Optical encoding of microbeads for gene screening: alternatives to microarrays

Rapid access to genetic information is central to the revolution presently occurring in the pharmaceutical industry, particularly In relation to novel drug target identification and drug development. Genetic variation, gene expression, gene function and gene structure are just some of the important research areas requiring efficient methods of DNA screening. Here, we highlight state-of-the-art techniques and devices for gene screening that promise cheaper and higher-throughput yields than currently achieved with DNA microarrays. We include an overview of existing and proposed bead-based strategies designed to dramatically increase the number of probes that can be interrogated in one assay. We focus, in particular, on the issue of encoding and/or decoding (bar-coding) large bead-based libraries for HTS.

Diagnostic performance of Antineutrophil Cytoplasmic Antibody tests for Idiopathic Vasculitides: Metaanalysis with a focus on antimyeloperoxidase antibodies

Objective. The diagnostic value of tests for antimyeloperoxidase antibodies (anti-MPO) for systemic vasculitis is less established than that for cytoplasmic antineutrophil cytoplasmic antibody (cANCA)/antiproteinase 3 antibodies (anti-PR3). Controversy exists regarding the optimal utilization of indirect immunofluorescence (IIF) ANCA testing versus antigen-specific ANCA testing. To summarize the pertinent data, we conducted a metaanalysis examining the diagnostic value of ANCA testing systems that include assays for anti-MPO. Methods. We performed a structured Medline search and reference list review. Target articles in the search strategy were those reporting the diagnostic value of immunoassays for anti-MPO for the spectrum of systemic necrotizing vasculitides that includes Wegener's granulomatosis, microscopic polyangiitis, the Churg-Strauss syndrome, and isolated pauci-immune necrotizing or crescentic glomerulonephritis, regardless of other types of ANCA tests. Inclusion criteria required specification of a consecutive or random patient selection method and the use of acceptable criteria for the diagnosis of vasculitis exclusive of ANCA test results. Weighted pooled summary estimates of sensitivity and specificity were calculated for anti-MPO alone, anti-MPO + perinuclear ANCA (pANCA), and anti-MPO/pANCA + anti-PR3/cANCA. Results. Of 457 articles reviewed, only 7 met the selection criteria. Summary estimates of sensitivity and specificity (against disease controls only) of assays for anti-MPO for the diagnosis of systemic necrotizing vasculitides were 37.1% (confidence interval 26.6% to 47.6%) and 96.3% (CI 94.1% to 98.5%), respectively. When the pANCA pattern by IIF was combined with anti-MPO testing, the specificity improved to 99.4%, with a lower sensitivity, 31.5%. The combined ANCA testing system (anti-PR3/cANCA + anti-MPO/pANCA) increased the sensitivity to 85.5% with a specificity of 98.6%. Conclusion. These results suggest that while anti-MPO is relatively specific for the diagnosis of systemic vasculitis, the combination system of immunoassays for anti-MPO and IIF for pANCA is highly specific and both tests should be used together given the high diagnostic precision required for these conditions. Because patients with ANCA associated vasculitis have either anti-MPO with pANCA or anti-PR3 with cANCA, and rarely both, a combined ANCA testing system including anti-PR3/cANCA and anti-MPO/pANCA is recommended to optimize the diagnostic performance of ANCA testing. (J Rheumatol 2001;28:1584-90)

Neurology of ciguatera

Ciguatera is a widespread ichthyosarcotoxaemia with dramatic and clinically important neurological features. This severe form of fish poisoning may present with either acute or chronic intoxication syndromes and constitutes a global health problem. Ciguatera poisoning is little known in temperate countries as a potentially global problem associated with human ingestion of large carnivorous fish that harbour the bioaccumulated ciguatoxins of the photosynthetic dinoflagellate Gambierdiscus toxicus. This neurotoxin is stored in the viscera of fish that have eaten the dinoflagellate and concentrated it upwards throughout the food chain towards progressively larger species, including humans. Ciguatoxin accumulates in all fish tissues, especially the liver and viscera, of at risk species. Both Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins are heat stable polyether toxins and pose a health risk at concentrations above 0.1 ppb. The presenting signs of ciguatera are primarily neurotoxic in more than 80% of cases. Such include the pathognomonic features of postingestion paraesthesiae, dysaesthesiae, and heightened nociperception. Other sensory abnormalities include the subjective features of metallic taste, pruritis, arthralgia, myalgia, and dental pain. Cerebellar dysfunction, sometimes diphasic, and weakness due to both neuropathy and polymyositis may be encountered. Autonomic dysfunction leads to hypotension, bradycardia, and hypersalivation in severe cases. Ciguatoxins are potent, lipophilic sodium channel activator toxins which bind to the voltage sensitive (site 5) sodium channel on the cell membranes of all excitable tissues. Treatment depends on early diagnosis and the early administration of intravenous mannitol. The early identification of the neurological features in sentinel patients has the potential to reduce the number of secondary cases in cluster outbreaks.

Media stress intensity analysis for vertical stirred mills

Experimental work has been carried out to investigate the effect of major operating variables on milling efficiency of calcium carbonate in laboratory and pilot size Tower and Sala Agitated (SAM) mills. The results suggest that the stirrer speed, media size and slurry density affect the specific energy consumption required to achieve the given product size. Media stress intensity analysis developed for high-speed horizontal mills was modified to include the effect of gravitational force in the vertical stirred mills such as the Tower and SAM units. The results suggest that this approach can be successfully applied for both mill types. For a given specific energy input, an optimum stress intensity range existed, for which the finest product was achieved. Finer product and therefore higher milling efficiency was obtained with SAM in the range of operating conditions tested. (C) 2001 Elsevier Science Ltd. All rights reserved.

n-fold Weibull multiplicative model

In this paper we study the n-fold multiplicative model involving Weibull distributions and examine some properties of the model. These include the shapes for the density and failure rate functions and the WPP plot. These allow one to decide if a given data set can be adequately modelled by the model. We also discuss the estimation of model parameters based on the WPP plot. (C) 2001 Elsevier Science Ltd. All rights reserved.

Role of LMP1 in immune control of EBV infection

The Epstein-Barr virus (EBV) encoded latent membrane protein (LMP1) plays a crucial role in the long-term persistence of this virus within the cells of the immune system. Not only is this protein critical for the transformation of resting B cells by EBV, it also displays pleiotropic effects on various cellular proteins expressed in the host cell. These include up-regulation of expression of B cell activation antigens, adhesion molecules and various components of the antigen processing pathway. Here we discuss how LMP1 acts like an expression 'switch' which, depending on the stage of EBV infection, manoeuvres various pathways that either modulate the immune system towards or against its survival.

Work-related stress and occupational therapy

Occupational stress and burnout have been studied extensively in the human services. It has been suggested that healthcare professionals in particular are at risk of stress owing to the caring nature of their work. Articles related to occupational therapy and work-related stress were reviewed in regard to practice in Australia, Canada, the United Kingdom, the United States and Sweden. Although the empirical literature is relatively weak for occupational therapy, it has been argued that occupational therapists in health care share risk factors with other healthcare professionals. These risk factors include repeated exposure to distress and difficult behaviour, prolonged interventions and uncertain outcome. Issues such as professional status, staffing issues and the nature of the profession have been identified as additional risk factors for occupational therapists. However, empirical studies that enable burnout rates of occupational therapists to be compared with those of related occupational groups suggest that this may not be the case. Occupational therapists may in fact be protected from some stress and burnout factors. Further research is recommended to clarify the nature of stress experienced by occupational therapists and to identify both risk and protective factors characteristic of the profession. Copyright © 2001 Whurr Publishers Ltd.

Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF(35)-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine,tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.

Characterization of mouse Frizzled-3 expression in hair follicle development and identification of the human homolog in keratinocytes

Frizzled genes encode a family of Wnt ligand receptors, which have a conserved cysteine-rich Wnt binding domain and include both transmembrane and secreted forms. Work by others has shown that experimental perturbation of Wnt signaling results in aberrant hair formation, hair growth, and hair structure. To date, however, there is no information on the contribution of individual Frizzled proteins to hair development. We now report that Frizzled-3 expression in skin is restricted to the epidermis and to the developing hair follicle. Northern analysis on total mouse skin mRNA revealed a single Frizzled-3 transcript of 3.7 kb. Reverse transcription-polymerase chain reaction and in situ hybridization analysis revealed Frizzled-3 expression in epidermal and hair follicle keratinocytes. Frizzled-3 transcripts are first detected in discrete foci in the developing epidermis of 13 d embryos and later in the hair follicle placodes of 15 d embryos, suggesting a role for this Frizzled isoform in follicle development. In 17 d embryos and id old newborn mice Frizzled-3 expression is limited to suprabasal keratinocytes and is not seen in pelage follicles until 3 d postpartum. In 7 d old neonatal skin, Frizzled-3 is expressed throughout the epidermis and in the outer cell layers of hair follicles. We have also identified the mRNA encoding human Frizzled-3 in epidermal keratinocytes and in the HaCaT keratinocyte cell line. Human Frizzled-3 mRNA encodes a 666 amino acid protein with 97.8% identity to the mouse protein. The human Frizzled-3 gene was mapped using a radiation-hybrid cell line panel to the short arm of chromosome 8 between the markers WI-1172 and WI-8496 near the loci for the Hypotrichosis of Marie Unna and Hairless genes.

The cystine knot motif in toxins and implications for drug design

The cystine knot structural motif is present in peptides and proteins from a variety of species, including fungi, plants, marine molluscs. insects and spiders. It comprises an embedded ring formed by two disulfide bonds and their connecting backbone segments which is threaded by a third disulfide bond. It is invariably associated with nearby beta-sheet structure and appears to be a highly efficient motif for structure stabilization. Because of this stability it makes an ideal framework for molecular engineering applications. In this review we summarize the main structural features of the cystine knot motif, focussing on toxin molecules containing either the inhibitor cystine knot or the cyclic cystine knot. Peptides containing these motifs are 26-48 residues long and include ion channel blockers, haemolytic agents, as well as molecules having antiviral and antibacterial activities. The stability of peptide toxins containing the cystine knot motif, their range of bioactivities and their unique structural scaffold can be harnessed for molecular engineering applications and in drug design. Applications of cystine knot molecules for the treatment of pain. and their potential use in antiviral and antibacterial applications are described. (C) 2000 Elsevier Science Ltd. All rights reserved.

Searching for missing pieces of the sex-determination puzzle

Little is known of the mechanisms whereby the mammalian indifferent gonad develops into a testis or ovary. In XY individuals, Sry, the mammalian testis-determining gene, is expressed in the pre-Sertoli cells, which then differentiate into Sertoli cells. Other cell types, which include the germ cells, the steroidogenic cells and the connective tissue cells, must then be instructed to develop in a male-specific manner. Although some genes involved in sex-determination and differentiation processes have been identified, we know little of how they interact and cooperate to orchestrate the development of a testis or ovary. We have initiated an expression-screening program designed to identify additional genes, known or novel, which play a role in these processes. This approach is based on our belief that many of the genes we seek will be expressed in a sex-specific manner during the period of sex-determination and differentiation. Most of the genes identified previously are transcription factors and so we aim, in particular, to find genes involved in cell-to-cell communication, signal transduction, and transcriptional regulation, downstream of the differentiation of Sertoli cells. We have used a suppression subtractive-hybridization method to generate male- and female-enriched probes and libraries. Clones are validated as being sex-specific in their expression patterns by array screening and in situ hybridization. Here we report on our progress to date and the general applicability of the approach for studies in other systems. J. Exp. Zool. 290:517-522, 2001. (C) 2001 Wiley-Liss, Inc.

Adaptation to chronic eccentric exercise in humans: the influence of contraction velocity

We compared changes in muscle fibre composition and muscle strength indices following a 10 week isokinetic resistance training programme consisting of fast (3.14 rad(.)s(-1)) or slow (0.52 rad(.)s(-1)) velocity eccentric muscle contractions. A group of 20 non-resistance trained subjects were assigned to a FAST (n = 7), SLOW (n = 6) or non-training CONTROL (n = 7) group. A unilateral training protocol targeted the elbow flexor muscle group and consisted of 24 maximal eccentric isokinetic contractions (four sets of six repetitions) performed three times a week for 10 weeks. Muscle biopsy samples were obtained from the belly of the biceps brachii. Isometric torque and concentric and eccentric torque at 0.52 and 3.14 rad(.)s(-1) were examined at 0, 5 and 10 weeks. After 10 weeks, the FAST group demonstrated significant [mean (SEM)] increases in eccentric [29.6 (6.4)%] and concentric torque [27.4 (7.3) %] at 3.14 rad(.)s(-1), isometric torque [21.3 (4.3)%] and eccentric torque [25.2 (7.2) %] at 0.52 rad(.)s(-1). The percentage of type I fibres in the FAST group decreased from [53.8 (6.6)% to 39.1 (4.4)%] while type lib fibre percentage increased from [5.8 (1.9)% to 12.9 (3.3)%; P < 0.05]. In contrast. the SLOW group did not experience significant changes in muscle fibre type or muscle torque. We conclude that neuromuscular adaptations to eccentric training stimuli may be influenced by differences in the ability to cope with chronic exposure to relatively fast and slow eccentric contraction velocities. Possible mechanisms include greater cumulative damage to contractile tissues or stress induced by slow eccentric muscle contractions.

Long-term metabolic and skeletal muscle adaptations to short-sprint training: Implications for sprint training and tapering

The adaptations of muscle to sprint training can be separated into metabolic and morphological changes. Enzyme adaptations represent a major metabolic adaptation to sprint training, with the enzymes of all three energy systems showing signs of adaptation to training and some evidence of a return to baseline levels with detraining. Myokinase and creatine phosphokinase have shown small increases as a result of short-sprint training in some studies and elite sprinters appear better able to rapidly breakdown phosphocreatine (PCr) than the sub-elite. No changes in these enzyme levels have been reported as a result of detraining. Similarly, glycolytic enzyme activity (notably lactate dehydrogenase, phosphofructokinase and glycogen phosphorylase) has been shown to increase after training consisting of either long (> 10-second) or short (< 10-second) sprints. Evidence suggests that these enzymes return to pre-training levels after somewhere between 7 weeks and 6 months of detraining. Mitochondrial enzyme activity also increases after sprint training, particularly when long sprints or short recovery between short sprints are used as the training stimulus. Morphological adaptations to sprint training include changes in muscle fibre type, sarcoplasmic reticulum, and fibre cross-sectional area. An appropriate sprint training programme could be expected to induce a shift toward type Ha muscle, increase muscle cross-sectional area and increase the sarcoplasmic reticulum volume to aid release of Ca2+. Training volume and/or frequency of sprint training in excess of what is optimal for an individual, however, will induce a shift toward slower muscle contractile characteristics. In contrast, detraining appears to shift the contractile characteristics towards type IIb, although muscle atrophy is also likely to occur. Muscle conduction velocity appears to be a potential non-invasive method of monitoring contractile changes in response to sprint training and detraining. In summary, adaptation to sprint training is clearly dependent on the duration of sprinting, recovery between repetitions, total volume and frequency of training bouts. These variables have profound effects on the metabolic, structural and performance adaptations from a sprint-training programme and these changes take a considerable period of time to return to baseline after a period of detraining. However, the complexity of the interaction between the aforementioned variables and training adaptation combined with individual differences is clearly disruptive to the transfer of knowledge and advice from laboratory to coach to athlete.

A modified pore-filling isotherm for liquid-phase adsorption in activated carbon

This article modifies the usual form of the Dubinin-Radushkevich pore-filling model for application to liquid-phase adsorption data, where large molecules are often involved. In such cases it is necessary to include the repulsive part of the energy in the micropores, which is accomplished here by relating the pore potential to the fluid-solid interaction potential. The model also considers the nonideality of the bulk liquid phase through the UNIFAC activity coefficient model, as well as structural heterogeneity of the carbon. For the latter the generalized adsorption integral is used while incorporating the pore-size distribution obtained by density functional theory analysis of argon adsorption data. The model is applied here to the interpretation of aqueous phase adsorption isotherms of three different esters on three commercial activated carbons. Excellent agreement between the model and experimental data is observed, and the fitted Lennard-Jones size parameter for the adsorbate-adsorbate interactions compares well with that estimated from known critical properties, supporting the modified approach. On the other hand, the model without consideration of bulk nonideality, or when using classical models of the characteristic energy, gives much poorer bts of the data and unrealistic parameter values.

Solution techniques for transport problems involving steep concentration gradients: application to noncatalytic fluid solid reactions

Some efficient solution techniques for solving models of noncatalytic gas-solid and fluid-solid reactions are presented. These models include those with non-constant diffusivities for which the formulation reduces to that of a convection-diffusion problem. A singular perturbation problem results for such models in the presence of a large Thiele modulus, for which the classical numerical methods can present difficulties. For the convection-diffusion like case, the time-dependent partial differential equations are transformed by a semi-discrete Petrov-Galerkin finite element method into a system of ordinary differential equations of the initial-value type that can be readily solved. In the presence of a constant diffusivity, in slab geometry the convection-like terms are absent, and the combination of a fitted mesh finite difference method with a predictor-corrector method is used to solve the problem. Both the methods are found to converge, and general reaction rate forms can be treated. These methods are simple and highly efficient for arbitrary particle geometry and parameters, including a large Thiele modulus. (C) 2001 Elsevier Science Ltd. All rights reserved.