Diversity and transmission of koala retrovirus: a case study in three captive koala populations.

Koala retrovirus is a recently endogenized retrovirus associated with the onset of neoplasia and infectious disease in koalas. There are currently twelve described KoRV subtypes (KoRV-A to I, K-M), most of which were identified through recently implemented deep sequencing methods which reveal an animals' overall KoRV profile. This approach has primarily been carried out on wild koala populations around Australia, with few investigations into the whole-population KoRV profile of captive koala colonies to date. This study conducted deep sequencing on 64 captive koalas of known pedigree, housed in three institutions from New South Wales and South-East Queensland, to provide a detailed analysis of KoRV genetic diversity and transmission. The final dataset included 93 unique KoRV sequences and the first detection of KoRV-E within Australian koala populations. Our analysis suggests that exogenous transmission of KoRV-A, B, D, I and K primarily occurs between dam and joey. Detection of KoRV-D in a neonate sample raises the possibility of this transmission occurring in utero. Overall, the prevalence and abundance of KoRV subtypes was found to vary considerably between captive populations, likely due to their different histories of animal acquisition. Together these findings highlight the importance of KoRV profiling for captive koalas, in particular females, who play a primary role in KoRV exogenous transmission.

Characterization of antigens adsorbed to anionic PLG microparticles by XPS and TOF-SIMS.

The chemical composition of the surface of anionic PLG microparticles before and after adsorption of vaccine antigens was measured using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The interfacial distributions of components will reflect underlying interactions that govern properties such as adsorption, release, and stability of proteins in microparticle vaccine delivery systems. Poly(lactide-co-glycolide) microparticles were prepared by a w/o/w emulsification method in the presence of the anionic surfactant dioctyl sodium sulfosuccinate (DSS). Ovalbumin, lysozyme, a recombinant HIV envelope glyocoprotein and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with XPS and time-of-flight secondary mass used to analyze elemental and molecular distributions of components of the surface of lyophilized products. Protein (antigen) binding to PLG microparticles was measured directly by distinct elemental and molecular spectroscopic signatures consistent with amino acids and excipient species. The surface sensitive composition of proteins also included counter ions that support the importance of electrostatic interactions being crucial in the mechanism of adsorptions. The protein binding capacity was consistent with the available surface area and the interpretation of previous electron and atomic force microscope images strengthened by the quantification possible by XPS and the qualitative identification possible with TOF-SIMS. Protein antigens were detected and quantified on the surface of anionic PLG microparticles with varying degrees of efficiency under different adsorption conditions such as surfactant level, pH, and ionic strength. Observable changes in elemental and molecular composition suggest an efficient electrostatic interaction creating a composite surface layer that mediates antigen binding and release.

Elastic modulus measurements from individual lactose particles using atomic force microscopy.

The elastic modulus of pharmaceutical materials affects a number of pharmaceutical processes and subsequently formulation performance and is currently assessed by bulk methods, such as beam bending of compacts. Here we demonstrate the accurate measurement of the elastic modulus of alpha monohydrate lactose from the dominant (011) face of single crystals using atomic force microscopy (AFM) as 3.45+/-0.90GPa. The criteria to ensure this data is recorded within the elastic limit and can be modelled using Hertzian theory are established. We compare and contrast this AFM method to a permanent indentation technique based upon a much larger Berkovich pyramidal indenter on a lactose compact and the wider literature. Finally the AFM was utilized to study the elastic response of amorphous lactose, demonstrating that the physical state of the amorphous material changes under repeated loading and behaves in a more crystalline manner under repeated force measurements, suggesting a pressure induced phase transition. The AFM based approach demonstrated has the significant advantages of requiring minimal sample, no need for producing a compact, being non-destructive in that no permanent indent is required and providing a technique capable of detecting variations in material properties across a single particle or a number of particles.

Determination of the surface free energy of crystalline and amorphous lactose by atomic force microscopy adhesion measurement.

PURPOSE: This study was conducted to accurately measure the dispersive surface free energy of lactose solids in ordered and disordered states. METHODS: Atomic force microscopy (AFM) was used to determine the contact adhesion force between an AFM tip and lactose under low humidity (ca. 1% RH). The geometry of the tip contacting apex was characterized by scanning a porous aluminum film with ultrasharp spikes (radius 2-3 nm). A sphere vs. flat surface model was employed to relate the adhesion force determined to the surface energy based upon the Johnson-Kendal-Roberts theory. Spray-dried amorphous lactose in a compressed-disk form and single crystals of alpha-lactose monohydrate were prepared as model samples. RESULTS: The condition of the smooth sample surface and sphere-shaped tip used was shown to be appropriate to the application of the JKR model. The surface energy of crystalline [(0,-1,-1) face] and amorphous lactose was determined to be 23.3 +/- 2.3 and 57.4 +/- 7.9 mJ m(-2), respectively. CONCLUSIONS: We have demonstrated the capability of AFM to measure the dispersive surface free energy of pharmaceutical materials directly through a blank probe at the nanometer scale. These data, although consistent with results from more traditional methods, illustrate some unique attributes of this approach, namely, surface energies are directly derived from solid-solid interactions, measurements may be made on specific crystalline faces, and the potential exists to identify the submicron heterogeneity of organic solids in terms of their molecular energy states (such as ordered and disordered lactose).

Identifying and mapping surface amorphous domains.

PURPOSE: Undesirable amorphous material generation during formulation is implicated in a growing number of pharmaceutical problems. Due to the importance of interfacial properties in many drug delivery systems, it seems that surface amorphous material is particularly significant. Consequently, this study investigates a range of methods capable of detecting and mapping surface amorphous material. METHODS: A micron-sized localized surface domain of amorphous sorbitol is generated using a novel localized heating method. The domain is subsequently investigated using atomic force microscopy (AFM) imaging, nanomechanical measurements, and Raman microscopy 3-D profiling. RESULTS: AFM phase and height images reveal nanoscale-order variations within both crystalline and amorphous sorbitol domains. Nanomechanical measurements are able to quantitatively distinguish the amorphous and crystalline domains through local Young's modulus measurements. Raman microscopy also distinguishes the amorphous and crystalline sorbitol through variations in peak width. This is shown to allow mapping of the 3-D distribution of the amorphous phase and is hence complementary to the more surface sensitive AFM measurements. CONCLUSIONS: AFM and Raman microscopy map the distribution of amorphous material at the surface of a sorbitol crystal with submicron spatial resolution, demonstrating surface analysis methods for characterizing semicrystalline solids generated during pharmaceutical processing.

Progressing single biomolecule force spectroscopy measurements for the screening of DNA binding agents.

Recent studies have indicated that the force-extension properties of single molecules of double stranded (ds) DNA are sensitive to the presence of small molecule DNA binding agents, and also to their mode of binding. These observations raise the possibility of using this approach as a highly sensitive tool for the screening of such agents. However, particularly for studies employing the atomic force microscope (AFM), several non-trivial barriers hinder the progress of this approach to the non-specialist arena and hence also the full realization of this possibility. In this paper, we therefore address a series of key reproducibility and metrological issues associated with this type of measurement. Specifically, we present an improved immobilization method that covalently anchors one end (5' end) of a dual labelled (5'-thiol, 3'-biotin) p53 DNA molecule onto a gold substrate via gold-thiol chemistry, whilst the biotinylated 3' end is available for 'pick-up' using a streptavidin modified AFM tip. We also show that co-surface immobilization of DNA with 6-mercapto-1-hexanol (MCH) can also lead to a further increase the measured contour length. We demonstrate the impact of these improved protocols through the observation of the cooperative transition plateau in a DNA fragment of approximately 118 bp, a significantly smaller fragment than previously investigated. The results of a comparative study of the effects of a model minor groove binder (Hoechst 33258) and an intercalating drug (proflavine), alone, as a mixture and under different buffer conditions, are also presented.