparable to that observed with a commercially available transfection agent, Lipofectamine 2000, but showed less cytotoxicity. PMID:21985221

  6. Genome-wide siRNA screen reveals coupling between mitotic apoptosis and adaptation

    PubMed Central

    Díaz-Martínez, Laura A; Karamysheva, Zemfira N; Warrington, Ross; Li, Bing; Wei, Shuguang; Xie, Xian-Jin; Roth, Michael G; Yu, Hongtao

    2014-01-01

    The antimitotic anti-cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint-dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome-wide small interfering RNA (siRNA) screen in taxol-treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro-apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest. PMID:25024437

  7. Understanding relations between breeding bird species and extreme weather

    NASA Astrophysics Data System (ADS)

    Allstadt, A.; Bateman, B.; Pidgeon, A. M.; Radeloff, V.; Vavrus, S. J.; Keuler, N.; Clayton, M.; Albright, T.; Thogmartin, W.; Heglund, P.

    2013-12-01

    Extreme weather events are increasing in frequency due to climate change. Extreme weather events like periods of drought or cold snaps may impose hardship on many animal and plant populations. However, little is known about biotic response to extreme events. For example, some species experience population size changes in association with extreme weather, and some do not. However the mechanisms responsible for observed declines in avian abundance following heat waves and drought are not clear. Our goal was to characterize the population changes of North American bird species in relation to temperature and precipitation extremes using North American Breeding Bird Survey (BBS) data. We derived standardized measures of extreme precipitation and air temperature based on phase 2 NASA Land Data Assimilation System (NLDAS-2), an hourly 1/8 degree resolution land surface forcing dataset, and modeled population responses, during the breeding season, of 363 bird species. Of those species in which a change was observed, many demonstrated decreases in total population size, suggesting either mortality or reproductive failure (or both) are the causative mechanisms of this decline. A greater proportion of population changes were associated with extreme conditions in the same year than in the previous year. Some species exhibited population decreases in areas of extreme weather and increases in areas with environmental conditions more favorable to breeding while overall abundance remained relatively constant, which might indicate movement. The patterns of bird population changes in relation to extreme weather events provide insight for planners as they consider modifications to our national protected area network that will limit threats posed by climate change to bird populations.

  8. Structure And Gene Silencing Activities of Monovalent And Pentavalent Cationic Lipid Vectors Complexed With Sirna

    SciTech Connect

    Bouxsein, N.F.; McAllister, C.S.; Ewert, K.K.; Samuel, C.E.; Safinya, C.R.; /UC, Santa Barbara

    2007-07-03

    Small interfering RNAs (siRNAs) of 19-25 bp mediate the cleavage of complementary mRNA, leading to post-transcriptional gene silencing. We examined cationic lipid (CL)-mediated delivery of siRNA into mammalian cells and made comparisons to CL-based DNA delivery. The effect of lipid composition and headgroup charge on the biophysical and biological properties of CL-siRNA vectors was determined. X-ray diffraction revealed that CL-siRNA complexes exhibited lamellar and inverted hexagonal phases, qualitatively similar to CL-DNA complexes, but also formed other nonlamellar structures. Surprisingly, optimally formulated inverted hexagonal 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) CL-siRNA complexes exhibited high toxicity and much lower target-specific gene silencing than lamellar CL-siRNA complexes even though optimally formulated, inverted hexagonal CL-DNA complexes show high transfection efficiency in cell culture. We further found that efficient silencing required cationic lipid/nucleic acid molar charge ratios (chg) nearly an order of magnitude larger than those yielding efficiently transfecting CL-DNA complexes. This second unexpected finding has implications for cell toxicity. Multivalent lipids (MVLs) require a smaller number of cationic lipids at a given chg of the complex. Consistent with this observation, the pentavalent lipid MVL5 exhibited lower toxicity and superior silencing efficiency over a large range in both the lipid composition and chg when compared to monovalent DOTAP. Most importantly, MVL5 achieved much higher total knockdown of the target gene in CL-siRNA complex regimes where toxicity was low. This property of CL-siRNA complexes contrasts to CL-DNA complexes, where the optimized transfection efficiencies of multivalent and monovalent lipids are comparable.

  9. Biomimetic nanoparticles for siRNA delivery in the treatment of leukaemia.

    PubMed

    Guo, Jianfeng; Cahill, Mary R; McKenna, Sharon L; O'Driscoll, Caitriona M

    2014-12-01

    Leukaemia is a bone marrow cancer occurring in acute and chronic subtypes. Acute leukaemia is a rapidly fatal cancer potentially causing death within a few weeks, if untreated. Leukaemia arises as a result of disruption to haematopoietic precursors, caused either by acquired gene fusions, gene mutations or inappropriate expression of the relevant oncogenes. Current treatment options have made significant progress, but the 5 year survival for acute leukaemia remains under 10% in elderly patients, and less than 50% for some types of acute leukaemia in younger adults. For chronic leukaemias longer survival is generally expected and for chronic myeloid leukaemia patients on tyrosine kinase inhibitors the median survival is not yet reached and is expected to exceed 10 years. Chemotherapy and haematopoietic stem cell transplantation (HSCT) for acute leukaemia provide the mainstay of therapy for patients under 65 and both carry significant morbidity and mortality. Alternative and superior therapeutic strategies for acute leukaemias are urgently required. Recent molecular-based knowledge of recurring chromosome rearrangements, in particular translocations and inversions, has resulted in significant advances in understanding the molecular pathogenesis of leukaemia. Identification of a number of unique fusion genes has facilitated the development of highly specific small interfering RNAs (siRNA). Although delivery of siRNA using multifunctional nanoparticles has been investigated to treat solid cancers, the application of this approach to blood cancers is at an early stage. This review describes current treatments for leukaemia and highlights the potential of leukaemic fusion genes as therapeutic targets for RNA interference (RNAi). In addition, the design of biomimetic nanoparticles which are capable of responding to the physiological environment of leukaemia and their potential to advance RNAi therapeutics to the clinic will be critically evaluated.

  10. Nanostructures to modulate vascular inflammation: Multifunctional nanoparticles for quantifiable siRNA delivery and molecular imaging

    NASA Astrophysics Data System (ADS)

    Kaneda, Megan Marie

    Early steps in the progression of inflammatory diseases such as atherosclerosis involve the recruitment of leukocytes to the vascular endothelium through the expression or up-regulation of adhesion molecules. These adhesion molecules are critical mediators of leukocyte attachment and subsequent extravasation through transendothelial migration. One of these adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) is particularly attractive as a marker of early atherosclerotic activity due to its low expression level on normal endothelium and up-regulation prior to and during the development of early lesions. With this in mind, the purpose of this thesis was to develop nanostructures for the detection and down-regulation of adhesion molecules by the vascular endothelium. To detect early inflammation we designed a perfluorocarbon nanoparticle (PFC-NP) probe, which was used for in vivo targeting of VCAM-1. Nanoparticles were detected ex vivo by the magnetic resonance (MR) signature from the fluorine core of the particle. Nanoparticles accumulated in tissues characterized by early inflammatory processes. To down-regulate VCAM-1 expression by vascular endothelial cells, cationic PFC-NP were produced through the addition of the cationic lipid 1,2-Dioleoyl-3-Trimethylammonium-Propane. Cationic PFC-NP were able to deliver anti-VCAM-1 siRNA to endothelial cells through a non-standard lipid raft mediated endocytic pathway. VCAM-1 levels were significantly reduced in treated cells indicating that this delivery mechanism may be advantageous for delivery of cargo into the cytoplasm. Using the fluorine signature from the core of the cationic PFC-NP, we were able to quantify and localize this siRNA delivery agent both in vitro and in vivo. The ability to quantify the local concentrations of these particles could be of great benefit for estimating local drug concentrations and developing new pharmacokinetic and pharmacodynamic paradigms to describe this new class of

  11. Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination

    PubMed Central

    Yao, Youli; Bilichak, Andriy; Golubov, Andrey; Kovalchuk, Igor

    2016-01-01

    ABSTRACT Small interfering RNAs (siRNAs) are involved in the regulation of plant development and response to stress. We have previously shown that mutants impaired in Dicer-like 2 (DCL2), DCL3 and DCL4, RDR2, RDR6 and NPRD1 are partially impaired in their response to stress and dcl2 and dcl3 plants are also impaired in transgenerational response to stress, including changes in homologous recombination frequency (HRF). Here, we have analyzed genome stability of dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl3 dcl4 and rdr6 mutants by measuring the non-induced and the stress-induced recombination frequency. We found that all mutants had the lower spontaneous HRF. The analysis of strand breaks showed that all tested Arabidopsis mutants had a higher level of spontaneous strand breaks, suggesting that the lower HRF is not due to the unusually low level of breaks. Exposure to methyl methane sulfonate (MMS) resulted in an increase in the level of strand breaks in wild-type plants and a decrease in mutants. All mutants had the higher methylation of cytosines at CpG sites under non-induced conditions. Exposure to MMS resulted in a decrease in methylation level in wild-type plants and an increase in methylation in all dcl mutants. The expression of several DNA repair genes was altered in dcl4 plants under non-induced and induced conditions. Our data suggest that siRNA biogenesis may be essential for the maintenance of the genome stability and stress response in Arabidopsis. PMID:26901311

  12. Loading and pre-loading processes generate a distinct siRNA population in Tetrahymena

    SciTech Connect

    Mochizuki, Kazufumi Kurth, Henriette M.

    2013-07-05

    Highlights: •The Tetrahymena Argonaute protein Twi1p binds to ∼28–30-nt siRNAs called scnRNAs. •The size of scnRNAs is determined during a pre-loading process. •The 5′ uracil bias of scnRNAs is attributed to pre-loading and loading processes. •The thermodynamic asymmetry of scnRNA duplex doesnot affect the guide strand decision. •scnRNAs may be produced non-sequentially from dsRNA substrates by Dicer. -- Abstract: The various properties of small RNAs, such as length, terminal nucleotide, thermodynamic asymmetry and duplex mismatches, can impact their sorting into different Argonaute proteins in diverse eukaryotes. The developmentally regulated 26- to 32-nt siRNAs (scnRNAs) are loaded to the Argonaute protein Twi1p and display a strong bias for uracil at the 5′ end. In this study, we used deep sequencing to analyze loaded and unloaded populations of scnRNAs. We show that the size of the scnRNA is determined during a pre-loading process, whereas their 5′ uracil bias is attributed to both pre-loading and loading processes. We also demonstrate that scnRNAs have a strong bias for adenine at the third base from the 3′ terminus, suggesting that most scnRNAs are direct Dicer products. Furthermore, we show that the thermodynamic asymmetry of the scnRNA duplex does not affect the guide and passenger strand decision. Finally, we show that scnRNAs frequently have templated uracil at the last base without a strong bias for adenine at the second base indicating non-sequential production of scnRNAs from substrates. These findings provide a biochemical basis for the varying attributes of scnRNAs, which should help improve our understanding of the production and turnover of scnRNAs in vivo.

  13. Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination.

    PubMed

    Yao, Youli; Bilichak, Andriy; Golubov, Andrey; Kovalchuk, Igor

    2016-07-02

    Small interfering RNAs (siRNAs) are involved in the regulation of plant development and response to stress. We have previously shown that mutants impaired in Dicer-like 2 (DCL2), DCL3 and DCL4, RDR2, RDR6 and NPRD1 are partially impaired in their response to stress and dcl2 and dcl3 plants are also impaired in transgenerational response to stress, including changes in homologous recombination frequency (HRF). Here, we have analyzed genome stability of dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl3 dcl4 and rdr6 mutants by measuring the non-induced and the stress-induced recombination frequency. We found that all mutants had the lower spontaneous HRF. The analysis of strand breaks showed that all tested Arabidopsis mutants had a higher level of spontaneous strand breaks, suggesting that the lower HRF is not due to the unusually low level of breaks. Exposure to methyl methane sulfonate (MMS) resulted in an increase in the level of strand breaks in wild-type plants and a decrease in mutants. All mutants had the higher methylation of cytosines at CpG sites under non-induced conditions. Exposure to MMS resulted in a decrease in methylation level in wild-type plants and an increase in methylation in all dcl mutants. The expression of several DNA repair genes was altered in dcl4 plants under non-induced and induced conditions. Our data suggest that siRNA biogenesis may be essential for the maintenance of the genome stability and stress response in Arabidopsis.

  14. Effect of siRNA PERK on fluoride-induced osteoblastic differentiation in OS732 cells.

    PubMed

    Lü, Peng; Li, Xining; Ruan, Lihong; Xu, Hui; Liu, Qinyi

    2014-06-01

    The purpose of this work is to study the action of fluoride on osteoblastic function through knocking down double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) mRNA in OS732 cells (human osteoblast-like cell line). The previous researches had demonstrated that fluoride induced endoplasmic reticulum (ER) stresses in other cells or tissues. PERK as one branch of UPR to combat ER stress played a role in mediating the proliferation and differentiation of osteoblast. The mechanism of skeletal fluorosis by which fluoride regulated osteoblast was not fully defined. We used the real-time PCR and small interfering RNA techniques to determine the expression PERK signaling and osteoblastic and osteoclastic differentiation-related factors and investigated the role of PERK signaling in fluoride-stimulated osteoblastic function. Cells transfected with 50 nM small interfering RNA (siRNA)-PERK showed effectively decreased protein and gene expression of PERK and reduced protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Meantime, cells transfected with siRNA significantly decreased the protein level of alkaline phosphatase (ALP) and nuclear factor kappa B ligand (RANKL) in cells under fluoride exposure. It suggested that knockdown of PERK expression hardly stimulated osteoblastic and osteoclastic early differentiation induced by fluoride. Conversely, there were littler effect of siRNA PERK on expression of Runt-related transcription factor 2 (RUNX2) and osteoprotegerin (OPG) in cells, but fluoride exposure markedly stimulated their expression. This study proved that the mechanism underlying fluoride induced osteoblastic and osteoclastic differentiation possible was due to activation of ALP and RANKL mediated by PERK in OS732 cells.

  15. Combination Anticancer Nanopreparations of Novel Proapoptotic Drug, TRAIL and siRNA

    NASA Astrophysics Data System (ADS)

    Riehle, Robert D.

    Development of drugs for the treatment of cancer is a challenging endeavor often hindered by the solubility and distribution of the drug in the body. Drug delivery systems have been used for many years to overcome these issues. Polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles in particular have shown utility as a nanosized drug delivery vehicle capable of incorporating poorly soluble drugs and preferentially delivering them to the tumor. Addition of PEG polymers to the surface prolongs the half-life of the particle in the blood by evading clearance by the reticuloendothelial system (RES) and increases tumor accumulation through the utilization of the enhanced permeability and retention (EPR) effect. Micelles have also been shown to successfully incorporate and protect modified siRNA, a notoriously challenging therapeutic to deliver. Additionally, co-delivery of multiple therapeutics in multifunctional micelles has emerged as an important area in combination therapy research. The main goal of this project was to develop a multifunctional PEG-PE micellar delivery system capable of delivering multiple therapeutics for increased anti-tumor activity. Previous studies have indicated the utility of a DM-PIT-1, a member of a class of novel PIP3-PH inhibitors, and its potential in the treatment of cancer. The PIP3-kinase (PI3K) pathway has been shown to have serious implications in cancer. Inhibiting this pathway has been shown to sensitize the cell to apoptosis. A second generation of more potent and druggable compounds has been developed based on the structure of DM- PIT-1. However, it has been difficult to develop successful compounds inhibiting PIP3 signaling while maintaining the physicochemical properties necessary for an effective drug. Many of these compounds are limited by their poor solubility and rapid clearance in vivo. Incorporating these compounds into PEG-PE micelles allows for increased solubility, prolonged half-life and tumor accumulation

  16. Solar-system abundances of the elements

    NASA Technical Reports Server (NTRS)

    Anders, E.; Ebihara, M.

    1982-01-01

    Elemental analyses of the Ogueil Cl meteorite and all previous Cl chondrite analyses were employed to develop a new solar system abundance table, including the standard deviation and number of analyses for each element. The table also comprises the abundances of radioactive and radiogenic nuclides at the present and 4.55 AE ago, as well as abundances by weight in a typical Cl chondrite. The new abundances were within 20% of those determined by Cameron (1982), except for 14 cases in the range 20-50%, and 5 over 50%. The solar abundances were compared with the Cl abundances, showing a total of only 7 disagreements. No significant discrepancies were detected in the major cosmochemical groups, and a smooth trend was found in the abundances of odd-A nuclides. The new set is interpreted as accurate to 10%, with the Cl chondrites matching the primordial solar system abundances to at most 10% deviation.

  17. 2’f-OMe-phosphorodithioate modified siRNAs show increased loading into the RISC complex and enhanced anti-tumour activity

    PubMed Central

    Wu, Sherry Y.; Yang, Xianbin; Gharpure, Kshipra M.; Hatakeyama, Hiroto; Egli, Martin; McGuire, Michael H.; Nagaraja, Archana S.; Miyake, Takahito M.; Rupaimoole, Rajesha; Pecot, Chad V.; Taylor, Morgan; Pradeep, Sunila; Sierant, Malgorzata; Rodriguez-Aguayo, Cristian; Choi, Hyun J.; Previs, Rebecca A.; Armaiz-Pena, Guillermo N.; Huang, Li; Martinez, Carlos; Hassell, Tom; Ivan, Cristina; Sehgal, Vasudha; Singhania, Richa; Han, Hee-Dong; Su, Chang; Kim, Ji Hoon; Dalton, Heather J.; Kowali, Chandra; Keyomarsi, Khandan; McMillan, Nigel A.J.; Overwijk, Willem W.; Liu, Jinsong; Lee, Ju-Seog; Baggerly, Keith A.; Lopez-Berestein, Gabriel; Ram, Prahlad T.; Nawrot, Barbara; Sood, Anil K.

    2014-01-01

    Improving small interfering RNA (siRNA) efficacy in target cell populations remains a challenge to its clinical implementation. Here, we report a chemical modification, consisting of phosphorodithioate (PS2) and 2’-O-Methyl (2’-OMe) MePS2 on one nucleotide that significantly enhances potency and resistance to degradation for various siRNAs. We find enhanced potency stems from an unforeseen increase in siRNA loading to the RNA-induced silencing complex, likely due to the unique interaction mediated by 2’-OMe and PS2. We demonstrate the therapeutic utility of MePS2 siRNAs in chemoresistant ovarian cancer mouse models via targeting GRAM Domain Containing 1B (GRAMD1B), a protein involved in chemoresistance. GRAMD1B silencing is achieved in tumors following MePS2-modified siRNA treatment, leading to a synergistic anti-tumor effect in combination with paclitaxel. Given the previously limited success in enhancing siRNA potency with chemically modified siRNAs, our findings represent an important advance in siRNA design with the potential for application in numerous cancer types. PMID:24619206

  18. Single-walled carbon nanotubes noncovalently functionalized with lipid modified polyethylenimine for siRNA delivery in vitro and in vivo.

    PubMed

    Siu, King S; Zheng, Xiufen; Liu, Yanling; Zhang, Yujuan; Zhang, Xusheng; Chen, Di; Yuan, Ken; Gillies, Elizabeth R; Koropatnick, James; Min, Wei-Ping

    2014-10-15

    siRNA can downregulate the expression of specific genes. However, delivery to specific cells and tissues in vivo presents significant challenges. Modified carbon nanotubes (CNTs) have been shown to protect siRNA and facilitate its entry into cells. However, simple and efficient methods to functionalize CNTs are needed. Here, noncovalent functionalization of CNTs is performed and shown to effectively deliver siRNA to target cells. Specifically, single-walled CNTs were functionalized by noncovalent association with a lipopolymer. The lipopolymer (DSPE-PEG) was composed of a phospholipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and poly(ethylene glycol) (PEG). Three different ratios of polyethylenimine (PEI) to DSPE-PEG were synthesized and characterized and the products were used to disperse CNTs. The resulting materials were used for siRNA delivery in vitro and in vivo. The structural, biophysical, and biological properties of DGI/C and their complexes formed with siRNA were investigated. Cytotoxicity of the materials was low, and effective gene silencing in B16-F10 cells was demonstrated in vitro. In addition, significant uptake of siRNA as well as gene silencing in the liver was found following intravenous injection. This approach provides a new strategy for siRNA delivery and could provide insight for the development of noncovalently functionalized CNTs for siRNA therapy.

  19. Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer

    PubMed Central

    Nourbakhsh, Mahnaz; Jaafari, Mahmoud Reza; Lage, Hermann; Abnous, Khalil; mosaffa, Fatemeh; Badiee, Ali; Behravan, Javad

    2015-01-01

    Objective(s): P-glycoprotein (P-gp) is an efflux protein, the overexpression of which has been associated with multidrug resistance in various cancers. Although siRNA delivery to reverse P-gp expression may be promising for sensitizing of tumor cells to cytotoxic drugs, the therapeutic use of siRNA requires effective carriers that can deliver siRNA intracellularly with minimal toxicity on target cells. We investigated a special class of PEGylated lipid-based nanoparticles (NP), named nanolipoparticles (NLPs), for siRNA-mediated P-gp downregulation. Materials and Methods: NLPs were prepared based on low detergent dialysis method. After characterization, we evaluated the effect of NLPs on siRNA delivery, and P-gp downregulation compared to oligofectamine™ (OFA) in vitro and in vivo. Results: Our results showed a significant decrease in P-gp expression and subsequent enhancement of chemosensitivity to doxorubicin in vitro. Although the effectiveness of NLPs for in vitro siRNA delivery compared to OFA was limited, the results of in vivo studies showed noticeable effectiveness of NLPs for systemic siRNA delivery. siRNA delivery using NLPs could downregulate MDR1 in tumor cells more than 80%, while OFA had a reverse effect on MDR1 expression in vivo. Conclusion: The results indicated that the prepared NLPs could be suitable siRNA delivery systems for tumor therapy. PMID:26019802

  20. Human Papillomavirus E6/E7-Specific siRNA Potentiates the Effect of Radiotherapy for Cervical Cancer in Vitro and in Vivo.

    PubMed

    Jung, Hun Soon; Rajasekaran, Nirmal; Song, Sang Yong; Kim, Young Deug; Hong, Sungyoul; Choi, Hyuck Jae; Kim, Young Seok; Choi, Jong-Sun; Choi, Yoon-La; Shin, Young Kee

    2015-05-29

    The functional inactivation of TP53 and Rb tumor suppressor proteins by the HPV-derived E6 and E7 oncoproteins is likely an important step in cervical carcinogenesis. We have previously shown siRNA technology to selectively silence both E6/E7 oncogenes and demonstrated that the synthetic siRNAs could specifically block its expression in HPV-positive cervical cancer cells. Herein, we investigated the potentiality of E6/E7 siRNA candidates as radiosensitizers of radiotherapy for the human cervical carcinomas. HeLa and SiHa cells were transfected with HPV E6/E7 siRNA; the combined cytotoxic effect of E6/E7 siRNA and radiation was assessed by using the cell viability assay, flow cytometric analysis and the senescence-associated β-galactosidase (SA-β-Gal) assay. In addition, we also investigated the effect of combined therapy with irradiation and E6/E7 siRNA intravenous injection in an in vivo xenograft model. Combination therapy with siRNA and irradiation efficiently retarded tumor growth in established tumors of human cervical cancer cell xenografted mice. In addition, the chemically-modified HPV16 and 18 E6/E7 pooled siRNA in combination with irradiation strongly inhibited the growth of cervical cancer cells. Our results indicated that simultaneous inhibition of HPV E6/E7 oncogene expression with radiotherapy can promote potent antitumor activity and radiosensitizing activity in human cervical carcinomas.

  1. Hematite Abundance Map at Echo

    NASA Technical Reports Server (NTRS)

    2004-01-01

    This image shows the hematite abundance map for a portion of the Meridiani Planum rock outcrop near where the Mars Exploration Rover Opportunity landed. It was acquired by the rover's miniature thermal emission spectrometer instrument from a spot called 'Echo.' Portions of the inner crater wall in this region appear rich in hematite (red). The sharp boundary from hematite-rich to hematite-poor (yellow and green) surfaces corresponds to a change in the surface texture and color. The hematite-rich surfaces have ripple-like forms suggesting wind transported hematite to these surfaces. The bounce marks produced during landing at the base of the slope on the left are low in hematite (blue). The hematite grains that originally covered the surface were pushed below the surface by the lander, exposing a soil that has less hematite.

  2. Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible Fluorinated Peptide Dendrimers for Cancer Therapy.

    PubMed

    Cai, Xiaojun; Zhu, Haofang; Zhang, Yanmei; Gu, Zhongwei

    2017-03-22

    RNA interference (RNAi) has a great promise in treating various acquired and hereditary diseases. However, it remains highly desirable to develop new delivery system to circumvent complex extra- and intracellular barriers for successful clinical translation. Here, we report on a versatile polymeric vector, bioreducible fluorinated peptide dendrimers (BFPD), for efficient and safe small interfering RNA (siRNA) delivery. In virtue of skillfully integrating all of the unique advantages of reversible cross-linking, fluorination, and peptide dendrimers, this novel vector can surmount almost all extra- and intracellular barriers associated with local siRNA delivery through highly improved physiological stability and serum resistance, significantly increased intratumoral enrichment, cellular internalization, successful facilitation of endosomal escape, and cytosolic siRNA release. BFPD polyplexes, carrying small interfering vascular endothelial growth factor (siVEGF), demonstrated excellent VEGF silencing efficacy (∼65%) and a strong capability for inhibiting HeLa cell proliferation. More importantly, these polyplexes showed superior performance in long-term enrichment in the tumor sites and had a high level of tumor growth inhibition. Furthermore, these polyplexes not only exhibited excellent in vivo antitumor efficacy but also demonstrated superior biocompatibility, compared with LPF2000, both in vivo and in vitro. These findings indicate that BFPD is an efficient and safe siRNA delivery system and has remarkable potential for RNAi-based cancer treatment.

  3. Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

    PubMed

    Grondin, Richard; Ge, Pei; Chen, Qingmin; Sutherland, Jessica E; Zhang, Zhiming; Gash, Don M; Stiles, David K; Stewart, Gregory R; Sah, Dinah W Y; Kaemmerer, William F

    2015-06-30

    One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

  4. Kindlin-2 siRNA inhibits vascular smooth muscle cell proliferation, migration and intimal hyperplasia via Wnt signaling.

    PubMed

    Wu, Xiaolin; Liu, Wenwei; Jiang, Hong; Chen, Jing; Wang, Jichun; Zhu, Rui; Li, Bin

    2016-02-01

    It is known that vascular smooth muscle cell (VSMC) proliferation and migration leads to intimal hyperplasia in cases of atherosclerosis and restenosis. In the present study, we investigated the effects of kindlin-2 on VSMC proliferation, migration and intimal hyperplasia, and the underlying mechanisms. The left common carotid artery of Sprague‑Dawley rats were subjected to balloon injury in order to induce intimal hyperplasia, and then transfected with kindlin-2 small interfering RNA (siRNA) lentivirus or negative control siRNA lentivirus. We noted that the degree of intimal hyperplasia 4 weeks after balloon injury was significantly reduced in arteries transfected with kindlin-2 siRNA lentivirus (P<0.05). In vitro, kindlin-2 siRNA suppressed VSMC proliferation and migration induced by Wnt3a (100 ng/ml). Western blot analyses and RT-qPCR revealed that kindlin-2 regulated Wnt/β-catenin signaling and thereby modulated the expression of β-catenin target genes, including c-myc and cyclin D1. This study demonstrated that kindlin-2 plays a critical role in VSMC proliferation, migration and intimal hyperplasia via Wnt signaling. Therefore, blocking the activity of kindlin-2 represents a novel therapeutic strategy for vascular injury.

  5. Nanoparticle delivery of Wnt-1 siRNA enhances photodynamic therapy by inhibiting epithelial-mesenchymal transition for oral cancer.

    PubMed

    Ma, Chuan; Shi, Leilei; Huang, Yu; Shen, Lingyue; Peng, Hao; Zhu, Xinyuan; Zhou, Guoyu

    2017-02-28

    Activation of the epithelial to mesenchymal transition (EMT) in photodynamic therapy (PDT) can lead to the recurrence and progression of tumors. To enhance the effects of PDT, it is essential to inhibit the Wnt/β-catenin signaling pathway involved in EMT progression. Herein, we used polyethylene glycol-polyethyleneimine-chlorin e6 (PEG-PEI-Ce6) nanoparticles to efficiently deliver Wnt-1 small interfering RNA (siRNA) to the cytoplasm of KB cells (oral squamous cell carcinoma) that were subjected to PDT. Wnt-1 siRNA effectively inhibited the Wnt/β-catenin signaling pathway, reducing the expression of Wnt-1, β-catenin and vimentin that are crucial to the EMT. Combined with Wnt-1 siRNA, PEG-PEI-Ce6 nanoparticle mediated PDT inhibited cell growth and enhanced the cancer cell killing effect remarkably. Our results show the promise of combination therapy of PEG-PEI-Ce6 nanoparticles for delivery of Wnt-1 siRNA along with PDT in the treatment of oral cancer.

  6. Effect of the guide strand 3'-end structure on the gene-silencing potency of asymmetric siRNA.

    PubMed

    Hong, Sun Woo; Park, June Hyun; Yun, Soyeong; Lee, Chang Han; Shin, Chanseok; Lee, Dong-ki

    2014-08-01

    siRNAs are short dsRNAs that mediate efficient target gene silencing in a sequence-specific manner. We previously developed a novel siRNA structure, called asiRNA (asymmetric siRNA), which alleviates the off-target effects associated with conventional siRNA structures without decreasing target gene silencing potency. In the present study, we explored the effect of the guide strand 3'-end structure on the gene silencing potency of asiRNA. Interestingly, asiRNAs with a 21 nt guide strand solely composed of RNA resulted in gene silencing that was more than 6-fold more efficient compared with the corresponding asiRNA guide strand harbouring a dTdT (deoxythymidine dinucleotide) at its 3'-end. We demonstrated that the molecular basis of potency of the asiRNA with a 21 nt guide strand composed solely of RNA was due to the enhanced formation of the RISC (RNA-induced silencing complex) and increased affinity towards hAgo2 (human Argonaute2). Our observations may assist researchers in designing new asiRNAs with high on-target silencing efficiency with low off-target effects, which is critical for applications in both basic research and therapeutic development.

  7. [CCR7 silence by siRNA inhibits proliferation, invasion and promotes apoptosis of human MG63 osteosarcoma cells].

    PubMed

    Zhang, Richun; Zhang, Hongtao; E, Zhen; Ma, Qiong; Yan, Shiju; Zhang, Enwei; Ma, Bao'an

    2016-12-01

    Objective To investigate the effect of siRNA-mediated chemokine receptor 7 (CCR7) silence on the proliferation, migration, invasion and apoptosis of human MG-63 osteosarcoma cells. Methods The study designed and synthesized siRNA targeting CCR7 (CCR7-siRNA). After MG63 cells were transfected with CCR7-siRNA, the expression of CCR7 was identified by Western blotting; cell apoptosis was detected by annexinV-FITC/PI double staining combined with flow cemetery; cell proliferation was tested by MTT assay; and cell migration and invasion abilities were examined by Transwell(TM) migration/invasion assays. Results CCR7 expression in MG63 cells was significantly inhibited after transfected with CCR7-siRNA. At the same time, cell proliferation, migration and invasion abilities were distinctly suppressed, and cell apoptosis rate increased. Conclusion Down-regulating CCR7 expression in MG63 cells could apparently inhibit cell proliferation, migration and invasion abilities of MG63 cells, and also induce cell apoptosis.

  8. A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis

    PubMed Central

    Ye, Ruiqiang; Chen, Zulong; Lian, Bi; Rowley, M. Jordan; Xia, Ning; Chai, Jijie; Li, Yan; He, Xin-Jian; Wierzbicki, Andrzej T.; Qi, Yijun

    2016-01-01

    SUMMARY DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. Twenty-four-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ~20 to 60 nt in length, often having same 5’ ends but differing in 3’ ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on distributive 3’-5’ exonucleases. Our findings suggest an alternative route for siRNA biogenesis: precursor transcripts are bound by AGO4 and subsequently subjected to 3’-5’ exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation. PMID:26711010

  9. Solid formulation of cell-penetrating peptide nanocomplexes with siRNA and their stability in simulated gastric conditions.

    PubMed

    Ezzat, Kariem; Zaghloul, Eman M; El Andaloussi, Samir; Lehto, Taavi; El-Sayed, Ramy; Magdy, Tarek; Smith, C I Edvard; Langel, Ulo

    2012-08-20

    Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect is obtained at low siRNA doses with a unique kinetic profile. Furthermore, the solid dispersion technique is utilized to formulate PF14/siRNA nanocomplexes into solid formulations that are as active as the freshly prepared nanocomplexes in solution. Importantly, the nanocomplexes are stable and active in mediating RNAi response after incubation with simulated gastric fluid (SGF) that is highly acidic. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.

  10. Comparing 2-nt 3' overhangs against blunt-ended siRNAs: a systems biology based study

    PubMed Central

    Ghosh, Preetam; Dullea, Robert; Fischer, James E; Turi, Tom G; Sarver, Ronald W; Zhang, Chaoyang; Basu, Kalyan; Das, Sajal K; Poland, Bradley W

    2009-01-01

    In this study, we formulate a computational reaction model following a chemical kinetic theory approach to predict the binding rate constant for the siRNA-RISC complex formation reaction. The model allowed us to study the potency difference between 2-nt 3' overhangs against blunt-ended siRNA molecules in an RNA interference (RNAi) system. The rate constant predicted by this model was fed into a stochastic simulation of the RNAi system (using the Gillespie stochastic simulator) to study the overall potency effect. We observed that the stochasticity in the transcription/translation machinery has no observable effects in the RNAi pathway. Sustained gene silencing using siRNAs can be achieved only if there is a way to replenish the dsRNA molecules in the cell. Initial findings show about 1.5 times more blunt-ended molecules will be required to keep the mRNA at the same reduced level compared to the 2-nt overhang siRNAs. However, the mRNA levels jump back to saturation after a longer time when blunt-ended siRNAs are used. We found that the siRNA-RISC complex formation reaction rate was 2 times slower when blunt-ended molecules were used pointing to the fact that the presence of the 2-nt overhangs has a greater effect on the reaction in which the bound RISC complex cleaves the mRNA. PMID:19594876

  11. Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1/PCTK1/Cdk16 Inhibits In Vivo Cancer Growth

    PubMed Central

    Yanagi, Teruki; Tachikawa, Kiyoshi; Wilkie-Grantham, Rachel; Hishiki, Asami; Nagai, Ko; Toyonaga, Ellen; Chivukula, Pad; Matsuzawa, Shu-ichi

    2016-01-01

    PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5 mg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells. PMID:27351680

  12. A combination of a DNA-chimera siRNA against PLK-1 and zoledronic acid suppresses the growth of malignant mesothelioma cells in vitro.

    PubMed

    Kawata, Eri; Ashihara, Eishi; Nakagawa, Yoko; Kiuchi, Takahiro; Ogura, Mai; Yao, Hisayuku; Sakai, Kazuki; Tanaka, Ruriko; Nagao, Rina; Yokota, Asumi; Takeuchi, Miki; Kimura, Shinya; Hirai, Hideyo; Maekawa, Taira

    2010-08-28

    Although novel agents effective against malignant mesothelioma (MM) have been developed, the prognosis of patients with MM is still poor. We generated a DNA-chimeric siRNA against polo-like kinase-1 (PLK-1), which was more stable in human serum than the non-chimeric siRNA. The chimeric PLK-1 siRNA inhibited MM cell proliferation through the induction of apoptosis. Next, we investigated the effects of zoledronic acid (ZOL) on MM cells, and found that ZOL also induced apoptosis in MM cells. Furthermore, ZOL augmented the inhibitory effects of the PLK-1 siRNA. In conclusion, combining a PLK-1 siRNA with ZOL treatment is an attractive strategy against MM.

  13. Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization.

    PubMed

    deRonde, Brittany M; Posey, Nicholas D; Otter, Ronja; Caffrey, Leah M; Minter, Lisa M; Tew, Gregory N

    2016-06-13

    Exploring the role of polymer structure for the internalization of biologically relevant cargo, specifically siRNA, is of critical importance to the development of improved delivery reagents. Herein, we report guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold containing tunable hydrophobic moieties that promote siRNA internalization. Structure-activity relationships using Jurkat T cells and HeLa cells were explored to determine how the length of the hydrophobic block and the hydrophobic side chain compositions of these PTDMs impacted siRNA internalization. To explore the hydrophobic block length, two different series of diblock copolymers were synthesized: one series with symmetric block lengths and one with asymmetric block lengths