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Find video protocols related to scientific articles indexed in Pubmed.
Preformulation study of highly purified inactivated polio vaccine, serotype 3.
J Pharm Sci
PUBLISHED: 09-14-2013
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To improve the effectiveness of the polio vaccination campaign, improvements in the thermal stability of the vaccine are being investigated. Here, inactivated polio vaccine, serotype 3 (IPV3) was characterized via a number of biophysical techniques. The size was characterized by transmission electronic microscopy and light scattering. The capsid protein conformation was evaluated by intrinsic fluorescence and circular dichroism (CD), and the D-antigen content by enzyme-linked immunosorbent assay (ELISA). The pH thermal stability of IPV3 (pH 3.0-8.0; 10°C-87.5°C) was evaluated by fluorescence, CD, and static light scattering. The transition temperatures reflect the responses, respectively, of tertiary structure, secondary structure, and size to applied thermal stress. The data were summarized as empirical phase diagrams, and the most stable conditions were found to be pH 7.0 with temperature lower than 40°C. CD detected a higher transition temperature for capsid protein than that for RNA. The effects of certain excipients on IPV3 thermal stability and antigen content were evaluated. The results of their effects, based on intrinsic fluorescence and ELISA, were in good agreement, suggesting the feasibility of applying intrinsic fluorescence as a high-throughput tool for formulation development. The study improves the understanding of IPV3 thermal stability, and provides a starting point for future formulation development of IPV3 and other serotypes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:140-151, 2014.
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Development of a Stable Virus-Like Particle Vaccine Formulation against Chikungunya Virus and Investigation of the Effects of Polyanions.
J Pharm Sci
PUBLISHED: 08-12-2013
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Chikungunya virus (CHIKV) is an alphavirus that infects millions of people every year, especially in the developing world. The selective expression of recombinant CHIKV capsid and envelope proteins results in the formation of self-assembled virus-like particles (VLPs) that have been shown to protect nonhuman primates against infection from multiple strains of CHIKV. This study describes the characterization, excipient screening, and optimization of CHIKV VLP solution conditions toward the development of a stable parenteral formulation. The CHIKV VLPs were found to be poorly soluble at pH 6 and below. Circular dichroism, intrinsic fluorescence, and static and dynamic light scattering measurements were therefore performed at neutral pH, and results consistent with the formation of molten globule structures were observed at elevated temperatures. A library of generally recognized as safe excipients was screened for their ability to physically stabilize CHIKV VLPs using a high-throughput turbidity-based assay. Sugars, sugar alcohols, and polyanions were identified as potential stabilizers and the concentrations and combinations of select excipients were optimized. The effects of polyanions were further studied, and while all polyanions tested stabilized CHIKV VLPs against aggregation, the effects of polyanions on conformational stability varied. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:4305-4314, 2013.
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Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2).
Biomacromolecules
PUBLISHED: 10-13-2011
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Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins.
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Biophysical characterization and conformational stability of Ebola and Marburg virus-like particles.
J Pharm Sci
PUBLISHED: 04-28-2011
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The filoviruses, Ebola virus and Marburg virus, cause severe hemorrhagic fever with up to 90% human mortality. Virus-like particles of EBOV (eVLPs) and MARV (mVLPs) are attractive vaccine candidates. For the development of stable vaccines, the conformational stability of these two enveloped VLPs produced in insect cells was characterized by various spectroscopic techniques over a wide pH and temperature range. Temperature-induced aggregation of the VLPs at various pH values was monitored by light scattering. Temperature/pH empirical phase diagrams (EPDs) of the two VLPs were constructed to summarize the large volume of data generated. The EPDs show that both VLPs lose their conformational integrity above about 50°C-60°C, depending on solution pH. The VLPs were maximally thermal stable in solution at pH 7-8, with a significant reduction in stability at pH 5 and 6. They were much less stable in solution at pH 3-4 due to increased susceptibility of the VLPs to aggregation. The characterization data and conformational stability profiles from these studies provide a basis for selection of optimized solution conditions for further vaccine formulation and long-term stability studies of eVLPs and mVLPs.
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The seroprevalence of pandemic influenza H1N1 (2009) virus in China.
PLoS ONE
PUBLISHED: 02-17-2011
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Mainland China experienced pandemic influenza H1N1 (2009) virus (pH1N1) with peak activity during November-December 2009. To understand the geographic extent, risk factors, and attack rate of pH1N1 infection in China we conducted a nationwide serological survey to determine the prevalence of antibodies to pH1N1.
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Identifying stabilizers of plasmid DNA for pharmaceutical use.
J Pharm Sci
PUBLISHED: 04-23-2010
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To better address the need for developing stable formulations of plasmid DNA-based biopharmaceuticals, 37 compounds from a generally regarded as safe library were examined for their potential use as stabilizers. A plasmid DNA-based therapeutic vaccine, BHT-DNA, was used as a model system. Initial studies were performed to compare the biophysical properties of BHT-DNA plasmid from bulk drug substance and finished drug product. An agarose gel electrophoresis-based assay was then employed in excipient compatibility studies for the drug product by monitoring supercoiled plasmid DNA content in various formulations. After incubation at 40 °C for 30 days, eight out of the 37 excipients tested were able to better retain the supercoil content compared to the control. Sodium citrate appeared to be the most effective stabilizer and its protective capability plateaued at an ionic strength of about 0.4. Several other excipients including malic acid, ethanol, and Pluronic F-68 were also identified as promising stabilizers for BHT-DNA plasmid DNA. Additionally, compounds, including ferrous chloride, ascorbic acid, human serum albumin, and PEG 1000, which significantly destabilized the supercoiled plasmid DNA were identified. These data may also be applicable to other plasmid DNA-based pharmaceuticals for storage stability improvement, due to chemical and structural similarities of these macromolecules.
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SecretP: a new method for predicting mammalian secreted proteins.
Peptides
PUBLISHED: 01-04-2010
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In contrast to a large number of classically secreted proteins (CSPs) and non-secreted proteins (NSPs), only a few proteins have been experimentally proved to enter non-classical secretory pathways. So it is difficult to identify non-classically secreted proteins (NCSPs), and no methods are available for distinguishing the three types of proteins simultaneously. In order to solve this problem, a data mining has been taken firstly, and mammalian proteins exported via ER-Golgi-independent pathways are collected through extensive literature searches. In this paper, a support vector machine (SVM)-based ternary classifier named SecretP is proposed to predict mammalian secreted proteins by using pseudo-amino acid composition (PseAA) and five additional features. When distinguishing the three types of proteins, SecretP yielded an accuracy of 88.79%. Evaluating the performance of our method by an independent test set of 92 human proteins, 76 of them are correctly predicted as NCSPs. When performed on another public independent data set, the prediction result of SecretP is comparable to those of other existing computational methods. Therefore, SecretP can be a useful supplementary tool for future secretome studies. The web server SecretP and all supplementary tables listed in this paper are freely available at http://cic.scu.edu.cn/bioinformatics/secretp/index.htm.
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Binding of Pseudomonas aeruginosa apobacterioferritin-associated ferredoxin to bacterioferritin B promotes heme mediation of electron delivery and mobilization of core mineral iron.
Biochemistry
PUBLISHED: 07-07-2009
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The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5-10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 +/- 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH, the heme in BfrB remains oxidized, and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR, and the apo form of P. aeruginosa bacterioferritin-associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to assemble iron-sulfur clusters in Bfd before the iron stored in BfrB can be mobilized and utilized.
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Towards development of stable formulations of a live attenuated bacterial vaccine: a preformulation study facilitated by a biophysical approach.
Hum Vaccin
PUBLISHED: 05-08-2009
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Development of optimal formulation conditions stabilizing live attenuated bacterial vaccines is impeded by traditional methods used for viability measurement. To facilitate preformulation studies of such vaccines, spectroscopic techniques capable of providing real-time and high throughput information have been employed to obtain a global stability profile for a live attenuated Ty21a bacterial typhoid vaccine over a wide range of pH (4 to 8) and temperature (10 to 85 degrees C). Using the data obtained from fluorescence and circular dichroism techniques, an empirical phase diagram (EPD) has been subsequently constructed, which suggests that Ty21a cells exist in at least four apparent physical phases related to different viability states, with the most stable phase at pH 6 and 7 at temperatures below 30 degrees C. A slightly basic pH (pH 8) appears to decrease the fluidity of the cell membrane, whereas acidic pH conditions are detrimental to membrane integrity over the entire temperature range. Based on the above stability profile, a fluorescence-based high throughput screening assay has been developed to test the stabilizing effects of various compounds at different concentrations. Amongst other promising stabilizers, 10% sucrose and 0.15 M glutamic acid display the greatest protective effects, with an increase of about 10 degrees C in the transition temperature of Ty21a cells. Preliminary studies have also been performed on foam dried formulations as an alternative approach to further stabilize Ty21a cells. The data show that 10% sucrose and trehalose both increase the in-process and storage stabilities of the cells.
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Using auto covariance method for functional discrimination of membrane proteins based on evolution information.
Amino Acids
PUBLISHED: 04-24-2009
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Membrane transporters are critical in living cells. Therefore, the discrimination of the types of membrane proteins based on their functions is of great importance both for helping genome annotation and providing a supplementary role to experimental researchers to gain insight into membrane proteins function. There are a lot of computational methods to facilitate the identification of the functional types of membrane proteins. However, in these methods, the local sequence environment was not integrated into the constructed model. In this study, we described a new strategy to predict the functional types of membrane proteins using a model based on auto covariance and position-specific scoring matrix. The novelty of the presented approach is considering the distribution of different positions of functional conservation sites in protein sequences. Thereby, this model adequately takes into account the long-range correlation between such sites during sequential evolution. Fivefold cross-validation test shows that this method greatly improves the prediction accuracy and achieves an acceptable prediction accuracy of 87.51%. The result indicates that the current approach might be an effective tool for predicting the functional types of membrane proteins only using the primary sequences. The code and dataset used in this article are freely available at http://cic.scu.edu.cn/bioinformatics/predict_membrane.zip.
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Adsorption of recombinant poxvirus L1-protein to aluminum hydroxide/CpG vaccine adjuvants enhances immune responses and protection of mice from vaccinia virus challenge.
Vaccine
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The stockpiling of live vaccinia virus vaccines has enhanced biopreparedness against the intentional or accidental release of smallpox. Ongoing research on future generation smallpox vaccines is providing key insights into protective immune responses as well as important information about subunit-vaccine design strategies. For protein-based recombinant subunit vaccines, the formulation and stability of candidate antigens with different adjuvants are important factors to consider for vaccine design. In this work, a non-tagged secreted L1-protein, a target antigen on mature virus, was expressed using recombinant baculovirus technology and purified. To identify optimal formulation conditions for L1, a series of biophysical studies was performed over a range of pH and temperature conditions. The overall physical stability profile was summarized in an empirical phase diagram. Another critical question to address for development of an adjuvanted vaccine was if immunogenicity and protection could be affected by the interactions and binding of L1 to aluminum salts (Alhydrogel) with and without a second adjuvant, CpG. We thus designed a series of vaccine formulations with different binding interactions between the L1 and the two adjuvants, and then performed a series of vaccination-challenge experiments in mice including measurement of antibody responses and post-challenge weight loss and survival. We found that better humoral responses and protection were conferred with vaccine formulations when the L1-protein was adsorbed to Alhydrogel. These data demonstrate that designing vaccine formulation conditions to maximize antigen-adjuvant interactions is a key factor in smallpox subunit-vaccine immunogenicity and protection.
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Epidemiological and virological characteristics of pandemic influenza A (H1N1) school outbreaks in China in 2009.
PLoS ONE
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During the 2009 pandemic influenza H1N1 (2009) virus (pH1N1) outbreak, school students were at an increased risk of infection by the pH1N1 virus. However, the estimation of the attack rate showed significant variability.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.