Fractionation of follicle stimulating hormone charge isoforms in their native form by preparative electrophoresis technology

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflow(TM) preparative electrophoresis technology is described. Gradiflow(TM) electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency. (C) 2005 Elsevier B.V. All rights reserved.

Adhesion of microbes using 3-aminopropyl triethoxy silane and specimen stabilisation techniques for analytical transmission electron microscopy

A variety of adhesive support-films were tested for their ability to adhere various biological specimens for transmission electron microscopy. Support films primed with 3-amino-propyl triethoxy silane (APTES), poly-L-lysine, carbon and ultraviolet-B (UV-B)-irradiated carbon were tested for their ability to adhere a variety of biological specimens including axenic cultures of Bacillus subtilis and Escherichia coli and wild-type magnetotactic bacteria. The effects of UV-B irradiation on the support film in the presence of air and electrostatic charge on primer deposition were tested and the stability of adhered specimens on various surfaces was also compared. APTES-primed UV-B-irradiated Pioloform(TM) was consistently the best adhesive, especially for large cells, and when adhered specimens were UV-B irradiated they became remarkably stable under an electron beam. This assisted the acquisition of in situ phase-contrast lattice images from a variety of biominerals in magnetotactic bacteria, in particular metastable greigite magnetosomes. Washing tests indicated that specimens adhering to APTES-primed UV-B-irradiated Pioloform(TM) were covalently coupled. The electron beam stability was hypothesised to be the result of mechanical strengthening of the specimen and support film and the reduced electrical resistance in the specimen and support film due to their polymerization and covalent coupling.

Comparison of palmtop-computer-assisted brief cognitive-behavioral treatment to cognitive-behavioral treatment for panic disorder

In the present study, the authors sought to determine whether the efficiency and cost-effectiveness of cognitive-behavioral treatment (CBT) for panic disorder could be improved by adjunctive computer-assisted therapy. Eighteen participants who met Diagnostic and Statistical Manual of Mental Disorders (3rd ed., revised; American Psychiatric Association, 1987) criteria for panic disorder were randomly assigned to a 12-session CBT (CBT12) condition (D. H. Barlow & M. G. Craske, 1989) or to a 4-session computer-assisted CBT (CBT4-CA) condition. Palmtop computers, with a program developed to incorporate basic principles of CBT, were used by CBT4-CA clients whenever they felt anxious or wanted to practice the therapy techniques and were used by all participants as a momentary assessment tool. CBT4-CA clients carried the computer at all times and continued to use it for 8 weeks after termination of therapy. Analyses of clinically significant change showed superiority of CBT12 at posttest on some measures; however, there were no differences at follow-up.

Development of ligase-assisted spacer addition for the measurement of microsatellites

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capability as a genotyping method.

Birth of Koalas Phascolarctos cinereus at Lone Pine Koala Sanctuary following artificial insemination

This paper documents the successful development of an artificial insemination (AI) programme for the Koala Phascolurctos cinereus. The protocols for trials involving two methods to induce ovulation and two insemination techniques are described. In Trial 1, interrupted coitus using a 'teaser'♂ successfully induced ovulation in nine Koalas. Five ♀♀ were inseminated while conscious using a modified 'foley catheter' (Cook insemination catheter) resulting in the births of two offspring. The other four ♀♀ were anaesthetized and inseminated using a technique which allowed visualization of the most cranial portion of the urogenital sinus, where semen was deposited using a 3.5 Fr. 'Tom-cat catheter' (urogen-itoscopic insemination). Three of the four ♀♀ inseminated by this technique produced pouch young. Microsatellite analysis of DNA from the pouch young excluded the teaser ♀♀ as possible sires, confirming that all offspring were sired by donor sperm. In Trial 2, eight ♀♀ were induced to ovulate by injecting them with 250 International Units of human chorionic gonadotrophin (hCG). A luteal phase was confirmed in all eight ♀♀ but only one gave birth following urogenitoscopic insemination. The Koala pouch young in this study are the first of any marsupial to be conceived and born following A1 procedures. Details of the A1 procedures used are presented and the significance of A1 to the conservation biology of P. cinereus discussed.

Taxonomy of Australian Funnel-web spiders using rp-HPLC/ESI-MS profiling techniques

The complex nature of venom from spider species offers a unique natural source of potential pharmacological tools and therapeutic leads. The increased interest in spider venom molecules requires reproducible and precise identification methods. The current taxonomy of the Australian Funnel-web spiders is incomplete, and therefore, accurate identification of these spiders is difficult. Here, we present a study of venom from numerous morphologically similar specimens of the Hadronyche infensa species group collected from a variety of geographic locations in southeast Queensland. Analysis of the crude venoms using online reversed-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (rp-HPLC/ESI-MS) revealed that the venom profiles provide a useful means of specimen identification, from the species level to species variants. Tables defining the descriptor molecules for each group of specimens were constructed and provided a quick reference of the relationship between one specimen and another. The study revealed that the morphologically similar specimens from the southeast Queensland region are a number of different species/species variants. Furthermore, the study supports aspects of the current taxonomy with respect to the H. infensa species group. Analysis of Australian Funnel-web spider venom by rp-HPLC/ESI-MS provides a rapid and accurate method of species/species variant identification. (c) 2006 Elsevier Ltd. All rights reserved.

Computationally efficient solution techniques for adsorption problems involving steep gradients in bidisperse particles

A piecewise uniform fitted mesh method turns out to be sufficient for the solution of a surprisingly wide variety of singularly perturbed problems involving steep gradients. The technique is applied to a model of adsorption in bidisperse solids for which two fitted mesh techniques, a fitted-mesh finite difference method (FMFDM) and fitted mesh collocation method (FMCM) are presented. A combination (FMCMD) of FMCM and the DASSL integration package is found to be most effective in solving the problems. Numerical solutions (FMFDM and FMCMD) were found to match the analytical solution when the adsorption isotherm is linear, even under conditions involving steep gradients for which global collocation fails. In particular, FMCMD is highly efficient for macropore diffusion control or micropore diffusion control. These techniques are simple and there is no limit on the range of the parameters. The techniques can be applied to a variety of adsorption and desorption problems in bidisperse solids with non-linear isotherm and for arbitrary particle geometry.

From Midden to Sieve: The Impact of Differential Recovery and Quantification Techniques on Interpretations of Shellfish Remains in Australian Coastal Archaeology

Experimental mechanical sieving methods are applied to samples of shellfish remains from three sites in southeast Queensland, Seven Mile Creek Mound, Sandstone Point and One-Tree, to test the efficacy of various recovery and quantification procedures commonly applied to shellfish assemblages in Australia. There has been considerable debate regarding the most appropriate sieve sizes and quantification methods that should be applied in the recovery of vertebrate faunal remains. Few studies, however, have addressed the impact of recovery and quantification methods on the interpretation of invertebrates, specifically shellfish remains. In this study, five shellfish taxa representing four bivalves (Anadara trapezia, Trichomya hirsutus, Saccostrea glomerata, Donax deltoides) and one gastropod (Pyrazus ebeninus) common in eastern Australian midden assemblages are sieved through 10mm, 6.3mm and 3.15mm mesh. Results are quantified using MNI, NISP and weight. Analyses indicate that different structural properties and pre- and postdepositional factors affect recovery rates. Fragile taxa (T. hirsutus) or those with foliated structure (S. glomerata) tend to be overrepresented by NISP measures in smaller sieve fractions, while more robust taxa (A. trapezia and P. ebeninus) tend to be overrepresented by weight measures. Results demonstrate that for all quantification methods tested a 3mm sieve should be used on all sites to allow for regional comparability and to effectively collect all available information about the shellfish remains.

Wolbachia infections and arthropod reproduction

Wolbachia is an intracellular bacterium that is almost exclusively maternally transmitted, and its reproductive effects favor transmission of the intracellular bacterial agent at the expense of the arthropod population that is not infected. Wolbachia can cause cytoplasmic incompatibility, parthenogenesis and feminization in many arthropods.

New techniques for biopsy and culture of human olfactory epithelial neurons

Objective: To improve the success of culturing olfactory neurons from human nasal mucosa by investigating the intranasal distribution of the olfactory epithelium and devising new techniques for growing human olfactory epithelium in vitro. Design: Ninety-seven biopsy specimens were obtained from 33 individuals, aged 21 to 74 years, collected from 6 regions of the nasal cavity. Each biopsy specimen was bisected, and 1 piece was processed for immunohistochemistry or electron microscopy while the other piece was dissected further for explant culture. Four culture techniques were performed, including whole explants and explanted biopsy slices. Five days after plating, neuronal differentiation was induced by means of a medium that contained basic fibroblast growth factor. After another 5 days, cultures were processed for immunocytochemical analysis. Results: The probability of finding olfactory epithelium in a biopsy specimen ranged from 30% to 76%, depending on its location. The dorsoposterior regions of the nasal septum and the superior turbinate provided the highest probability, but, surprisingly, olfactory epithelium was also found anteriorly and ventrally on both septum and turbinates. A new method of culturing the olfactory epithelium was devised. This slice culture technique improved the success rate for generating olfactory neurons from 10% to 90%. Conclusions: This study explains and overcomes most of the variability in the success in observing neurogenesis in cultures of adult human olfactory epithelium. The techniques presented here make the human olfactory epithelium a useful model for clinical research into certain olfactory dysfunctions and a model for the causes of neurodevelopmental and neurodegenerative diseases.

Pore structure tailoring of pillared clays with cation doping techniques

Techniques and mechanism of doping controlled amounts of various cations into pillared clays without causing precipitation or damages to the pillared layered structures are reviewed and discussed. Transition metals of great interest in catalysis can be doped in the micropores of pillared clay in ionic forms by a two-step process. The micropore structures and surface nature of pillared clays are altered by the introduced cations, and this results in a significant improvement in adsorption properties of the clays. Adsorption of water, air components and organic vapors on cation-doped pillared clays were studied. The effects of the amount and species of cations on the pore structure and adsorption behavior are discussed. It is demonstrated that the presence of doped Ca2+ ions can effectively aides the control of modification of the pillared clays of large pore openings. Controlled cation doping is a simple and powerful tool for improving the adsorption properties of pillared clay.

New techniques in nutritional assessment: body composition methods

New techniques in air-displacement plethysmography seem to have overcome many of the previous problems of poor reproducibility and validity. These have made body-density measurements available to a larger range of individuals, including children, elderly and sick patients who often have difficulties in being submerged underwater in hydrodensitometry systems. The BOD POD air-displacement system (BOD POD body composition system; Life Measurement Instruments, Concord, CA, USA) is more precise than hydrodensitometry, is simple and rapid to operate (approximately 1 min measurements) and the results agree closely with those of hydrodensitometry (e.g. +/-3.4% for estimation of body fat). Body line scanners employing the principles of three-dimensional photography are potentially able to measure the surface area and volume of the body and its segments even more rapidly (approximately 10 s), but the validity of the measurements needs to be established. Advances in i.r. spectroscopy and mathematical modelling for calculating the area under the curve have improved precision for measuring enrichment of (H2O)-H-2 in studies of water dilution (CV 0.1-0.9% within the range of 400-1000 mu l/l) in saliva, plasma and urine. The technique is rapid and compares closely with mass spectrometry (bias 1 (SD 2) %). Advances in bedside bioelectrical-impedance techniques are making possible potential measurements of skinfold thicknesses and limb muscle mass electronically. Preliminary results suggest that the electronic method is more reproducible (intra-and inter-individual reproducibility for measuring skinfold thicknesses) and associated with less bias (+ 12%), than anthropometry (+ 40%). In addition to these selected examples, the 'mobility' or transfer of reference methods between centres has made the distinction between reference and bedside or field techniques less distinct than in the past.