GeneMCL in microarray analysis

Accurately and reliably identifying the actual number of clusters present with a dataset of gene expression profiles, when no additional information on cluster structure is available, is a problem addressed by few algorithms. GeneMCL transforms microarray analysis data into a graph consisting of nodes connected by edges, where the nodes represent genes, and the edges represent the similarity in expression of those genes, as given by a proximity measurement. This measurement is taken to be the Pearson correlation coefficient combined with a local non-linear rescaling step. The resulting graph is input to the Markov Cluster (MCL) algorithm, which is an elegant, deterministic, non-specific and scalable method, which models stochastic flow through the graph. The algorithm is inherently affected by any cluster structure present, and rapidly decomposes a graph into cohesive clusters. The potential of the GeneMCL algorithm is demonstrated with a 5730 gene subset (IGS) of the Van't Veer breast cancer database, for which the clusterings are shown to reflect underlying biological mechanisms. (c) 2005 Elsevier Ltd. All rights reserved.

Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5±3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.

Development and application of diazirines in biological and synthetic macromolecular systems

Many different reagents and methodologies have been utilised for the modification of synthetic and biological macromolecular systems. In addition, an area of intense research at present is the construction of hybrid biosynthetic polymers, comprised of biologically active species immobilised or complexed with synthetic polymers. One of the most useful and widely applicable techniques available for functionalisation of macromolecular systems involves indiscriminate carbene insertion processes. The highly reactive and non-specific nature of carbenes has enabled a multitude of macromolecular structures to be functionalised without the need for specialised reagents or additives. The use of diazirines as stable carbene precursors has increased dramatically over the past twenty years and these reagents are fast becoming the most popular photophors for photoaffinity labelling and biological applications in which covalent modification of macromolecular structures is the basis to understanding structure-activity relationships. This review reports the synthesis and application of a diverse range of diazirines in macromolecular systems.

Serum proteomic abnormality predating screen detection of ovarian cancer

Ovarian cancer is characterized by vague, non-specific symptoms, advanced stage at diagnosis and poor overall survival. A nested case control study was undertaken on stored serial serum samples from women who developed ovarian cancer and healthy controls (matched for serum processing and storage conditions as well as attributes such as age) in a pilot randomized controlled trial of ovarian cancer screening. The unique feature of this study is that the women were screened for up to 7 years. The serum samples underwent prefractionation using a reversed-phase batch extraction protocol prior to MALDI-TOF MS data acquisition. Our exploratory analysis shows that combining a single MS peak with CA125 allows statistically significant discrimination at the 5% level between cases and controls up to 12 months in advance of the original diagnosis of ovarian cancer. Such combinations work much better than a single peak or CA125 alone. This paper demonstrates that mass spectra from the low molecular weight serum proteome carry information useful for early detection of ovarian cancer. The next step is to identify the specific biomarkers that make early detection possible.

DNA cleavage and antitumour activity of platinum(II) and copper(II) compounds derived from 4-methyl-2-N-(2-pyridylmethyl)aminophenol: spectroscopic, electrochemical and biological investigation

The reaction of the redox-active ligand, Hpyramol (4-methyl-2-N-(2-pyridylmethyl)aminophenol) with K2PtCl4 yields monofunctional square-planar [Pt(pyrimol)Cl], PtL-Cl, which was structurally characterised by single-crystal X-ray diffraction and NMR spectroscopy. This compound unexpectedly cleaves supercoiled double-stranded DNA stoichiometrically and oxidatively, in a non-specific manner without any external reductant added, under physiological conditions. Spectro-electrochemical investigations of PtL-Cl were carried out in comparison with the analogue CuL-Cl as a reference compound. The results support a phenolate oxidation, generating a phenoxyl radical responsible for the ligand-based DNA cleavage property of the title compounds. Time-dependent in vitro cytotoxicity assays were performed with both PtL-Cl and CuL-Cl in various cancer cell lines. The compound CuL-Cl overcomes cisplatin-resistance in ovarian carcinoma and mouse leukaemia cell lines, with additional activity in some other cells. The platinum analogue, PtL-Cl also inhibits cell-proliferation selectively. Additionally, cellular-uptake studies performed for both compounds in ovarian carcinoma cell lines showed that significant amounts of Pt and Cu were accumulated in the A2780 and A2780R cancer cells. The conformational and structural changes induced by PtL-Cl and CuL-Cl on calf thymus DNA and phi X174 supercoiled phage DNA at ambient conditions were followed by electrophoretic mobility assay and circular dichroism spectroscopy. The compounds induce extensive DNA degradation and unwinding, along with formation of a monoadduct at the DNA minor groove. Thus, hybrid effects of metal-centre variation, multiple DNA-binding modes and ligand-based redox activity towards cancer cell-growth inhibition have been demonstrated. Finally, reactions of PtL-Cl with DNA model bases (9-Ethylguanine and 5'-GMP) followed by NMR and MS showed slow binding at Guanine-N7 and for the double stranded self complimentary oligonucleotide d(GTCGAC)(2) in the minor groove.

An in vitro assessment of the effects of broad-spectrum antibiotics on the human gut microflora and concomitant isolation of a Lactobacillus plantarum with anti-Candida activities

Chemostat culture was used to determine the effects of the antimicrobial agents tetracycline and nystatin on predominant components of the human gut microflora. Their addition to mixed culture systems caused a non-specific, and variable, decrease in microbial populations, although tetracycline allowed an increase in numbers of yeasts. Both had a profound inhibitory effect upon populations seen as important for gut health (bifidobacteria, lactobacilli). However, a tetracycline resistant Lactobacillus was enriched from the experiments. A combination of genotypic and phenotypic characterisations confirmed its identity as Lactobacillus plantarum. This strain exerted powerful inhibitory effects against Candida albicans. Because of its ability to resist the effects of tetracycline, this organism may be useful as a probiotic for the improved management of yeast related conditions such as thrush and irritable bowel syndrome. (C) 2004 Elsevier Ltd. All rights reserved.

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using alpha-mannosidases from Penicillium citrinum, Aspergillus phoenicis and almond

Recombinant Penicillium citrinum alpha-1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of alpha-D-mannopyranosyl-(1-->2)-D-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with alpha1-->2 linkages present at 98.5% of the total linkages formed. Non-specific alpha-mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45-50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 1-->2, 1-->3 and 1-->6 isomers. alpha-1,2-linkage specific mannosidase from P. citrinum and alpha-1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in alpha-D-mannopyranosyl-(1-->3)-D-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as 'decoys' of receptors of type-1 fimbriae carried by enterobacteria.

Do children with Williams syndrome really have good vocabulary knowledge? Methods for comparing cognitive and linguistic abilities in developmental disorders

The comparison of cognitive and linguistic skills in individuals with developmental disorders is fraught with methodological and psychometric difficulties. In this paper, we illustrate some of these issues by comparing the receptive vocabulary knowledge and non-verbal reasoning abilities of 41 children with Williams syndrome, a genetic disorder in which language abilities are often claimed to be relatively strong. Data from this group were compared with data from typically developing children, children with Down syndrome, and children with non-specific learning difficulties using a number of approaches including comparison of age-equivalent scores, matching, analysis of covariance, and regression-based standardization. Across these analyses children with Williams syndrome consistently demonstrated relatively good receptive vocabulary knowledge, although this effect appeared strongest in the oldest children.

Thalassaemia: its diagnosis and interpretation in past skeletal populations

The diagnosis of thalassaemia in archaeological populations has long been hindered by a lack of pathogonomic features, and the non-specific nature of cribra orbitalia and porotic hyperostosis. In fact, clinical research has highlighted more specific diagnostic criteria for thalassaemia major and intermedia based on changes to the thorax (‘rib-within-a-rib’ and costal osteomas). A recent re-examination of 364 child skeletons from Romano-British Poundbury Camp, Dorset revealed children with general ‘wasting’ of the bones and three children who demonstrated a variety of severe lesions (e.g. zygomatic bone and rib hypertrophy, porotic hyperostosis, rib lesions, osteopenia and pitted diaphyseal shafts) that are inconsistent with dietary deficiency alone, and more consistent with a diagnosis of genetic anaemia. Two of these children displayed rib lesions typical of those seen in modern cases of thalassaemia. The children of Poundbury Camp represent the first cases of genetic anaemia identified in a British archaeological population. As thalassaemia is a condition strongly linked to Mediterranean communities, the presence of this condition in a child from England, found within a mausoleum, suggests that they were born to wealthy immigrant parents living in this small Roman settlement in Dorset. This paper explores the diagnostic criteria for genetic anaemia in the archaeological literature and what its presence in ancient populations can contribute to our knowledge of past human migration.

Gap junction-mediated spontaneous Ca(2+) waves in differentiated cholinergic SN56 cells

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca(2+) waves. These waves were unaffected by tetrodotoxin (1microM), but blocked by removal of extracellular Ca(2+), or addition of non-specific Ca(2+) channel inhibitors (Cd(2+) (0.1mM) or Ni(2+) (1mM)). Combined application of antagonists of NMDA receptors (AP5; 100microM), AMPA/kainate receptors (NBQX; 20microM), nicotinic AChR receptors (hexamethonium; 100microM) or inotropic purinoceptors (brilliant blue; 100nM) was also without effect. However, Ca(2+) waves were fully prevented by carbenoxolone (200microM), halothane (3mM) or niflumic acid (100microM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd(2+), Ni(2+) or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca(2+) waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.

Effects of plant pathogens on population dynamics and community composition in grassland ecosystems: two case studies

Grassland ecosystems comprise a major portion of the earth’s terrestrial surface, ranging from high-input cultivated monocultures or simple species mixtures to relatively unmanaged but dynamic systems. Plant pathogens are a component of these systems with their impact dependent on many interacting factors, including grassland species population dynamics and community composition, the topics covered in this paper. Plant pathogens are affected by these interactions and also act reciprocally by modifying their nature. We review these features of disease in grasslands and then introduce the 150-year long-term Park Grass Experiment (PGE) at Rothamsted Research in the UK. We then consider in detail two plant-pathogen systems present in the PGE, Tragopogon pratensis-Puccinia hysterium and Holcus lanata-Puccinia coronata. These two systems have very different life history characteristics: the first, a biennial member of the Asteraceae infected by its host-specific, systemic rust; the second, a perennial grass infected by a host-non-specific rust. We illustrate how observational, experimental and modelling studies can contribute to a better understanding of population dynamics, competitive interactions and evolutionary outcomes. With Tragopogon pratensis-Puccinia hysterium, characterised as an “outbreak” species in the PGE, we show that pathogen-induced mortality is unlikely to be involved in host population regulation; and that the presence of even a short-lived seed-bank can affect the qualitative outcomes of the host-pathogen dynamics. With Holcus lanata-Puccinia coronata, we show how nutrient conditions can affect adaptation in terms of host defence mechanisms, and that co-existence of competing species affected by a common generalist pathogen is unlikely.

Effects of action observation on corticospinal excitability: muscle specificity, direction, and timing of the mirror response

Many human behaviours and pathologies have been attributed to the putative mirror neuron system, a neural system that is active during both the observation and execution of actions. While there are now a very large number of papers on the mirror neuron system, variations in the methods and analyses employed by researchers mean that the basic characteristics of the mirror response are not clear. This review focuses on three important aspects of the mirror response, as measured by modulations in corticospinal excitability: (1) muscle specificity, (2) direction, and (3) timing of modulation. We focus mainly on electromyographic (EMG) data gathered following single-pulse transcranial magnetic stimulation (TMS), because this method provides precise information regarding these three aspects of the response. Data from paired-pulse TMS paradigms and peripheral nerve stimulation (PNS) are also considered when we discuss the possible mechanisms underlying the mirror response. In this systematic review of the literature, we examine the findings of 85 TMS and PNS studies of the human mirror response, and consider the limitations and advantages of the different methodological approaches these have adopted in relation to discrepancies between their findings. We conclude by proposing a testable model of how action observation modulates corticospinal excitability in humans. Specifically, we propose that action observation elicits an early, non-specific facilitation of corticospinal excitability (at around 90 ms from action onset), followed by a later modulation of activity specific to the muscles involved in the observed action (from around 200 ms). Testing this model will greatly advance our understanding of the mirror mechanism and provide a more stable grounding on which to base inferences about its role in human behaviour.

Exploring quercetin and luteolin derivatives as antiangiogenic agents

The formation of new blood vessels from the pre-existing vasculature (angiogenesis) is a crucial stage in cancer progression and, indeed, angiogenesis inhibitors are now used as anticancer agents, clinically. Here we have explored the potential of flavonoid derivatives as antiangiogenic agents. Specifically, we have synthesised methoxy and 4-thio derivatives of the natural flavones quercetin and luteolin, two of which (4-thio quercetin and 4-thio luteolin) had never been previously reported. Seven of these compounds showed significant (P<0.05) antiangiogenic activity in an in vitro scratch assay. Their activity ranged from an 86% inhibition of the vascular endothelium growth factor (VEGF)-stimulated migration (observed for methoxyquercetin at 10 µM and for luteolin at 1 µM) to a 36% inhibition (for thiomethoxy quercetin at 10 µM). Western blotting studies showed that most (4 out of 7) compounds inhibited phosphorylation of the VEGF receptor-2 (VEGFR2), suggesting that the antiangiogenic activity was due to an interference with the VEGF/VEGFR2 pathway. Molecular modelling studies looking at the affinity of our compounds towards VEGFR and/or VEGF confirmed this hypothesis, and indeed the compound with the highest antiangiogenic activity (methoxyquercetin) showed the highest affinity towards VEGFR and VEGF. As reports from others have suggested that structurally similar compounds can elicit biological responses via a non-specific, promiscuous membrane perturbation, potential interactions of the active compounds with a model lipid bilayer were assessed via DSC. Luteolin and its derivatives did not perturb the model membrane even at concentrations 10 times higher than the biologically active concentration and only subtle interactions were observed for quercetin and its derivatives. Finally, cytotoxicity assessment of these flavonoid derivatives against MCF-7 breast cancer cells demonstrated also a direct anticancer activity albeit at generally higher concentrations than those required for an antiangiogenic effect (10 fold higher for the methoxy analogues). Taken together these results show promise for flavonoid derivatives as antiangiogenic agents.

Treatment of child anxiety in the context of maternal anxiety disorder: a randomised controlled trial and economic analysis

Background Cognitive–behavioural therapy (CBT) for childhood anxiety disorders is associated with modest outcomes in the context of parental anxiety disorder. Objectives This study evaluated whether or not the outcome of CBT for children with anxiety disorders in the context of maternal anxiety disorders is improved by the addition of (i) treatment of maternal anxiety disorders, or (ii) treatment focused on maternal responses. The incremental cost-effectiveness of the additional treatments was also evaluated. Design Participants were randomised to receive (i) child cognitive–behavioural therapy (CCBT); (ii) CCBT with CBT to target maternal anxiety disorders [CCBT + maternal cognitive–behavioural therapy (MCBT)]; or (iii) CCBT with an intervention to target mother–child interactions (MCIs) (CCBT + MCI). Setting A NHS university clinic in Berkshire, UK. Participants Two hundred and eleven children with a primary anxiety disorder, whose mothers also had an anxiety disorder. Interventions All families received eight sessions of individual CCBT. Mothers in the CCBT + MCBT arm also received eight sessions of CBT targeting their own anxiety disorders. Mothers in the MCI arm received 10 sessions targeting maternal parenting cognitions and behaviours. Non-specific interventions were delivered to balance groups for therapist contact. Main outcome measures Primary clinical outcomes were the child’s primary anxiety disorder status and degree of improvement at the end of treatment. Follow-up assessments were conducted at 6 and 12 months. Outcomes in the economic analyses were identified and measured using estimated quality-adjusted life-years (QALYs). QALYS were combined with treatment, health and social care costs and presented within an incremental cost–utility analysis framework with associated uncertainty. Results MCBT was associated with significant short-term improvement in maternal anxiety; however, after children had received CCBT, group differences were no longer apparent. CCBT + MCI was associated with a reduction in maternal overinvolvement and more confident expectations of the child. However, neither CCBT + MCBT nor CCBT + MCI conferred a significant post-treatment benefit over CCBT in terms of child anxiety disorder diagnoses [adjusted risk ratio (RR) 1.18, 95% confidence interval (CI) 0.87 to 1.62, p = 0.29; adjusted RR CCBT + MCI vs. control: adjusted RR 1.22, 95% CI 0.90 to 1.67, p = 0.20, respectively] or global improvement ratings (adjusted RR 1.25, 95% CI 1.00 to 1.59, p = 0.05; adjusted RR 1.20, 95% CI 0.95 to 1.53, p = 0.13). CCBT + MCI outperformed CCBT on some secondary outcome measures. Furthermore, primary economic analyses suggested that, at commonly accepted thresholds of cost-effectiveness, the probability that CCBT + MCI will be cost-effective in comparison with CCBT (plus non-specific interventions) is about 75%. Conclusions Good outcomes were achieved for children and their mothers across treatment conditions. There was no evidence of a benefit to child outcome of supplementing CCBT with either intervention focusing on maternal anxiety disorder or maternal cognitions and behaviours. However, supplementing CCBT with treatment that targeted maternal cognitions and behaviours represented a cost-effective use of resources, although the high percentage of missing data on some economic variables is a shortcoming. Future work should consider whether or not effects of the adjunct interventions are enhanced in particular contexts. The economic findings highlight the utility of considering the use of a broad range of services when evaluating interventions with this client group. Trial registration Current Controlled Trials ISRCTN19762288. Funding This trial was funded by the Medical Research Council (MRC) and Berkshire Healthcare Foundation Trust and managed by the National Institute for Health Research (NIHR) on behalf of the MRC–NIHR partnership (09/800/17) and will be published in full in Health Technology Assessment; Vol. 19, No. 38.

Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.