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.

Differential patterns of Male and Female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis

Comparisons among loci with differing modes of inheritance can reveal unexpected aspects of population history. We employ a multilocus approach to ask whether two types of independently assorting mitochondrial DNAs (maternally and paternally inherited: F- and M-mtDNA) and a nuclear locus (ITS) yield concordant estimates of gene flow and population divergence. The blue mussel, Mytilus edulis, is distributed on both North American and European coastlines and these populations are separated by the waters of the Atlantic Ocean. Gene flow across the Atlantic Ocean differs among loci, with F-mtDNA and ITS showing an imprint of some genetic interchange and M-mtDNA showing no evidence for gene flow. Gene flow of F-mtDNA and ITS causes trans-Atlantic population divergence times to be greatly underestimated for these loci, although a single trans-Atlantic population divergence time (1.2 MYA) can be accommodated by considering all three loci in combination in a coalescent framework. The apparent lack of gene flow for M-mtDNA is not readily explained by different dispersal capacities of male and female mussels. A genetic barrier to M-mtDNA exchange between North American and European mussel populations is likely to explain the observed pattern, perhaps associated with the double uniparental system of mitochondrial DNA inheritance.

Selection bias in gene extraction on the basis of microarray gene-expression data

In the context of cancer diagnosis and treatment, we consider the problem of constructing an accurate prediction rule on the basis of a relatively small number of tumor tissue samples of known type containing the expression data on very many (possibly thousands) genes. Recently, results have been presented in the literature suggesting that it is possible to construct a prediction rule from only a few genes such that it has a negligible prediction error rate. However, in these results the test error or the leave-one-out cross-validated error is calculated without allowance for the selection bias. There is no allowance because the rule is either tested on tissue samples that were used in the first instance to select the genes being used in the rule or because the cross-validation of the rule is not external to the selection process; that is, gene selection is not performed in training the rule at each stage of the cross-validation process. We describe how in practice the selection bias can be assessed and corrected for by either performing a cross-validation or applying the bootstrap external to the selection process. We recommend using 10-fold rather than leave-one-out cross-validation, and concerning the bootstrap, we suggest using the so-called. 632+ bootstrap error estimate designed to handle overfitted prediction rules. Using two published data sets, we demonstrate that when correction is made for the selection bias, the cross-validated error is no longer zero for a subset of only a few genes.

Periodic or Bounded Solutions of Carathéodory Systems of Ordinary Differential Equations

In this paper we extend the guiding function approach to show that there are periodic or bounded solutions for first order systems of ordinary differential equations of the form x1 =f(t,x), a.e. epsilon[a,b], where f satisfies the Caratheodory conditions. Our results generalize recent ones of Mawhin and Ward.

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection

This communication describes an improved one-step solid-phase extraction method for the recovery of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) from human plasma with reduced coextraction of endogenous plasma constituents, compared to that of the authors' previously reported method. The magnitude of the peak caused by endogenous plasma components in the chromatogram that eluted immediately before the retention time of M3G has been reduced (similar to 80%) significantly (p < 0.01) while achieving high extraction efficiencies for the compounds of interest, viz morphine, M6G, and M3G (93.8 +/- 2.5, 91.7 +/- 1.7, and 93.1 +/- 2.2%, respectively). Furthermore, when the improved solid-phase extraction method was used, the extraction cartridge-derived late-eluting peak (retention time 90 to 100 minutes) reported in our previous method, was no longer present in the plasma extracts. Therefore the combined effect of reducing the recovery of the endogenous components of plasma that chromatographed just before the retention time of M3G and the removal of the late-eluting, extraction cartridge-derived peak has resulted in a decrease in the chromatographic run-time to 20 minutes, thereby increasing the sample throughput by up to 100%.

Pilot-scale extraction of PHB from recombinant E-coli by homogenization and centrifugation

A new method of poly-beta-hydroxybutyrate (PHB) extraction from recombinant E. coli is proposed, using homogenization and centrifugation coupled with sodium hypochlorite treatment. The size of PHB granules and cell debris in homogenates was characterised as a function of the number of homogenization passes. Simulation was used to develop the PHB and cell debris fractionation system, enabling numerical examination of the effects of repeated homogenization and centrifuge-feedrate variation. The simulation provided a good prediction of experimental performance. Sodium hypochlorite treatment was necessary to optimise PHB fractionation. A PHB recovery of 80% at a purity of 96.5% was obtained with the final optimised process. Protein and DNA contained in the resultant product were negligible. The developed process holds promise for significantly reducing the recovery cost associated with PHB manufacture.

Solid-phase extraction method with high-performance liquid chromatography and electrochemical detection for the quantitative analysis of oxycodone in human plasma

A sensitive and reproducible solid-phase extraction (SPE) method for the quantification of oxycodone in human plasma was developed. Varian Certify SPE cartridges containing both C-8 and benzoic acid functional groups were the most suitable for the extraction of oxycodone and codeine (internal standard), with consistently high (greater than or equal to 80%) and reproducible recoveries. The elution mobile phase consisted of 1.2 ml of butyl chloride-isopropanol (80:20, v/v) containing 2% ammonia. The quantification limit for oxycodone was 5.3 pmol on-column. Within-day and inter-day coefficients of variation were 1.2% and 6.8% respectively for 284 nM oxycodone and 9.5% and 6.2% respectively for 28.4 nM oxycodone using 0.5-ml plasma aliquots. (C) 1998 Elsevier Science BN. All rights reserved.

Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphoryl

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific alpha subunits and a common signal-transducing beta subunit (h beta c). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of h beta c. We report here a comprehensive screen of the entire h beta c molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of h beta c that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most h beta c mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant beta subunits were constitutively tyrosine phosphorylated. Taken together; these results highlight key regions involved in h beta c activation, dissociate h beta c tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by h beta c. (C) 1998 by The American Society of Hematology.

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography

A simple method for the measurement of pindolol enantiomers by HPLC is presented. Alkalinized serum or urine is extracted with ethyl acetate and the residue remaining after evaporation of the organic layer is then derivatised with (S)-(-)-alpha-methylbenzyl isocyanate. The diastereoisomers of derivatised pindolol and metoprolol (internal standard) are separated by high-performance liquid chromatography (HPLC) using a C-18 silica column and detected using fluorescence (excitation lambda: 215 nm, emission lambda: 320 nm). The assay displays reproducible linearity for pindolol enantiomers with a correlation coefficient of r(2) greater than or equal to 0.998 over the concentration range 8-100 ng ml(-1) for plasma and 0.1-2.5 mu g ml(-1) for urine. The coefficient of variation for accuracy and precision of the quality control samples for both plasma and urine are consistently

mRNA differential display of 2-amino-1-methyl-6-phenylinlidazo[4,5-b]pyridine-induced rat mammary gland tumors

The mRNA differential display technique was used to compare mRNAs between normal mammary gland and turner-derived epithelial cells from female Sprague-Dawley rat mammary gland tumors induced by the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and promoted by a high-fat diet (23.5% corn oil). Two genes, beta-casein and transferrin, were identified as differentially expressed. The expression of these genes was examined across a bank of rat mammary gland tumors derived from animals on a low-fat diet (5% corn oil) or the high-fat diet. Carcinomas had over a 10- and 50-fold lower expression of beta-casein and transferrin, respectively than normal mammary gland. In addition, carcinomas from animals on the high-fat diet showed on average a 5-fold higher expression of beta-casein, and transferrin than carcinomas from animals on the low-fat diet. The results indicate the process of mammary gland tumorigenesis alters the expression of certain genes in the mammary gland, and that the level of dietary fat further modulates the expression of these genes.

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gem in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen In a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction In the alcohol-affected brain.

A reproducible method for automated extraction of brain volumes from 3D human head MR images

An automated method for extracting brain volumes from three commonly acquired three-dimensional (3D) MR images (proton density, T1 weighted, and T2-weighted) of the human head is described. The procedure is divided into four levels: preprocessing, segmentation, scalp removal, and postprocessing. A user-provided reference point is the sole operator-dependent input required, The method's parameters were first optimized and then fixed and applied to 30 repeat data sets from 15 normal older adult subjects to investigate its reproducibility. Percent differences between total brain volumes (TBVs) for the subjects' repeated data sets ranged from .5% to 2.2%. We conclude that the method is both robust and reproducible and has the potential for wide application.