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- Formulation Approach for the Development of a Stable, Lyophilized Formaldehyde-Containing Vaccine. [JOURNAL ARTICLE]
- Eur J Pharm Biopharm 2013 May 11.
Formaldehyde has been used in the inactivation of a number of viral and bacterial toxins used in vaccines. In some cases, a small amount of formaldehyde may be necessary in order to prevent reversion back to the toxic state during storage. When a lyophilized preparation is required, care must be taken to ensure that formaldehyde is not lost during the process in order to ensure safety of the product. A design of experiments (DOE) approach was taken to devise a stable, lyophilized, vaccine formulation. A formaldehyde - inactivated bacterial toxin was used as a model antigen. Entrapment of formaldehyde in an amorphous matrix and/or interactions with amorphous components was found to be required for complete recovery of formaldehyde during lyophilization. In formulations consisting of sucrose and citrate, formaldehyde could be recovered across a wide range of excipient concentrations. Stability of the antigen was dependent on formaldehyde concentration, with antigen stability decreasing with increasing formaldehyde concentration. This is in contrast to the risk of reversion which increases with decreasing concentrations of formaldehyde. Finally, variations in temperatures during annealing, primary drying, and secondary drying had no impact on formaldehyde recovery.
- Using 20-million-year-old amber to test the super-Arrhenius behaviour of glass-forming systems. [Journal Article]
- Nat Commun 2013.:1783.
Fossil amber offers the opportunity to investigate the dynamics of glass-forming materials far below the nominal glass transition temperature. This is important in the context of classical theory, as well as some new theories that challenge the idea of an 'ideal' glass transition. Here we report results from calorimetric and stress relaxation experiments using a 20-million-year-old Dominican amber. By performing the stress relaxation experiments in a step-wise fashion, we measured the relaxation time at each temperature and, above the fictive temperature of this 20-million-year-old glass, this is an upper bound to the equilibrium relaxation time. The results deviate dramatically from the expectation of classical theory and are consistent with some modern ideas, in which the diverging timescale signature of complex fluids disappears below the glass transition temperature.
- Construction and analysis of a genetically tuneable lytic phage display system. [JOURNAL ARTICLE]
- Appl Microbiol Biotechnol 2013 May 3.
The Bacteriophage λ capsid protein gpD has been used extensively for fusion polypeptides that can be expressed from plasmids in Escherichia coli and remain soluble. In this study, a genetically controlled dual expression system for the display of enhanced green fluorescent protein (eGFP) was developed and characterized. Wild-type D protein (gpD) expression is encoded by λ Dam15 infecting phage particles, which can only produce a functional gpD protein when translated in amber suppressor strains of E. coli in the absence of complementing gpD from a plasmid. However, the isogenic suppressors vary dramatically in their ability to restore functional packaging to λDam15, imparting the first dimension of decorative control. In combination, the D-fusion protein, gpD::eGFP, was supplied in trans from a multicopy temperature-inducible expression plasmid, influencing D::eGFP expression and hence the availability of gpD::eGFP to complement for the Dam15 mutation and decorate viable phage progeny. Despite being the worst suppressor, maximal incorporation of gpD::eGFP into the λDam15 phage capsid was imparted by the SupD strain, conferring a gpDQ68S substitution, induced for plasmid expression of pD::eGFP. Differences in size, fluorescence and absolute protein decoration between phage preparations could be achieved by varying the temperature of and the suppressor host carrying the pD::eGFP plasmid. The effective preparation with these two variables provides a simple means by which to manage fusion decoration on the surface of phage λ.
- Configurational assignments of conformationally restricted bis-monoterpene hydroquinones: Utility in exploration of endangered plants. [JOURNAL ARTICLE]
- Biochim Biophys Acta 2013 Apr 26.
BACKGROUND:Endangered plant species are an important resource for new chemistry. Lindera melissifolia is native to the Southeastern U.S. and scarcely populates the edges of lakes and ponds. Quantum mechanics (QM) used in combination with NMR/ECD is a powerful tool for the assignment of absolute configuration in lieu of X-ray crystallography.
METHODS:The EtOAc extract of L. melissifolia was subject to chromatographic analysis by VLC and HPLC. Spin-spin coupling constant (SSCC) were calculated using DFT at the MPW1PW91/6-31G(d,p) level for all staggered rotamers. ECD calculations employed Amber* force fields followed by PM6 semi-empirical optimizations. Hetero- and homo-nuclear coupling constants were extracted from 1D (1)H, E.COSY and HETLOC experiments.
RESULTS:Two meroterpenoids, melissifolianes A (1) and B (2) were purified and their 2-D structures elucidated using NMR and HRESIMS. The relative configuration of 1 was established using the combination of NOE-based distance restraints and the comparisons of experimental and calculated SSCCs. The comparison of calculated and experimental ECD assigned the absolute configuration of 1. The relative configuration of a racemic mixture, melissifoliane B (2) was established utilizing J-based analysis combined with QM and NMR techniques. GENERAL
SIGNIFICANCE:QM-NMR/ECD combined approaches are of utility for unambiguous assignment of 3-D structures, especially with limited plant material and when a molecule is conformationally restricted. Conservation of an endangered plant species can be supported through identification of its new chemistry and utilization of that chemistry for commercial purposes.
- Multiple site-selective insertions of noncanonical amino acids into sequence-repetitive polypeptides. [Journal Article]
- Chembiochem 2013 May 27; 14(8):968-78.
A simple and efficient method is described for the introduction of noncanonical amino acids at multiple, defined sites within recombinant polypeptide sequences. Escherichia coli MRA30, a bacterial host strain with attenuated activity of release factor 1 (RF1), was assessed for its ability to support incorporation of a diverse range of noncanonical amino acids in response to multiple encoded amber (TAG) codons within genes derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pair and the noncanonical amino acid. Elastin-mimetic protein polymers were prepared in which noncanonical amino acid derivatives were incorporated at up to 22 specific sites within the polypeptide sequence with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments from thermolysin digestion. The data suggest that this multisite suppression approach permits the preparation of protein-based materials in which novel chemical functionalities can be introduced at precisely defined positions within the polypeptide sequence.
- Cover image, volume 34, issue 15. [Journal Article]
- J Comput Chem 2013 Jun 5; 34(15):i-ii.
Developable domains characteristic of columnar mesophases are seen when mixed-valent diruthenium tetracarboxylates are observed under a polarizing optical microscopy by Maria Castro, Adrian Roitberg, and Fabio Cukiernik on page 1283. Molecular dynamics simulations with an extended AMBER force field provide insights about the relative contributions of different intermolecular interactions to the supramolecular architecture of these coordination polymers in the liquid crystalline phase. The cover shows two snapshot views from the molecular dynamics calculation, which are overlaid onto an image from the microscope.
- Stability of propranolol in extemporaneously compounded suspensions. [Journal Article]
- Can J Hosp Pharm 2013 Mar; 66(2):118-24.
Propranolol is a drug of choice for many diseases occurring in neonates and infants, an age group for which oral suspensions are required almost exclusively. Many adult and elderly patients for whom propranolol is prescribed are also unable to swallow solid dosage forms. In Canada, propranolol is not commercially available in a liquid dosage form, and existing recipes for extemporaneously compounded suspensions of propranolol (1 mg/mL) are limited by concerns regarding diabetes mellitus in certain subpopulations, the need for a more concentrated suspension for patients taking larger doses, and the tediousness of compounding.To evaluate the stability of propranolol suspensions in a sugar-free, commercially available vehicle after storage at room temperature and under refrigeration for up to 120 days.Suspensions of propranolol (2 and 5 mg/mL) were prepared in the sugar-free vehicle (Ora-Blend SF), placed in 100-mL amber plastic prescription bottles, and stored at 25°C and 4°C. Samples were collected from each bottle once weekly for 120 days, stored frozen, and analyzed by a validated, stability-indicating high-performance liquid chromatography - ultraviolet detection method. A suspension was considered stable if it maintained at least 90% of its initial concentration of propranolol. Physical compatibility was evaluated in terms of colour, taste, precipitation, and pH.Propranolol suspensions 2 mg/mL and 5 mg/mL stored at 25°C maintained at least 94.7% of their initial concentration for 120 days, and suspensions stored at 4°C maintained at least 93.9% of their initial concentration for 120 days. There were no notable changes in pH, and all samples remained physically unchanged except for a slight change in colour, around day 70, of suspensions stored at room temperature.Propranolol suspensions (2 mg/mL and 5 mg/mL) prepared in Ora-Blend SF and stored in plastic prescription bottles at either 25°C or 4°C are expected to remain stable for 120 days.
- Ribozyme-based tRNA switches for post-transcriptional control of amino acid identity in protein synthesis. [JOURNAL ARTICLE]
- J Am Chem Soc 2013 Apr 23.
Protein mutants are studied in a variety of contexts in the life sciences. However, individual mutations need to be generated in order to transcribe and translate the respective protein variants. Here we introduce a novel strategy for controlling the incorporation of different amino acids in response to an amber stop codon by utilizing switchable designer tRNAs in Escherichia coli.