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There is a bus stop right in front of the B and B which made it so easy to get around the city. Rooms large and wonderfully decorated. Location at Logan Circle offers the option of many restaurants and cafes. Good breakfast and great conversation around the table. We will definitely return.
- Official Description (provided by the hotel):
- Enjoy old-world charm with modern conveniences at our Victorian Bed and Breakfast. We are centrally located 8 blocks north of the White House at historic Logan Circle. Metro access is a 10-minute walk, or 10-minutes by bus to the Smithsonian and National Mall. Washington's Convention Center is a 10-15 minute walk. There are 4 guestrooms, each with private bath. Some rooms can accommodate extra guests. Well-behaved children welcome, and off-street parking is available. Daily maid service on request. ... more less
- Also Known As:
- Chester Arthur House Bed And Breakfast At Logan Circle
- Chester Arthur House b & b At Logan Circle Hotel Washington Dc


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We loved the park and did spend a whole day there. We liked the pool-area and the playgrounds.
It was a very hot day, and it was almost to hot to be outside, therefor we were very pleased to find a indoor playing area, that was really fantastic. Very nice decorated, and the kids loved it (and it was nok like the indoor plastic monkey tonkey playgrounds, you find everywhere).
Also a nice area with the animals. The thing about the park is, that there is so many different things to do, so you don't get tired or bored.
Only negativ thing is, that the food stinks. I can't believe it is so difficult to make food, that is not junk.
I would recomend this park for parents and kids under 10.
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Synonyms of uninhabited in English:
1 much of this land was uninhabited
2 an uninhabited hut
vacant, empty, unoccupied, untenanted, tenantless, unfilled, to let, free;
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Lazareth, a motorcycle and automobile tuner that hails from France, is known for producing some of the most outrageous custom creations in all of Europe. Its latest, known as the Wazuma GT, can be seen above. It's got four wheels but really only three contact points, the minimum amount of bodywork possible, two seats and an engine.
Lazareth Wazuma GT: Geneva 2014
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Orlando Mini Maker Faire 2013
Application for the 2013 Mini Maker Faire in Orlando, Florida.This app provides a schedule of events, list of makers that will be exhibiting
OTA RootKeeper
First versions of Voodoo OTA Root Keeper. Also serves as temporary un-root (see statement) Distributed as free of charge - no warranty. Ope


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A two-million-channel 40-MHz-bandwidth, digital spectrum analyzer is described. Its synchronization and configuration control are discussed. The two-million-point matrix discrete Fourier transform at the core of the analyzer, which transforms the time-domain input signal into the frequency domain, is examined. The real-adjust stage and frequency-domain processing modules are also highlighted. The analyzer system will serve as a prototype processor for the sky survey portion of NASA's search for extraterrestrial intelligence program and for other applications in the deep space network


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Applications are now being accepted for the 2016 Cox Communications Scholarship program. more
By Lori Stabile
Google and Apple Inc. have some bright ideas about the future of health care. more
By Scott Moritz, Olga Kharif and Gerrit De Vynck
Eight University of Massachusetts technology projects will each receive $25,000 in Tech Development Fund Awards, University of Massachusetts President Martin T. Meehan said Thursday. more
By Emily Gowdey-Backus
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Twitter: @FlashGowdey | 4/7/16
Medrobotics Corp. believes it has changed the field of invasive surgery, with the first-ever surgical tool that can snake through the human body, without disturbing other anatomy, to reach previously difficult-to-access regions. The … more
Hope Valley Industries in North Kingstown, which makes automotive accessories such as floor mats and mudflaps, could use a few more CNC programmers. To that end, says Julie Zito, the firm's director of human resources, the company plans to … more
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PROVIDENCE – A new computer science initiative, Computer Science for RI, to help every public school in Rhode Island teach computer science classes by December 2017, was launched last week by Gov. Gina M. Raimondo. Raimondo, whose budget … more
In 2012, then FBI Director Robert Mueller warned, "There are only two types of companies: those that have been hacked, and those that will be." Earlier this year, a federal court judge offered a more grim proclamation that the only two types of … more
The vast reach of cybercriminals and the vulnerability of networked systems threaten businesses big and small, say cybercrime specialists and others who participated in last week's Cybersecurity Summit put on by Providence Business News. … more
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Two adults and two juveniles have been charged in connection with a theft Wednesday night in Hammonton that occurred after the juveniles went to a friend's house.
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Theory in Contemporary Art Since 1985 / Edition 1by Simon Leung
View All Available Formats & Editions
Pub. Date: 06/28/2004
Publisher: Wiley
- Provides the first truly comprehensive and international anthology of theory in contemporary art of the last two decades.
- Brings together a broad selection of important contributions to the fields of
Theory in Contemporary Art since 1985 is a groundbreaking anthology that captures the essence and the edge of the contemporary art scene.
- Provides the first truly comprehensive and international anthology of theory in contemporary art of the last two decades.
- Brings together a broad selection of important contributions to the fields of contemporary art, theory, and culture from established and emergent art voices, including scholars, curators, critics, and artists from around the globe.
- Focuses on key theoretical and aesthetic issues in contemporary art, such as cultural/multicultural theory, identity politics, AIDS, post-colonialism, globalization, and spectatorship.
- Includes editorial material and 44 illustrations.
Product Details
- 9780631228677
- Publisher:
- Wiley
- Publication date:
- 06/28/2004
- Edition description:
- Older Edition
- Pages:
- 472
- Product dimensions:
- 6.80(w) x 9.70(h) x 1.00(d)
Table of Contents
Notes on Contributors.
Text, Figure, and Plate Credits.
How To Use This Book.
Part I: Contemporary Art Practices and Models:.
1. The Intellectual Field: a world apart: Pierre Bourdieu (deceased 2001, Collège de France).
2. When Form Has Become Attitude – And Beyond: Thierry de Duve (University of Lille 3, France and the Hogeschool Sint Lucas Beeldende Kunst, Gent).
3. One Place After Another: Notes on Site Specificity: Miwon Kwon (UCLA).
4. The Curator's Moment: Michael Brenson (independent scholar).
5. How to Provide an Artistic Service: An Introduction: Andrea Fraser (artist).
6. Conversation Pieces: The Role of Dialogue in Socially Engaged Art: Grant Kester (University of California, San Diego).
7. 'yBa' as Critique: The Socio-Political Inferences of the Mediated: James Gaywood (independent scholar).
8. Video Projection: The Space Between Screens: Liz Kotz (University of Minnesota).
Part II: Culture/Identities/Political Fields:.
9. The War on Culture: Carole S. Vance (Columbia University).
10. Feminist Fundamentalism: Women against Images: Carole S. Vance (Columbia University).
11. AIDS: Cultural Analysis/Cultural Activism: Douglas Crimp (University of Rochester).
12. Architecture of the Evicted: Rosalyn Deutsche (Barnard College, Columbia University).
13. Gender is Burning: Questions of Appropriation and Subversion: Judith Butler (University of California, Berkeley).
14. Cornered: A Video Installation Project: Adrian Piper (Professor of Philosophy, Wellesley College).
15. The Mythology of Difference: Vulgar Identity Politics at the Whitney: Charles A. Wright, Jr. (independent scholar).
16. Haunted TV: Avital Ronell (New York University).
Part III: Post-colonial Critiques:.
17. The Marco Polo Syndrome: Some Problems around Art and Eurocentrism: Gerardo Mosquera (New Museum of Contemporary Art, New York).
18. In the 'Heart of Darkness': Olu Oguibe (University of Connecticut).
19. The Syncretic Turn: Cross-Cultural Practices in the Age of Multiculturalism: Jean Fisher (Middlesex University).
20. Authenticity, Reflexivity & Spectacle: or, the rise of New Asia is not the End of the World: Lee Weng Choy (The Substation, an independent contemporary art center in Singapore).
21. All-Owning Spectatorship: Trinh T. Minh-Ha (University of California, Berkeley).
Part IV: Rethinking Aesthetics:.
22. A Note on Gerhard Richter's October 18, 1977: Benjamin Buchloh (Barnard College, Columbia University).
23. Notes on Surface: toward a genealogy of flatness: David Joselit (Yale University).
24. Ruins, Fragmentation, and the Chinese Modern/Postmodern: Wu Hung (The University of Chicago).
25. Function and Field: Nana Last (Rice University).
26. 1989: Juli Carson (UCLA).
Part V: Theories after Postmodernism:.
27. Postmodernism and Periphery: Nelly Richard (Universidad Arcis, Santiago de Chile).
28. Looking for Trouble: Kobena Mercer (Goldsmith's).
29. Repossessing Popular Culture: Laura Kipnis (Northwestern University).
30. The Lightness of Theory: John Rajchman (Columbia University).
31. Informe Without Conclusion: Rosalind Krauss (Columbia University).
32. The Database: Lev Manovich (University of California, San Diego).
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Monday, May 28, 2012
Crunchy Vanilla Clusters
Crunchy Vanilla Clusters (adapted from Southern Living)
1 ( 3 ounce) pkg ramen noodles
3/4 cup pecan pieces
1/2 cup sliced almonds
2 Tbsp butter, melted
1 ( 12 ounce) pkg white chocolate chips
Gently crush ramen noodles before open package. Open package and discard flavoring packet. Saute crushed noodles, pecans and almonds in butter in a large skillet over medium heat until toasted - avoid crushing noodles too find while sauteing. Pour noodle mixture into a large bowl to cool. Melt white chocolate according to package directions. ( I used the microwave) Pour melted chocolate over cooled noodle mixture, tossing gently to coat. Drop candy mixture by rounded tablespoonfuls onto wax paper; let stand until firm - about 45 minutes. Store in an airtight container at room temperature. Makes about 3 dozen (we also like ours cold - stored in fridge )
4 comments:
- Val said...
My kiddies would love love love these. Such a fun recipe.
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text:
Dirac: A Scientific Biographyby Helge Kragh, Kragh Helge
View All Available Formats & Editions
Pub. Date: 07/31/2005
Publisher: Cambridge University Press
This first full-length biography of Paul Adrien Maurice Dirac offers a comprehensive account of his physics in its historical context, including less known areas such as cosmology and classical electron theory. It is based extensively on unpublished sources, including Dirac's correspondence with Bohr, Heisenberg, Pauli, Schrödinger, Gamow and others. Dirac was undoubtedly one of the most brilliant and influential physicists of the twentieth century. Between 1925 and 1934, the Nobel Prize laureate revolutionized physics with his brilliant contributions to quantum theory. This work examines Dirac's successes and failures, and pays particular attention to his opposition to modern quantum electrodynamics; an opposition based on aesthetic objections.
Product Details
- 9780521017565
- Publisher:
- Cambridge University Press
- Publication date:
- 07/31/2005
- Pages:
- 400
- Product dimensions:
- 6.14(w) x 9.21(h) x 0.83(d)
Table of ContentsPreface; 1. Early years; 2. Discovery of quantum mechanics; 3. Relativity and spinning electrons; 4. Travels and thinking; 5. The dream of philosophers; 6. Quanta and fields; 7. Fifty years of a physicist's life; 8. 'The so-called quantum electrodynamics'; 9. Electrons and ether; 10. Just a disappointment; 11. Adventures in cosmology; 12. The purest soul; 13. Philosophy in physics; 14. The principle of mathematical beauty; Appendices; Bibliography of P. A. M. Dirac; Notes and references; General bibliography; Index of names; Index of subjects.
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Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo.
Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and cationic-cholesterol modified gelatin (cCMG), were synthesized using different cholesterol derivatives such as the cholesterol-isocyanate (Ch-I) for aCMG and amino-modified cholesterol for cCMG. One was anionic and the other cationic, and therefore they differed in terms of their zeta potential. The aCMG and cCMG were characterized for their size, zeta potential, and in their ability to form micelles. Cytotoxicity was also evaluated. The modified human gelatins were then investigated as a carrier of antigenic proteins for inducing cellular immunity both in vitro in DC 2.4 cells, a murine dendritic cell line, as well as in vivo. The mechanism of entry of the polymeric micelles into the cells was also evaluated.
It was found that only cCMG successfully complexed with the model antigenic protein, fluorescein-isothiocyanate ovalbumin (OVA) and efficiently delivered and processed proteins in DC 2.4 cells. It was hypothesized that cCMG enter the cells predominantly by a caveolae-mediated pathway that required tyrosine kinase receptors on the cell surface. Animal testing using mice showed that the cationic cholesterol-modified gelatin complexed with OVA produced significantly high antibody titers against OVA: 2580-fold higher than in mice immunized with free OVA.
Conclusively, cCMG has shown to be very effective in stimulating an immune response due to its high efficiency, stability, and negligible cytotoxicity.
Vaccine administration has always been an attractive option in the treatment of a broad range of infectious diseases or even cancers by inducing effective immune responses. Delivering antigenic proteins to dendritic cells (DCs), which play a pivotal role in immunology, helps in activating antigen-specific cytotoxic T lymphocytes by processing antigenic proteins to the peptides that are important in achieving target-specific immunity by eliminating infected cells and pathogens directly from the body.1 Nanocarriers show great potential in their efficiency to deliver proteins and induce an optimal immune response safely. Several groups have worked on various antigen-delivery carriers.2–4 Polymer-derived nanoparticles are used extensively for vaccine delivery and have been approved for human use.5
Although many synthetic polymers are being used for biomedical applications, natural polymers have always remained an attractive choice because they have good biocompatibility, low toxicity, and can be fabricated readily by simple chemistry. Among the strategies used to synthesize polymeric nanocarriers by natural polymers, researchers have introduced hydrophobic segments such as cholesterol,6 tocopherol,7 and deoxycholic acid,8 thus making the polymers amphiphilic in nature. Such amphiphilic polymers have the ability to self-aggregate and hence act as efficient carriers. Thus, it is possible to design nanocarriers made of only natural materials by choosing natural hydrophobic segments.
Among the many natural polymers, gelatin represents a good raw material as a delivery system because of its affinity to cells.9–12 However, there are some drawbacks. Cross-linking with chemical compounds that are usually toxic is needed typically to prepare a gelatin nanocarrier.13 Transmittable diseases or allergic reactions such as anaphylaxis have been reported from gelatins originating from animal sources.14,15 Lastly, nanoparticles prepared from conventional gelatins pose a significant challenge to analytical characterization because of their heterogeneous nature, as they are made up of a mixture of differently sized peptides that are heterogeneous in molecular weight (MW).16 However, of the many nanoparticles developed, only a few possess properties that allow them to be used further in vivo to test their therapeutic abilities.
In this study, we used recombinant human gelatin (rhG) derived from human collagen. This overcomes the above limitations as it is homogeneous in MW, nontoxic, and is being considered as an alternative to conventional gelatin extracted from bovine or porcine sources.17,18 We report the synthesis of two types of rhG derivatives by using two kinds of cholesterol derivatives. They have the ability to form micelles but differ in terms of their zeta (ζ)-potential. They are referred to here as anionic cholesterol-modified gelatin (aCMG) and cationic cholesterol-modified gelatin (cCMG).
Recombinant human gelatin (MW 89.8 kDa) was purchased from Neosilk (Hiroshima, Japan). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was obtained from Dojindo Laboratories (Kumamoto, Japan). Ethylenediamine was purchased from Tokyo Chemical Industry Co, Limited (Tokyo, Japan) and ovalbumin (OVA), fluorescein isothiocyanate-labeled OVA (FITC-OVA), chlorpromazine, nystatin and cytochalasin D were obtained from Sigma-Aldrich (St Louis, MO). 4′,6-Diamidino-2-phenylindole (DAPI) and Vectashield mounting medium were purchased from Vector Laboratories (Burlingame, CA). LysoTracker™ Red DND-99 was obtained from Invitrogen (Carlsbad, CA). OVA-DQ™ was purchased from Molecular Probes (Eugene, OR). Cholesterol, hexamethylene diisocyanate, toluene (dry), pyridine (dry), triethylamine (TEA), hexane, dimethylsulfoxide (DMSO) (dehydrated), dichloromethane, 4% paraformaldehyde in phosphate-buffered saline (PBS), pyrene, and genistein were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
The immature dendritic cell line DC 2.4 was kindly provided by Dr Kenji Kono (Osaka Prefecture University, Osaka, Japan) with the permission of Dr KL Rock (Harvard Medical School, Boston, MA). The cells were cultured at 37°C in RPMI-1640 medium with l-glutamine (Gibco BRL, Invitrogen) and supplemented with 10% fetal bovine serum (FBS), 100 μM nonessential amino acids, 50 μM 2-mercaptoethanol, and antibiotics.
Six- to 10-week-old female C57BL/6NCrSlc mice (Japan SLC Inc, Shizuoka, Japan) were maintained at the animal facilities of RIKEN Brain Science Institute (Saitama, Japan) according to the guidelines of the institution. The mice were kept in an air-conditioned room and fed standard laboratory food and water ad libitum. All experiments were approved by the RIKEN Animal Experiments Committee.
Cholesteryl N-6-(isocyanatohexyl carbamate) or cholesterol isocyanate (Ch-I) was synthesized as reported previously.6 Briefly, cholesterol (7.80 g, 20.12 mmol) was reacted with 1,6-hexyldiisocyanate (64 mL, 0.39 mol) in 200 mL of dry toluene containing 15 mL of pyridine at 80°C for 48 hours. The reaction was monitored using thin-layer chromatography (TLC). After the removal of solvent in vacuo, 500 mL of hexane was added to the residue and kept overnight at −20°C. The precipitates thus obtained were then separated and dried in vacuo to give a yield of 3.88 g (49.74%). Proton nuclear magnetic resonance (1H NMR; 400 MHz, CDCl3, 25°C): δ = 0.60 (s, 3H, cholesterol-18 H3), 0.78–2.30 (m, 40H, cholesterol), 1.30–1.56 (m, 8H), 3.09 (m, 2H), 3.22 (t,3J (H,H) = 6.8 Hz, 2H), 4.42–4.51 (b, 1H, cholesterol), and 5.30 (m, 1H, cholesterol).
Cholesteryl N-6-(3-(2-aminoethyl)ureido)hexyl carbamate or amino-modified cholesterol (Ch-A) was synthesized as follows. A solution of Ch-I (200 mg, 0.360 mmol) in dichloromethane (10 mL) synthesized in the previous step was added dropwise to a vigorously stirred solution of ethylenediamine (0.481 mL, 7.2 mmol) in an excess of dichloromethane (25 mL) at room temperature (RT). The disappearance of cholesteryl N-(6-isocyanatohexyl)carbamate was monitored using TLC. The solvent was removed in vacuo and the compound was extracted using a 1:1 chloroform-to-water ratio giving a yield of 144.57 mg (72.2%). 1H NMR (400 MHz, [D6] DMSO, 25°C, TMS): δ = 0.65 (s, 3H, cholesterol), 0.84–2.33 (m, 40H, cholesterol), 1.41–1.55 (m, 8H), 1.22–1.23 (m, 2H), 2.92–3.00 (m, 8H), 4.27–4.32 (m, 1H, cholesterol), 5.34 (b, 1H, cholesterol), 5.80–5.85 (m, 2H), 7.03 (m, 1H). HR-ESI-MS: m/z calculated for (C37H66N4O3) ([M–H]−) 614.5134; found, 614.5208.
aCMG was synthesized from Ch-I and rhG as follows. rhG (100 mg, 0.0273 mmol, corresponding to the lysine moieties and N-terminal) was dissolved in DMSO (15 mL) and reacted with Ch-I (7.56 mg, 0.0136 mmol) in DMSO (5 mL) containing TEA (1 mL, 7.16 mmol) at 50°C. The reaction was monitored using TLC until the disappearance of Ch-I was confirmed (developed using chloroform). It was then dialyzed against distilled water and freeze-dried to give a yield of 66 mg (66%).
cCMG was synthesized using an EDC-coupling method wherein Ch-A (23.22 mg, 0.0378 mmol) was reacted with rhG (100 mg, 0.0756 mmol, corresponding to the Asp and Glu moieties and the C-terminal) in 15 mL DMSO (Ch-A and rhG were dissolved separately in DMSO). To this mixture, an EDC-HCl solution (144.89 mg, 0.756 mmol), prepared freshly in 8 mL of DMSO, was added to the rhG and Ch-A mixture to obtain a 10-fold excess in the amounts of Asp and Glu. It was then allowed to react at room temperature overnight and dialyzed against distilled water to remove solvent and excess reagents including any byproducts such as isourea formed during the reaction, and lyophilized. Yield was 56 mg (56%).
The conjugation of cholesterol with rhG was confirmed by MALDI-TOF/MS. All mass measurements were performed using a microflex MALDI-TOF mass spectrometer (Bruker Daltonics Inc, Billerica, MA).
Polymeric micelles were prepared by hydration of aCMG or cCMG in distilled water or PBS; they dispersed easily to give a uniform suspension on vortexing. The CMC values were estimated by fluorescence spectroscopy using pyrene as the fluorescence probe as described previously.19 Briefly, the concentration of pyrene was kept constant at 0.6 μM while the concentrations of the polymeric micelles were varied from 1 × 10−7 to 1 mg/mL. The fluorescence spectra were recorded using a fluorescence spectrophotometer (JASCO FP-6500; Jasco Corp, Tokyo, Japan) with an excitation wavelength of 320 nm. Fluorescent light emissions were monitored at 372 and 383 nm. The CMC was estimated as the cross-point when extrapolating the intensity ratio I372/I383 at low- and high-concentration regions.
The morphology of the polymeric micelles was analyzed by SEM (JEM-6330; JEOL Instruments, Tokyo, Japan). SEM measurements were carried out at an accelerating voltage of 15 kV. The samples were prepared by dropping 10 μL of the micelle solution (1 mg/mL) onto a carbon seal attached to a metallic specimen holder, followed by air-drying for a day. The samples were then sputter-coated with gold before observation.
The cytotoxicity of the polymeric micelles was determined using a WST cell viability assay. DC 2.4 cells were seeded in a 96-well plate at a concentration of 2 × 104 cells/well and cultured in complete medium for 24 hours at 37°C under 5% humidified CO2. The culture medium was then replaced with fresh complete medium containing aCMG, cCMG, or rhG at predetermined concentrations and incubated under the same conditions for 24 hours. Then the cells were washed twice with PBS, and complete medium was added to the cells. Aliquots of 10 μL of cell counting kit-8 solution (CCK-8; Dojindo Laboratories) were then added to the medium and shaken for 30 seconds to mix the solution thoroughly and then incubated at 37°C for 30 minutes. The absorbance of each well was measured using a microplate reader (Model-680; Bio-Rad, Hercules, CA) at 450 nm. The percentage of cell viability was calculated from the absorbance values.
To prepare the polymeric micelle and protein complexes, aqueous solutions of FITC-OVA and polymeric micelles were mixed, vortexed gently, and incubated at RT for 30 minutes. Native rhG served as a control because it does not form a complex with OVA.
The mean hydrodynamic size and polydispersity index of rhG, aCMG, and cCMG were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). Measurements were carried out at 25°C. The ζ-potential (in mV) was also determined using the Zetasizer Nano ZS. The size and ζ-potential are reported as the mean ± standard deviation (SD) of 14 subruns.
Dual-color fluorescence-activated cell sorting (FACS) analysis was performed to quantify the uptake of FITC-OVA by the polymeric micelles in DC 2.4 cells. The cells were seeded at a concentration of 1 × 106, cultured in RPMI-1640 medium supplemented with 10% FBS and incubated with the samples for 1 hour at 4°C and 37°C. The cells were then washed with PBS and collected in PBS containing 0.25 μg/mL of 7-amino-actinomycin D (7-AAD) to stain the dead cells. Uptake of FITC-OVA was analyzed using a BD FACSCalibur (Becton-Dickinson, Tokyo, Japan). Results are expressed as the mean fluorescence intensity. Specific uptake was assessed by subtracting the mean fluorescence intensity of the cells incubated at 4°C from that recorded at 37°C.
For fluorescence microscopy, the DC 2.4 cells were seeded at a concentration of 1 × 105 cells and cultured for 24 hours in a 35-mm glass-bottomed dish containing RPMI-1640 medium supplemented with or without 10% FBS. For analyzing the cell uptake and subcellular location of FITC-OVA, free FITC-OVA (15 μg) or test samples (80 μg) complexed with FITC-OVA (15 μg) were used. For evaluating antigen processing, cCMG (80 μg) complexed with OVA-DQ™ (15 μg) was added gently to the cells and incubated at 37°C. For cellular uptake and antigen processing, after incubation for the indicated time periods, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature, after which the nuclei were stained with DAPI. For the subcellular localization of FITC-OVA, after incubation for the required time periods, LysoTracker Red™ (50 nM) was added 20 minutes before fixation. The cells were observed using a fluorescence microscope (Carl Zeiss, Göttingen, Germany).
To investigate the mechanism of uptake of the polymeric micelles, DC 2.4 cells were seeded at a concentration of 1 × 105 cells in a 35 mm culture dish and cultured for 48 hours. The cells were then incubated with four endocytosis inhibitors: nystatin (25 μg/mL), genistein (300 μM), chlorpromazine (15 μg/mL) and cytochalasin D (10 μg/mL) for 1 hour at 37°C, after which cCMG (80 μg) complexed with FITC-OVA (20 μg) was added and incubated for 4 hours. Cells incubated with only cCMG complexed with FITC-OVA served as controls. The cells were washed twice with PBS, collected in 1% bovine serum albumin (BSA) in PBS and the inhibition of FITC-OVA fluorescence was analyzed by flow cytometry (Cytomics™ FC500; Beckman-Coulter, Miami, FL). Results are expressed as the percentage of fluorescent positive cells.
To test the efficiency of in vivo antigen delivery, 6-week-old female C57BL/6 mice were administered with 3.3 mg/kg of free OVA or polymeric micelles complexed with OVA on days 0 and 14 by intraperitoneal injection. Blood samples were collected from the tail vein on days 7 and 21 for antibody titration. On day 28, whole blood samples were collected by heart puncture with the animal under deep diethyl ether anesthesia and the spleens were harvested before allowing the mouse to succumb. The spleens were prepared individually as single-cell suspensions in 3 mL RPMI 1640 supplemented with l-glutamine, 10% FBS and 100 μg/mL each of penicillin, streptomycin, and gentamycin. The erythrocytes were removed by suspension in a lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA-2Na). Splenocytes were washed with complete RPMI medium and live cells counted using a trypan blue exclusion method.
Specific antibody titers against OVA were determined by performing an endpoint enzyme-linked immunosorbent assay (ELISA). NUNC MaxiSorp 96-well ELISA plates (Nalge Nunc International, Roskilde, Denmark) were coated with 5 μg/mL of OVA in 0.1 M NaHCO3 (Nacalai Tesque, Kyoto, Japan), incubated overnight at 4°C and washed with PBS–Tween (PBS-T). The plates were blocked with 3% BSA in PBS for 1 hour at room temperature and washed with PBS-T. After serial dilution in 1% BSA in PBS beginning at a 40-fold dilution, serum samples (100 μL) were added into the wells and the plates were incubated for 1 hour at room temperature. The plates were washed with PBS-T and 100 μL of peroxidase-conjugated goat anti-mouse IgG (heavy chain- and light chain-specific, diluted 32000-fold in 1% BSA in PBS; Jackson ImmunoResearch Laboratories Inc, West Grove, WA) was added to each well. The plates were incubated for 1 hour at room temperature and then washed with PBS-T. Finally, colorimetric signals were generated using 1 Step Ultra TMB-ELISA solution (Pierce Biotechnology Inc, Rockford, IL). The reaction was stopped with 1 N H2SO4 after 15 minutes and the absorbance at 450 nm was measured using a Wallac ARVO counter (Perkin Elmer, Norwalk, CT). The antibody titer was determined as the highest dilution below the cutoff value indicated by the mean optical density value of the background ±2 SD.
All the animal experimental data are expressed as the mean ± SD. Multiple comparisons were performed using one-way analysis of variance (ANOVA) with post hoc analysis followed by Bonferroni's test.
The aCMG and cCMG were synthesized as described in Figures 1 and and22 and confirmed by MALDI-TOF data (Figure 3A). Compositions, MW, and the exact amount of substituted cholesterol in these derivatives are listed in Table 1. In the case of aCMG, the NH2 groups of 36 lysine residues and one N-terminal in rhG were targeted for modification by Ch-I, of which 6.7 residues were modified (Table 1). Table 1 also shows the mean hydrodynamic size of formed polymeric micelles (485 nm) and a net increase in the negative charges resulting in a ζ-potential of −2 mV. It was expected that aCMG would have both hydrophobic cholesterol-modified and hydrophilic-unmodified regions in forming micelles and would attain an overall negative charge as a result of the ionization of the remaining COOH groups in rhG. On the other hand, in the case of cCMG, COOH groups of 78 residues of Asp and Glu and one C-terminal were targeted for modification by Ch-A resulting in 9.99 modified residues. The hydrodynamic size of formed polymeric micelles was 350 nm and there was a net increase in the positive charges resulting in a +6 mV ζ-potential. It was also expected that cCMG would have both hydrophobic cholesterol-modified and hydrophilic-unmodified regions in forming polymeric micelles and would attain an overall positive charge from ionization of the remaining NH2 groups in rhG.
The CMCs of aCMG and cCMG were estimated to be 1.7 × 10−2 mg/mL and 2.0 × 10−3 mg/mL, respectively. The native rhG did not have the ability to form micelles and so no CMC was observed (Figure S1). It is known that the hydrophobic nature of cholesterol increases its propensity to form strong hydrophobic interactions.20,21 The introduction of cholesterol allows polymeric micelles to be generated and used in biological fluids,20,21 which makes them amphiphilic in nature, and thus preventing gelatin from being dissolved rapidly in an aqueous environment.
SEM images further elucidated the formation of micelles. Figure 3B shows the SEM images of anionic and cationic polymeric micelles. The hydrodynamic size of the polymeric micelles by DLS was larger than that observed by SEM. This discrepancy in size can be attributed to the shrinking of the particles during air-drying prior to SEM analysis.
It was also found that aCMG as well as cCMG showed no cytotoxicity at a maximum concentration of ~1 μM (80 μg) (Figure 3C).
FITC-OVA was used to form a complex with the polymeric micelles. Table 1 shows the changes in ζ-potential. In the case of cCMG, at a polymer-to-protein weight ratio (w/w) of 1.0, the polymeric micelle–protein complex was negatively charged. On increasing the concentration of cCMG with respect to protein, the ζ-potential became more positively charged. Here, the anionic protein interacted with cCMG through electrostatic interactions, which are considered to be the main driving force for the binding of the protein. The positively charged amino groups in cCMG appeared to be very effective in the formation of a complex with FITC-OVA. On the other hand, the ζ-potential of aCMG remained constant even in the presence of FITC-OVA. Similarly, the ζ-potential of rhG remained constant at all proportions with FITC-OVA. The formation of complexes was elucidated further by measuring size distributions using DLS (Table 1). The mean hydrodynamic size of cCMG complexed with FITC-OVA increased significantly from 350 to 483 nm whereas the size of aCMG as well as rhG remained the same, which can be attributed to their failure in forming a complex with the protein. Strong complex formation of cCMG was also demonstrated by native polyacrylamide gel electrophoresis analysis (Figure S2).
Following treatment of DC 2.4 cells with cCMG, flow cytometry analysis revealed a large population of cells with intense fluorescence whereas few cells were positive for fluorescence among the free FITC-OVA, rhG- and aCMG-treated cells (Figure 4A). Cells incubated with cCMG at 4°C revealed a much lower uptake than at 37°C (Figure S3), as is known for polymer-based nanoparticles.22 This result demonstrated that the cellular uptake was caused by an active process of internalization and not by a passive one. Figure 3B also shows a marked increase in the uptake of the protein by cCMG, caused by interactions between the cationic charges on the surface of the polymeric micelles and the anionic domains present on the cell surface. The appearance of strong fluorescence at certain points after 2 hours and its distribution throughout the cytosol after 24 hours was observed by fluorescence microscopy (Figure S4).
To investigate whether the proteins had escaped lysosomal degradation, the DC 2.4 cells were stained with LysoTracker™ Red, which accumulates in late endosomes and lysosomes. In the case of DC 2.4 cells treated with rhG and aCMG (Figure 5A), after 24 hours the protein was detected along with the lysosomes that were obvious in the colocalization signals of FITC-OVA (green) and LysoTracker™ (red) that together appeared yellow. In contrast, DC 2.4 cells treated with cCMG showed strong green as well as red fluorescence. Thus, the protein delivered by cCMG was not trapped in lysosomes. Except for some locations (partial colocalizations indicated by white circles in Figure 5A), there was no colocalization of signals, confirming that FITC-OVA and lysosomes were present at different locations. This showed that the proteins had been released efficiently into the cell cytosol.
OVA-DQ™ was employed to visualize the processing of antigen into peptides. This is a self-quenching ovalbumin conjugate that emits bright green fluorescence upon enzymatic degradation into single peptides. Figure 5B shows that there was bright green fluorescence after 5 hours of incubation with cCMG. This became more diffuse and spread throughout the cell after 24 hours. This result indicates the efficient processing of OVA into peptides by DC 2.4 cells.
To investigate the mechanism of uptake of cCMG by the DC 2.4 cells, we used four chemical inhibitors that block endocytosis pathways. These were: chlorpromazine, a potent inhibitor of clathrin-mediated endocytosis;23 nystatin and genistein, which both block caveolin-mediated endocytosis;24 and cytochalasin D, an actin inhibitor that blocks the macropinocytosis pathway.25 Of these inhibitors, chlorpromazine and genistein reduced the uptake to about 67% and 40%, respectively, when compared with the control untreated cells and showing 98% fluorescent positive cells (Figure 6A). The result from chlorpromazine inhibition demonstrated that cCMG was internalized into the DCs via a clathrin-mediated pathway and ultimately entered the lysosomal compartments (partial colocalization; Figure 5A).26 To enter efficiently via this pathway, it is important for the compound to possess properties that can escape lysosomes efficiently, as in the case of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) lipids.26 In addition to this pathway, the result from genistein inhibition indicated the presence of a caveolin-mediated pathway, which is known to avoid the lysosomes.27 This was considered to be the major uptake mechanism (strong green fluorescence, Figure 5A). Interestingly, treatment with nystatin failed to inhibit the endocytosis of cCMG. It has been reported that the uptake of polyethyleneimine, a well-studied cationic polymer that utilizes the caveolin pathway for endocytosis, is inhibited by nystatin28 as well as by genistein.26 Nystatin is a sterol-binding drug.24 Hence, when cells are pretreated with it there is a depletion of cholesterol. It is known that treatment with cholesterol-depleting drugs cannot block Kirsten Ras (KRas)-mediated activation,29 which results in an efficient uptake of cCMG even after treatment. Considering that genistein inhibits the protein tyrosine kinases,24 we conclude that the uptake of cCMG was mediated by a caveolin-mediated pathway through specific interactions with tyrosine kinase receptors (Figure 6B).
The induction of humoral immune responses by cCMG-OVA was investigated using mice. Serum samples were collected once a week from C57BL/6 mice immunized intraperitoneally with OVA or cCMG-OVA and OVA-specific antibody titers were measured using an endpoint ELISA. A high antibody titer in mice immunized with cCMG-OVA was observed as early as 7 days after the first immunization. On day 21, the OVA-specific antibody titer in these mice was >2580-fold higher than that in mice immunized with free OVA (Figure 7). Antigen doses as low as 1 μg were sufficient to induce immunity (data not shown). These results are consistent with our in vitro data. They illustrate the stability of the micelles, which is another important factor to consider when delivering proteins via nanoparticles in vivo. If there had been no significant increase in the serum IgG levels in mice after immunization with cCMG, it could have been attributed to the instability of the polymeric micelles upon their interaction with the body's serum proteins and other enzymes, ultimately leading to their degradation. This was observed only in the case of free OVA, where there was only a minimal production of serum antibodies against OVA and not in the case of cCMG.
The successful delivery of cCMG as well as the efficient production of antibodies in vivo derived from the combined effect of size, a strong complex formation with protein, efficient release of the protein into the cytoplasm by caveolin-mediated endocytosis, and the processing of protein for antigen presentation by the DCs. Antigen presentation is generally enhanced by antigen carriers that lie within the size range of 0.1–10 μm, which apparently increases cross-presentation of the antigen to T-cells.30 Understanding the subcellular localization of an antigen by polymeric micelles is crucial for their application in drug delivery. Escape of the antigen from lysosomes is considered an important step in the success of vaccine therapy. An increase in the cytosolic delivery of the protein antigen facilitates cross presentation of the antigen loaded onto MHC-I molecules to activate T-cells. This is achieved when the antigen is processed by proteases present in the cytosolic-based proteasomes.31 We believe this to hold true in the case of cCMG.
In conclusion, we have shown for the first time the successful synthesis of cationic cholesterol-modified gelatin and have evaluated its efficiency in inducing humoral immunity in mice. We used a simple method of loading protein into the polymeric micelles via complex formation through electrostatic interaction. The ability of cCMG to deliver an antigen with high efficiency, stability, and negligible cytotoxicity should open new doors in the field of drug delivery, particularly for proteins. This should bring us a step closer to translating our work from academic research to practical applications.
This work was supported by a grant from the Program for the Promotion of Basic and Applied Research for Innovations in Bio-oriented Industry.
The authors report no conflicts of interest in this work.
Critical micelle concentration (CMC) values of aCMG (1.7 × 10−2 mg/mL) and cCMG (2.0 × 10−3 mg/mL) analyzed by fluorescence spectrometry with the fluorescence intensity ratio I1/I3 of pyrene as a function of the concentration of modified gelatin.
Note: The native rhG does not form micelles and so does not have any CMC.
Abbreviations: aCMG, anionic cholesterol-modified gelatin; cCMG, cationic cholesterol-modified gelatin.
Confirmation of complex formation with OVA by aCMG or cCMG, as determined by native polyacrylamide gel electrophoresis analysis.
Notes: The gel was stained with Coomassie Brilliant Blue (stacking gel −3% and separating gel −12.5%). Lane 1, OVA; Lane 2, aCMG with OVA; Lane 3, cCMG with OVA.
Abbreviations: aCMG, anionic cholesterol-modified gelatin; cCMG, cationic cholesterol-modified gelatin; OVA, ovalbumin.
FACS analysis of the cellular uptake of free FITC-OVA, rhG, aCMG, and cCMG at 4 and 37°C.
Abbreviations: aCMG, anionic cholesterol-modified gelatin; cCMG, cationic cholesterol-modified gelatin; FACS, fluorescence-activated cell-sorting; FITC-OVA, fluorescein isothiocyanate-labeled ovalbumin; MFI; mean fluorescence intensity; rhG, recombinant human gelatin.
Fluorescence microscopy results of a time-course study on the uptake of cCMG by DC 2.4 cells after 2 and 24 hours.
Note: Intracellular FITC-OVA (green) was observed.
Abbreviations: cCMG, cationic cholesterol-modified gelatin; DCs, dendritic cells; FITC-OVA, fluorescein isothiocyanate-labeled ovalbumin.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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text: Supreme Court Allows Hobby Lobby to Refuse Birth Control CoverageMichelle Dean6/30/14 10:43amFiled to: Supreme CourtLunaciesHobby Lobby67719EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink The Supreme Court's biggest decision of the term is here. In the cases known as Hobby Lobby, they've ruled 5-4 that closely held corporations need not provide contraceptive coverage because it would substantially burden them under the Religious Freedom Restoration Act. Justice Alito wrote the majority opinion.[Image by Jim Cooke.]Reply677 repliesLeave a reply


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I was all set to complain until I looked at the post by Kevin Parker here. Duh! It even shows the method of putting in the redirects in the settings page. Don't I feel silly! :)
But once you READ the screen and put in the proper links in the proper method it works flawlessly. Thank you!


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July 7, 2012
I just started climbing in the late fall of 2011. I started going to the VE gym in Minneapolis very often around February of 2012. Currently I am able to climb 5.6s to 5.9s, but haven't been able to do a 5.10 yet (still trying though!) Like my technique instructor said "If you're not flyin', You're not tryin'!" I think the best thing I've improved so far is just my endurance, I can climb for hours now! Hooray :D
VE in Minneapolis :)
I like to climb! It was love at first hand-to-rock when I got the guts to come in last fall (2011) I love the blend of physical and mental challenge, its unlike anything else I'm into. :D
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Afro Samurai is a Japanese manga series written and illustrated by Takashi Okazaki. It has also been adapted to an anime, a made-for-TV film, and a video game.
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Does the number 1 headband give the power of a god?
In Afro Samurai, it seems everybody is fighting to get the number 1 headband. In most accounts about the number 1 headband, they say you become "like a god." But when Afro faced Justice, in the end, ...


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Foam pipe insulation for the rails, 1" XPS sheet foam insulation from HD, cut to fit the deck and bottom.
\nDON'T TOUCH THE VENT PLUG. Just make sure it's clear of debris (sand, salt crust, wax...) and cut a hole in the sheet foam over the vent plug.
\nMitchell... you're one funny f'er, brother! That had me laughing at my desk!
\nMikey... I had a Sunova Nitro for several months, and that board absolutely rocks! Amazing tech. Made me wish I was a better surfer, cause that thing could do stuff way beyond my ability.
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Thread: Traveling with a Firewire
Mar 13, 2012, 05:05 PM
Last edited by LBCrew; Mar 13, 2012 at 05:15 PM.
'; pd[120927] = '
Mar 13, 2012, 06:12 PM
Junior Member
- Join Date
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Pipe foam, bubble wrap, and forget about any factory support should your beautiful Sunova suffer a delam... It will in no way be any fault of the factory, they will just leave you HANGing MAN....
Removing the vent plug will only allow the 1lb eps to fully breath while minimizing the chance that the lamination would pull from the foam, just make sure the plug is back in before you splash...
'; // next/previous post info pn[120912] = "120927,120916"; pn[0] = ",120912"; pn[120916] = "120912,120918"; pn[120918] = "120916,120922"; pn[120922] = "120918,120927"; pn[120927] = "120922,120912"; // cached usernames pu[0] = guestphrase; pu[10290] = "Mikey"; pu[10955] = "Mitchell"; pu[31127] = "skruslicky"; pu[12449] = "LBCrew"; pu[26577] = "ron4pres"; // -->
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Mar 13, 2012, 03:08 PM #1
Traveling with a Firewire
I am going to Panama this summer and want to bring my Sunova Nitro with me. Its construction is identical to a Firewire--1lb eps foam, balsa rails, balsa deck, pressure valve. Is there anything special I should know about packing it for the trip and handling the board in the tropics.
My flight is Baltimore to Miami, Miami to Panama City, and then a 2hr shuttle from Panama City to the hotel.
I avoided the Angry Beavers thread on purpose, out of fear that radical feminists will hijack it and shoot it all to smithereens.


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Large and clean rooms with kitchens, free laundry and parking, nice staff. Close to airport, and scenic day trips. Not so close to Portland neighborhoods, college and local flavor. Lots of traffic but next to mall with lots of free parking and rail service. Shuttle to mall offered but we didn't use it. Cascade mall has several places to eat and buy groceries and supplies - even a clinic that came in handy.
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Making the Case: Lohse for Cy Young
On Major League Baseball's Facebook page yesterday there was a picture posted that featured images of R.A. Dickey, Gio Gonzalez, Johnny Cueto, and Craig Kimbrell and asked who is this year's NL Cy Young Award winner. Cardinals' fans immediately noticing that Kyle Lohse is missing from the list of contenders, bumped from the image by two guys who have higher ERAs and a reliever. The question you now have to … Continue reading
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The Yes to Carrots Daily Moisture Body Lotion was created in response to customers demanding a larger size of their favorite daily moisturizer. This basic daily-use product will moisturize and smooth your skin with carrots and sweet potatoes for Beta-Carotene nourishment, and safflower seed oil for a big ol' dose of softness. This larger pump is the perfect bedside or bathroom addition!
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Ukraine Opposition Refuses to End Rallies in Yanukovych Snub
Ukraine's opposition urged protesters to stay on the streets, rebuffing a power-share offer from President Viktor Yanukovych, which they said fell short of their demands.
Opposition leaders Arseniy Yatsenyuk, Vitali Klitschko and Oleh Tyahnybok, addressing demonstrators after Yanukovych offered to give them the top cabinet jobs in return for ending protests, said they won't give up their demands for the president's resignation, snap elections and the annulment of anti-rally laws.
Yanukovych earlier today offered his biggest concessions yet since anti-government rallies erupted two months ago, saying he was ready to give the premiership and a deputy prime minister position to Yatsenyuk and Klitschko. The president is struggling to tame demonstrations that turned deadly this week as anti-protest laws triggered violent riots.
"We will not backtrack!" Klitschko said. "We are keeping our positions. There are no extremists here. We are peaceful people, who are fighting for their rights and demands. We are not listening to any provocations."
An extraordinary parliament session called for Jan. 28 will be crucial in deciding the fate of the country, Yatsenyuk said. Klitschko and Svoboda Party leader Oleh Tyahnybok urged activists to continue protests until all their demands are met.
Amnesty, Constitution
The government also offered an amnesty for protesters who agree to release administration buildings they have seized, Andriy Portnov, Yanukovych's deputy chief of staff, said in a separate statement on the president's website.
The two sides will start talks on changing the constitution and on repealing anti-protest laws that have ignited violence this week, according to Portnov. The clashes have left as many as six people dead and more than 1,000 injured.
"The reshuffle offered by Yanukovych isn't on protesters' agenda now," Vadym Omelchenko, the head of the Gorshenin Institute in Kiev, said by phone today.
After both sides called yesterday for foreign mediation to help find a resolution to the crisis, opposition leaders met again today with Yanukovych, claiming the president plans to initiate a state of emergency. Officials including Prime Minister Mykola Azarov have denied such plans in recent days.
Clashes Resume
Riots flared up again in Ukraine's capital last night, prompting the interior minister to say that peaceful efforts to end the unrest were useless.
After a day of calm, clashes resumed shortly after 10 p.m. yesterday near parliament as demonstrators threw Molotov cocktails and police deployed rubber bullets and stun grenades. Interior Minister Vitaliy Zakharchenko said the opposition had lost control over radical activists and warned peaceful protesters to vacate central Kiev or be deemed extremists.
Activists in Kiev, who seized the Agriculture Ministry yesterday, took over five floors and the basement of the Energy Ministry today, the Unian news service reported.
Protesters are also occupying buildings of governors picked by Yanukovych in the western cities of Lviv, Ternopil, Rivne, Lutsk, Ivano-Frankivsk, Khmelnytskyi and Chernihiv and are targeting administrative offices in at least five more of the nation's 24 regions, smashing their way in when police offered resistance, TV5 reported.