On surrogate methods for detecting lateral gene transfer

Surrogate methods for detecting lateral gene transfer are those that do not require inference of phylogenetic trees. Herein I apply four such methods to identify open reading frames (ORFs) in the genome of Escherichia coli K12 that may have arisen by lateral gene transfer. Only two of these methods detect the same ORFs more frequently than expected by chance, whereas several intersections contain many fewer ORFs than expected. Each of the four methods detects a different non-random set of ORFs. The methods may detect lateral ORFs of different relative ages; testing this hypothesis will require rigorous inference of trees. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science BN. All rights reserved.

Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency

Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require the use of radioactivity and usually rely upon subjective determination of the results. We have developed two competitive quantitative assays that use an enzyme-linked, amplicon hybridization assay (ELAHA) to detect the products of PCR-amplified regions of transgene from cells transduced with Moloney murine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved to be simple, rapid, and sensitive, avoiding the need for Southern hybridization, complex histochemical stains, or often subjective and time-consuming tissue culture and immunofluorescence assays. The PCR-ELAHA systems can rapidly detect proviral DNA from any retroviral vector carrying the common selective and marker genes neomycin phosphotransferase and green fluorescent protein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one or both qPCR targets.

Freeform fabrication of aluminum metal-matrix composites

A series of metal-matrix composites were formed by extrusion freeform, fabrication of a sinterable aluminum alloy in combination with silicon carbide particles and whiskers, carbon fibers, alumina particles, and hollow flyash cenospheres. Silicon carbide particles were most successful in that the composites retained high density with up to 20 vol% of reinforcement and the strength approximately doubles over the strength of the metal matrix alone. Comparison with simple models suggests that this unexpectedly high degree of reinforcement can be attributed to the concentration of small silicon carbide particles around the larger metal powder. This fabrication method also allows composites to be formed with hollow spheres that cannot be formed by other powder or melt methods.

Stream-bank shade and larval distribution of the Philippine malaria vector Anopheles flavirostris

The principal malaria vector in the Philippines, Anopheles flavirostris (Ludlow) (Diptera: Culicidae), is regarded as 'shade-loving' for its breeding sites, i.e. larval habitats. This long-standing belief, based on circumstantial observations rather than ecological analysis, has guided larval control methods such as 'stream-clearing' or the removal of riparian vegetation, to reduce the local abundance of An. flavirostris . We measured the distribution and abundance of An. flavirostris larvae in relation to canopy vegetation cover along a stream in Quezon Province, the Philippines. Estimates of canopy openness and light measurements were obtained by an approximation method that used simplified assumptions about the sun, and by hemispherical photographs analysed using the program hemiphot(C) . The location of larvae, shade and other landscape features was incorporated into a geographical information system (GIS) analysis. Early larval instars of An. flavirostris were found to be clustered and more often present in shadier sites, whereas abundance was higher in sunnier sites. For later instars, distribution was more evenly dispersed and only weakly related to shade. The best predictor of late-instar larvae was the density of early instars. Distribution and abundance of larvae were related over time (24 days). This pattern indicates favoured areas for oviposition and adult emergence, and may be predictable. Canopy measurements by the approximation method correlated better with larval abundance than hemispherical photography, being economical and practical for field use. Whereas shade or shade-related factors apparently have effects on larval distribution of An. flavirostris , they do not explain it completely. Until more is known about the bionomics of this vector and the efficacy and environmental effects of stream-clearing, we recommend caution in the use of this larval control method.

Lexicographic decomposition of chains and the concept of a planar chain

In an earlier paper [Journal of Mathematical Economics, 37 (2002) 17-38], we proved that if a preference relation on a commodity space is non-representable by a real-valued function then that chain is necessarily a long chain, a planar chain, an Aronszajn-like chain or a Souslin chain. In this paper, we study the class of planar chains, the simplest example of which is the Debreu chain (R-2, <(l)). (C) 2002 Elsevier Science B.V. All rights reserved.

Abnormal extracellular matrix protein transport associated with increased apoptosis of vascular smooth muscle cells in Marfan syndrome and bicuspid aortic valve thoracic aortic aneurysm

Background - Marfan syndrome (MS) is a genetic disorder caused by a mutation in the fibrillin gene FBN1. Bicuspid aortic valve (BAV) is a congenital heart malformation of unknown cause. Both conditions are associated with ascending aortic aneurysm and premature death. This study examined the relationship among the secretion of extracellular matrix proteins fibrillin, fibronectin, tenascin, and vascular smooth muscle cell (VSMC) apoptosis. The role of matrix metalloproteinase (MMP)- 2 in VSMC apoptosis was studied in MS aneurysm. Methods and Results - Aneurysm tissue was obtained from patients undergoing surgery ( MS: 4 M, 1 F, age 27 - 45 years; BAV: 3 M, 2 F, age 28 - 65 years). Normal aorta from subjects with nonaneurysm disease was also collected ( 4 M, 1 F, age 23 - 93 years). MS and BAV aneurysm histology showed areas of cystic medial necrosis (CMN) without inflammatory infiltrate. Immunohistochemical study of cultured MS and BAV VSMC showed intracellular accumulation and reduction of extracellular distribution of fibrillin, fibronectin, and tenascin. Western blot showed no increase in expression of fibrillin, fibronectin, or tenascin in MS or BAV VSMC and increased expression of MMP-2 in MS VSMCs. There was 4-fold increase in loss of cultured VSMC incubated in serum-free medium for 24 hours in both MS ( 27 +/- 8%) and BAV ( 32 +/- 14%) compared with control ( 7 +/- 5%). Conclusions - In MS and BAV there is alteration in both the amount and quality of secreted proteins and an increased degree of VSMC apoptosis. Up-regulation of MMP-2 might play a role in VSMC apoptosis in MS VSMC. The findings suggest the presence of a fundamental cellular abnormality in BAV thoracic aorta, possibly of genetic origin.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Selecting methods to solve multi-well master equations

There are several competing methods commonly used to solve energy grained master equations describing gas-phase reactive systems. When it comes to selecting an appropriate method for any particular problem, there is little guidance in the literature. In this paper we directly compare several variants of spectral and numerical integration methods from the point of view of computer time required to calculate the solution and the range of temperature and pressure conditions under which the methods are successful. The test case used in the comparison is an important reaction in combustion chemistry and incorporates reversible and irreversible bimolecular reaction steps as well as isomerizations between multiple unimolecular species. While the numerical integration of the ODE with a stiff ODE integrator is not the fastest method overall, it is the fastest method applicable to all conditions.

Comparison of three commercial sparse-matrix crystallization screens

Sparse-matrix sampling using commercially available crystallization screen kits has become the most popular way of determining the preliminary crystallization conditions for macromolecules. In this study, the efficiency of three commercial screening kits, Crystal Screen and Crystal Screen 2 (Hampton Research), Wizard Screens I and II (Emerald BioStructures) and Personal Structure Screens 1 and 2 (Molecular Dimensions), has been compared using a set of 19 diverse proteins. 18 proteins yielded crystals using at least one crystallization screen. Surprisingly, Crystal Screens and Personal Structure Screens showed dramatically different results, although most of the crystallization formulations are identical as listed by the manufacturers. Higher molecular weight polyethylene glycols and mixed precipitants were found to be the most effective precipitants in this study.

Multipole expansion of strongly focussed laser beams

Multipole expansion of an incident radiation field-that is, representation of the fields as sums of vector spherical wavefunctions-is essential for theoretical light scattering methods such as the T-matrix method and generalised Lorenz-Mie theory (GLMT). In general, it is theoretically straightforward to find a vector spherical wavefunction representation of an arbitrary radiation field. For example, a simple formula results in the useful case of an incident plane wave. Laser beams present some difficulties. These problems are not a result of any deficiency in the basic process of spherical wavefunction expansion, but are due to the fact that laser beams, in their standard representations, are not radiation fields, but only approximations of radiation fields. This results from the standard laser beam representations being solutions to the paraxial scalar wave equation. We present an efficient method for determining the multipole representation of an arbitrary focussed beam. (C) 2003 Elsevier Science Ltd. All rights reserved.

Practical considerations for the measurement of free light chains in serum

Background: A new immunoassay for free light chain measurements has been reported to be useful for the diagnosis and monitoring of monoclonal light chain diseases and nonsecretory myeloma. We describe experience with and some potential pitfalls of the assay. Methods: The assay was assessed for precision, sample type and stability, recovery, and harmonization of results between two analyzers on which the reagents are used. Free-light-chain concentrations were measured in healthy individuals (to determine biological variation), patients with monoclonal gammopathy of undetermined significance, myeloma patients after autologous stem cell transplants, and patients with renal disease. Results: Analytical imprecision (CV) was 6-11% for kappa and A free-light-chain measurement and 16% for the calculated kappa/lambda ratio. Biological variation was generally insignificant compared with analytical variation. Despite the same reagent source, values were not completely harmonized between assay systems and may produce discordant free-light-chain ratios. In some patients with clinically stable myeloma, or post transplantation, or with monoclonal gammopathy of undetermined significance, free-light-chain concentration and ratio were within the population reference interval despite the presence of monoclonal intact immunoglobulin in serum. In other patients with monoclonal gammopathy of undetermined significance, values were abnormal although there was no clinical evidence of progression to multiple myeloma. Conclusions: The use of free-light-chain measurements alone cannot differentiate some groups of patients with monoclonal gammopathy from healthy individuals. As with the introduction of any new test, it is essential that more scientific data about use of this assay in different subject groups are available so that results can be interpreted with clinical certainty. (C) 2003 American Association for Clinical Chemistry.

Orientation of the genetic variance-covariance matrix and the fitness surface for multiple male sexually selected traits

Stabilizing selection has been predicted to change genetic variances and covariances so that the orientation of the genetic variance-covariance matrix (G) becomes aligned with the orientation of the fitness surface, but it is less clear how directional selection may change G. Here we develop statistical approaches to the comparison of G with vectors of linear and nonlinear selection. We apply these approaches to a set of male sexually selected cuticular hydrocarbons (CHCs) of Drosophila serrata. Even though male CHCs displayed substantial additive genetic variance, more than 99% of the genetic variance was orientated 74.9degrees away from the vector of linear sexual selection, suggesting that open-ended female preferences may greatly reduce genetic variation in male display traits. Although the orientation of G and the fitness surface were found to differ significantly, the similarity present in eigenstructure was a consequence of traits under weak linear selection and strong nonlinear ( convex) selection. Associating the eigenstructure of G with vectors of linear and nonlinear selection may provide a way of determining what long-term changes in G may be generated by the processes of natural and sexual selection.

Existence and uniqueness of Q-processes with a given finite μ-invariant measure

Let Q be a stable and conservative Q-matrix over a countable state space S consisting of an irreducible class C and a single absorbing state 0 that is accessible from C. Suppose that Q admits a finite mu-subinvariant measure in on C. We derive necessary and sufficient conditions for there to exist a Q-process for which m is mu-invariant on C, as well as a necessary condition for the uniqueness of such a process.

Full S matrix calculation via a single real-symmetric Lanczos recursion: The Lanczos artificial boundary inhomogeneity method

We present an efficient and robust method for the calculation of all S matrix elements (elastic, inelastic, and reactive) over an arbitrary energy range from a single real-symmetric Lanczos recursion. Our new method transforms the fundamental equations associated with Light's artificial boundary inhomogeneity approach [J. Chem. Phys. 102, 3262 (1995)] from the primary representation (original grid or basis representation of the Hamiltonian or its function) into a single tridiagonal Lanczos representation, thereby affording an iterative version of the original algorithm with greatly superior scaling properties. The method has important advantages over existing iterative quantum dynamical scattering methods: (a) the numerically intensive matrix propagation proceeds with real symmetric algebra, which is inherently more stable than its complex symmetric counterpart; (b) no complex absorbing potential or real damping operator is required, saving much of the exterior grid space which is commonly needed to support these operators and also removing the associated parameter dependence. Test calculations are presented for the collinear H+H-2 reaction, revealing excellent performance characteristics. (C) 2004 American Institute of Physics.

SVMtm: Support vector machines to predict transmembrane segments

A new method has been developed for prediction of transmembrane helices using support vector machines. Different coding schemes of protein sequences were explored, and their performances were assessed by crossvalidation tests. The best performance method can predict the transmembrane helices with sensitivity of 93.4% and precision of 92.0%. For each predicted transmembrane segment, a score is given to show the strength of transmembrane signal and the prediction reliability. In particular, this method can distinguish transmembrane proteins from soluble proteins with an accuracy of similar to99%. This method can be used to complement current transmembrane helix prediction methods and can be Used for consensus analysis of entire proteomes . The predictor is located at http://genet.imb.uq.edu.au/predictors/ SVMtm. (C) 2004 Wiley Periodicals, Inc.