Spontaneous generation and survival of blood dendritic cells in mononuclear cell culture without exogenous cytokines

Studies on purified blood dendritic cells (DCs) are hampered by poor viability in tissue culture. We, therefore, attempted to study some of the interactions/relationships between DCs and other blood cells by culturing unseparated peripheral blood mononuclear cell (PBMC) preparations in vitro. Flow cytometric techniques were used to undertake a phenotypic and functional analysis of DCs within the cultured PBMC population. We discovered that both the CD11c(+) and CD11c(-) CD123(hi) DC subsets maintained their viability throughout the 3-day culture period, without the addition of exogenous cytokines. This viability was accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts increased (for CD123hi and CD11c+ subsets) after overnight culture of PBMCs. Single-cell lineage depletion experiments demonstrated the rapid and spontaneous emergence of new in vitro generated DCs from CD14(+)/CD16(+) PBMC radioresistant precursors, additional to the preexisting ex vivo DC population. Unlike monocyte-derived DCs, blood DCs increased dextran uptake with culture and activation. Finally, DCs obtained after culture of PBMCs for 3 days were as effective as freshly isolated DCs in stimulating an allogeneic mixed leukocyte reaction. (C) 2002 by The American Society of Hematology.

Analysis of lipoproteins using 2D diffusion-edited NMR spectroscopy and multi-way chemometrics

This study represents the first application of multi-way calibration by N-PLS and multi-way curve resolution by PARAFAC to 2D diffusion-edited H-1 NMR spectra. The aim of the analysis was to evaluate the potential for quantification of lipoprotein main- and subtractions in human plasma samples. Multi-way N-PLS calibrations relating the methyl and methylene peaks of lipoprotein lipids to concentrations of the four main lipoprotein fractions as well as 11 subfractions were developed with high correlations (R = 0.75-0.98). Furthermore, a PARAFAC model with four chemically meaningful components was calculated from the 2D diffusion-edited spectra of the methylene peak of lipids. Although the four extracted PARAFAC components represent molecules of sizes that correspond to the four main fractions of lipoproteins, the corresponding concentrations of the four PARAFAC components proved not to be correlated to the reference concentrations of these four fractions in the plasma samples as determined by ultracentrifugation. These results indicate that NMR provides complementary information on the classification of lipoprotein fractions compared to ultracentrifugation. (C) 2004 Elsevier B.V. All rights reserved.

Comparative analysis of prothrombin activators from the venom of Australian elapids

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Cost-effectiveness analysis of a kidney and cardiovascular disease treatment program in an Australian aboriginal population

The objective of the study was to assess, from a health service perspective, whether a systematic program to modify kidney and cardiovascular disease reduced the costs of treating end-stage kidney failure. The participants in the study were 1,800 aboriginal adults with hypertension, diabetes with microalbuminuria or overt albuminuria, and overt albuminuria, living on two islands in the Northern Territory of Australia during 1995 to 2000. Perindopril was the primary treatment agent, and other medications were also used to control blood pressure. Control of glucose and lipid levels were attempted, and health education was offered. Evaluation of program resource use and costs for follow-up periods was done at 3 and 4.7 years. On an intention-to-treat basis, the number of dialysis starts and dialysis-years avoided were estimated by comparing the fate of the treatment group with that of historical control subjects, matched for disease severity, who were followed in the before the treatment program began. For the first three years, an estimated 11.6 person-years of dialysis were avoided, and over 4.7 years, 27.7 person-years of dialysis were avoided. The net cost of the program was $1,210 more per person per year than status quo care, and dialyses avoided gave net savings of $1.0 million at 3 years and $3.4 million at 4.6 years. The treatment program provided significant health benefit and impressive cost savings in dialysis avoided. (C) 2005 by the National Kidney Foundation, Inc.

Crystallization and preliminary X-ray analysis of a Kunitz-type inhibitor, textilinin-1 from Pseudonaja textilis textilis

Textilinin-1 (Txln-1), a Kunitz-type serine protease inhibitor, is a 59-amino-acid polypeptide isolated from the venom of the Australian Common Brown snake Pseudonaja textilis textilis. This molecule has been suggested as an alternative to aprotinin, also a Kunitz-type serine protease inhibitor, for use as an anti-bleeding agent in surgical procedures. Txln-1 shares only 47% amino-acid identity to aprotinin; however, six cysteine residues in the two peptides are in conserved locations. It is therefore expected that the overall fold of these molecules is similar but that they have contrasting surface features. Here, the crystallization of recombinant textilinin-1 (rTxln-1) as the free molecule and in complex with bovine trypsin (229 amino acids) is reported. Two organic solvents, phenol and 1,4-butanediol, were used as additives to facilitate the crystallization of free rTxln-1. Crystals of the rTxln-1-bovine trypsin complex diffracted to 2.0 angstrom resolution, while crystals of free rTxln-1 diffracted to 1.63 angstrom resolution.

Longitudinal Genetic Analysis of Plasma Lipids

The consensus from published studies is that plasma lipids are each influenced by genetic factors, and that this contributes to genetic variation in risk of cardiovascular disease. Heritability estimates for lipids and lipoproteins are in the range .48 to .87, when measured once per study participant. However, this ignores the confounding effects of biological variation measurement error and ageing, and a truer assessment of genetic effects on cardiovascular risk may be obtained from analysis of longitudinal twin or family data. We have analyzed information on plasma high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, and triglycerides, from 415 adult twins who provided blood on two to five occasions over 10 to 17 years. Multivariate modeling of genetic and environmental contributions to variation within and across occasions was used to assess the extent to which genetic and environmental factors have long-term effects on plasma lipids. Results indicated that more than one genetic factor influenced HDL and LDL components of cholesterol, and triglycerides over time in all studies. Nonshared environmental factors did not have significant long-term effects except for HDL. We conclude that when heritability of lipid risk factors is estimated on only one occasion, the existence of biological variation and measurement errors leads to underestimation of the importance of genetic factors as a cause of variation in long-term risk within the population. In addition our data suggest that different genes may affect the risk profile at different ages.

Hydrothermal Stability Analysis of Carbonised Template Molecular Sieve Silica Membranes

For fuel cell CO clean up application, the presence of water with silica membranes greatly reduces their selectivity to CO. We show results of a new functional carbonised template membrane of around 13nm thickness which offered hydrothermal stability with no compromise to the membrane’s H2/CO permselectivity of 16. Lost permeance was also regenerated.

A matching pursuit-based signal complexity measure for the analysis of newborn EEG

This paper presents a new relative measure of signal complexity, referred to here as relative structural complexity, which is based on the matching pursuit (MP) decomposition. By relative, we refer to the fact that this new measure is highly dependent on the decomposition dictionary used by MP. The structural part of the definition points to the fact that this new measure is related to the structure, or composition, of the signal under analysis. After a formal definition, the proposed relative structural complexity measure is used in the analysis of newborn EEG. To do this, firstly, a time-frequency (TF) decomposition dictionary is specifically designed to compactly represent the newborn EEG seizure state using MP. We then show, through the analysis of synthetic and real newborn EEG data, that the relative structural complexity measure can indicate changes in EEG structure as it transitions between the two EEG states; namely seizure and background (non-seizure).

Analysis of Wolbachia protein synthesis in Drosophila in vivo

Intracellular Wolbachia infections are extremely common in arthropods and exert profound control over the reproductive biology of the host. However, very little is known about the underlying molecular mechanisms which mediate these interactions with the host. We examined protein synthesis by Wolbachia in a Drosophila host in vivo by selective metabolic labelling of prokaryotic proteins and subsequent analysis by 1D and 2D gel electrophoresis. Using this method we could identify the major proteins synthesized by Wolbachia in ovaries and testes of flies. Of these proteins the most abundant was of low molecular weight and showed size variation between Wolbachia strains which correlated with the reproductive phenotype they generated in flies. Using the gel systems we employed it was not possible to identify any proteins of Wolbachia origin in the mature sperm cells of infected flies.

16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects

Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the α-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggests that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized.

XSophe-Sophe-XeprView (R). A computer simulation software suite (v. 1.1.3) for the analysis of continuous wave EPR spectra

The XSophe-Sophe-XeprView((R)) computer simulation software suite enables scientists to easily determine spin Hamiltonian parameters from isotropic, randomly oriented and single crystal continuous wave electron paramagnetic resonance (CW EPR) spectra from radicals and isolated paramagnetic metal ion centers or clusters found in metalloproteins, chemical systems and materials science. XSophe provides an X-windows graphical user interface to the Sophe programme and allows: creation of multiple input files, local and remote execution of Sophe, the display of sophelog (output from Sophe) and input parameters/files. Sophe is a sophisticated computer simulation software programme employing a number of innovative technologies including; the Sydney OPera HousE (SOPHE) partition and interpolation schemes, a field segmentation algorithm, the mosaic misorientation linewidth model, parallelization and spectral optimisation. In conjunction with the SOPHE partition scheme and the field segmentation algorithm, the SOPHE interpolation scheme and the mosaic misorientation linewidth model greatly increase the speed of simulations for most spin systems. Employing brute force matrix diagonalization in the simulation of an EPR spectrum from a high spin Cr(III) complex with the spin Hamiltonian parameters g(e) = 2.00, D = 0.10 cm(-1), E/D = 0.25, A(x) = 120.0, A(y) = 120.0, A(z) = 240.0 x 10(-4) cm(-1) requires a SOPHE grid size of N = 400 (to produce a good signal to noise ratio) and takes 229.47 s. In contrast the use of either the SOPHE interpolation scheme or the mosaic misorientation linewidth model requires a SOPHE grid size of only N = 18 and takes 44.08 and 0.79 s, respectively. Results from Sophe are transferred via the Common Object Request Broker Architecture (CORBA) to XSophe and subsequently to XeprView((R)) where the simulated CW EPR spectra (1D and 2D) can be compared to the experimental spectra. Energy level diagrams, transition roadmaps and transition surfaces aid the interpretation of complicated randomly oriented CW EPR spectra and can be viewed with a web browser and an OpenInventor scene graph viewer.

Experimental Analysis of Froude Number Effect on Air Entrainment in the Hydraulic Jump

A hydraulic jump is characterized by strong energy dissipation and mixing, large-scale turbulence, air entrainment, waves and spray. Despite recent pertinent studies, the interaction between air bubbles diffusion and momentum transfer is not completely understood. The objective of this paper is to present experimental results from new measurements performed in rectangular horizontal flume with partially-developed inflow conditions. The vertical distributions of void fraction and air bubbles count rate were recorded for inflow Froude number Fr1 in the range from 5.2 to 14.3. Rapid detrainment process was observed near the jump toe, whereas the structure of the air diffusion layer was clearly observed over longer distances. These new data were compared with previous data generally collected at lower Froude numbers. The comparison demonstrated that, at a fixed distance from the jump toe, the maximum void fraction Cmax increases with the increasing Fr1. The vertical locations of the maximum void fraction and bubble count rate were consistent with previous studies. Finally, an empirical correlation between the upper boundary of the air diffusion layer and the distance from the impingement point was provided.