Encoding combinatorial libraries: A novel application of fluorescent silica colloids

A major challenge associated with using large chemical libraries synthesized on microscopic solid support beads is the rapid discrimination of individual compounds in these libraries. This challenge can be overcome by encoding the beads with 1 mum silica colloidal particles (reporters) that contain specific and identifiable combinations of fluorescent byes. The colored bar code generated on support beads during combinatorial library synthesis can be easily, rapidly, and inexpensively decoded through the use of fluorescence microscopy. All reporters are precoated with polyelectrolytes [poly(acrylic acid), PAA, poly(sodium 4-styrenesulfonate PSSS, polyethylenimine, PEI, and/or poly(diallyldimethylammonium chloride), PDADMAC] with the aim of enhancing surface charge, promoting electrostatic attraction to the bead, and facilitating polymer bridging between the bead and reporter for permanent adhesion. As shown in this article, reporters coated with polyelectrolytes clearly outperform uncoated reporters with regard to quantity of attached reporters per bead (54 +/- 23 in 2500 mum(2) area for PEI/PAA coated and 11 +/- 6 for uncoated reporters) and minimization of cross-contamination (1 red reporter in 2500 mum(2) area of green-labeled bead for PEI/PAA coated and 26 +/- 15 red reporters on green-labeled beads for uncoated reporters after 10 days). Examination of various polyelectrolyte systems shows that the magnitude of the xi -potential of polyelectrolyte-coated reporters (-64 mV for PDADMAC/PSSS and -42 mV for PEI/PAA-coated reporters) has no correlation with the number of reporters that adhere to the solid support beads (21 +/- 16 in 2500 mum(2) area for PDADMAC/PSSS and 54 +/- 23 for PEI/PAA-coated reporters). The contribution of polymer bridging to the adhesion has a far greater influence than electrostatic attraction and is demonstrated by modification of the polyelectrolyte multilayers using gamma irradiation of precoated reporters either in aqueous solution or in polyelectrolyte solution.

Small area population disease burden

Small area health statistics has assumed increasing importance as the focus of population and public health moves to a more individualised approach of smaller area populations. Small populations and low event occurrence produce difficulties in interpretation and require appropriate statistical methods, including for age adjustment. There are also statistical questions related to multiple comparisons. Privacy and confidentiality issues include the possibility of revealing information on individuals or health care providers by fine cross-tabulations. Interpretation of small area population differences in health status requires consideration of migrant and Indigenous composition, socio-economic status and rural-urban geography before assessment of the effects of physical environmental exposure and services and interventions. Burden of disease studies produce a single measure for morbidity and mortality - disability adjusted life year (DALY) - which is the sum of the years of life lost (YLL) from premature mortality and the years lived with disability (YLD) for particular diseases (or all conditions). Calculation of YLD requires estimates of disease incidence (and complications) and duration, and weighting by severity. These procedures often mean problematic assumptions, as does future discounting and age weighting of both YLL and YLD. Evaluation of the Victorian small area population disease burden study presents important cross-disciplinary challenges as it relies heavily on synthetic approaches of demography and economics rather than on the empirical methods of epidemiology. Both empirical and synthetic methods are used to compute small area mortality and morbidity, disease burden, and then attribution to risk factors. Readers need to examine the methodology and assumptions carefully before accepting the results.

A preliminary phylogeny of the scale insects (Hemiptera : Sternorrhyncha : Coccoidea) based on nuclear small-subunit ribosomal DNA

Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are a speciose and morphologically specialized group of plant-feeding bugs in which evolutionary relationships and thus higher classification are controversial. Sequences derived from nuclear small-subunit ribosomal DNA were used to generate a preliminary molecular phylogeny for the Coccoidea based on 39 species representing 14 putative families. Monophyly of the archaeococcoids (comprising Ortheziidae, Margarodidae sensu lato, and Phenacoleachia) was equivocal, whereas monophyly of the neococcoids was supported. Putoidae, represented by Puto yuccae, was found to be outside the remainder of the neococcoid clade. These data are consistent with a single origin (in the ancestor of the neococcoid clade) of a chromosome system involving paternal genome elimination in males. Pseudococcidae (mealybugs) appear to be sister to the rest of the neococcoids and there are indications that Coccidae (soft scales) and Kerriidae (lac scales) are sister taxa. The Eriococcidae (felt scales) was not recovered as a monophyletic group and the eriococcid genus Eriococcus sensu lato was polyphyletic. (C) 2002 Elsevier Science (USA). All rights reserved.

A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted.We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Limited ecoclinal variation found in Malva parviflora (small-flowered mallow) across the Mediterranean-climatic agricultural region of Western Australia

Malva parviflora L. populations were collected from 24 locations across the Mediterranean-climatic agricultural region of Western Australia and grown in Perth in a common garden experiment. Seventeen morphometric and taxonomic measurements were taken and genetic variation was investigated by performing principal components analysis (PCA). Taxonomic measurements confirmed that all plants used in the study were M. parviflora. Greater variation occurred within populations than between populations. Separation between populations was only evident between northern and southern populations along principal components 2 (PC2), which was due mainly to flowering time. Flowering time and consequently photoperiod were highly correlated with latitude and regression analysis revealed a close relationship (r(2) = 0.6). Additionally, the pollination system of M. parviflora was examined. Plants were able to self-pollinate without the need for external vectors and the pollen ovule ratio (31 +/- 1.3) revealed that M. parviflora is most likely to be an obligate inbreeder with a slight potential for outcrossing. The limited variation of M. parviflora enhances the likelihood of suitable control strategies being effective across a broad area.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Estimating Investment Risk in Small-Scale Plantation Rainforest Cabinet Timbers and Eucalypts

‘This book is a landmark opening and first attempt at such a process for defining farm forestry, as well as making a contribution to small-scale forestry.’