Orientation-dependent Dxz4 contacts shape the 3D structure of the inactive X chromosome.

The mammalian inactive X chromosome (Xi) condenses into a bipartite structure with two superdomains of frequent long-range contacts, separated by a hinge region. Using Hi-C in edited mouse cells with allelic deletions or inversions within the hinge, here we show that the conserved Dxz4 locus is necessary to maintain this bipartite structure. Dxz4 orientation controls the distribution of contacts on the Xi, as shown by a massive reversal in long-range contacts after Dxz4 inversion. Despite an increase in CTCF binding and chromatin accessibility on the Xi in Dxz4-edited cells, only minor changes in TAD structure and gene expression were detected, in accordance with multiple epigenetic mechanisms ensuring X silencing. We propose that Dxz4 represents a structural platform for frequent long-range contacts with multiple loci in a direction dictated by the orientation of its bank of CTCF motifs, which may work as a ratchet to form the distinctive bipartite structure of the condensed Xi.

Clusterin regulates ß-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway.

Although the mechanism of Aß action in the pathogenesis of Alzheimer's disease (AD) has remained elusive, it is known to increase the expression of the antagonist of canonical wnt signalling, Dickkopf-1 (Dkk1), whereas the silencing of Dkk1 blocks Aß neurotoxicity. We asked if clusterin, known to be regulated by wnt, is part of an Aß/Dkk1 neurotoxic pathway. Knockdown of clusterin in primary neurons reduced Aß toxicity and DKK1 upregulation and, conversely, Aß increased intracellular clusterin and decreased clusterin protein secretion, resulting in the p53-dependent induction of DKK1. To further elucidate how the clusterin-dependent induction of Dkk1 by Aß mediates neurotoxicity, we measured the effects of Aß and Dkk1 protein on whole-genome expression in primary neurons, finding a common pathway suggestive of activation of wnt-planar cell polarity (PCP)-c-Jun N-terminal kinase (JNK) signalling leading to the induction of genes including EGR1 (early growth response-1), NAB2 (Ngfi-A-binding protein-2) and KLF10 (Krüppel-like factor-10) that, when individually silenced, protected against Aß neurotoxicity and/or tau phosphorylation. Neuronal overexpression of Dkk1 in transgenic mice mimicked this Aß-induced pathway and resulted in age-dependent increases in tau phosphorylation in hippocampus and cognitive impairment. Furthermore, we show that this Dkk1/wnt-PCP-JNK pathway is active in an Aß-based mouse model of AD and in AD brain, but not in a tau-based mouse model or in frontotemporal dementia brain. Thus, we have identified a pathway whereby Aß induces a clusterin/p53/Dkk1/wnt-PCP-JNK pathway, which drives the upregulation of several genes that mediate the development of AD-like neuropathologies, thereby providing new mechanistic insights into the action of Aß in neurodegenerative diseases.

Astrocytes are important mediators of Aß-induced neurotoxicity and tau phosphorylation in primary culture.

Alzheimer's disease (AD) is pathologically characterised by the age-dependent deposition of ß-amyloid (Aß) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aß-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aß-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aß-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aß-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aß in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.

Transgenic mouse models of tauopathy in drug discovery.

Tauopathies, including Alzheimer's disease, are neurodegenerative diseases characterized by the deposition of hyperphosphorylated tau protein in the central nervous system, and are the major cause of dementia in later life. Considerable advances have been made in developing mouse models that recapitulate, to varying extents, the development of human tau pathology, and the learning and memory deficits characteristic of some tauopathies. Furthermore, such models have been used to show promising disease-modifying effects in pre-clinical testing of new therapeutics. Various strategies have been utilised to generate mouse models of tauopathies. Some of the most enlightening models developed to date either constitutively or inducibly express pathogenic tau mutations. These animals have been instrumental in defining critical disease-related mechanisms, including the observation that tangles are not the toxic form of tau in disease. Here, we discuss the strengths and weaknesses of well characterised transgenic models that emulate human tauopathy, and include a comprehensive listing of the main phenotypic characteristics of all reported tau transgenic rodents. We summarise the use of tau mice for the development and evaluation of new therapeutic approaches, and their utility in identifying novel drug targets. In addition, we review the parameters to be considered in the development of the next generation of mouse models of tauopathy, aimed at further increasing our understanding of disease aetiology and in evaluating novel treatments.

Multi-scale correlations in continuous genomic data.

Functional genomic quantities such as histone modifications, chromatin accessibility, and evolutionary constraint can now be measured in a nearly continuous fashion across the genome. The genome is highly heterogeneous, and the relationships between different functional annotations may be fluid. Here we present an approach for visualizing, quantifying, and determining the statistical significance of local and regional correlations between high-density continuous genomic datasets. We use wavelets to generate a multi-scale view of each component data set and calculate correlations between data types as a function of genome position over a continuous range of scales in sliding window fashion. We determine the statistical significance of correlations using a non-parametric sampling approach. We apply the wavelet correlation method to histone modification and chromatin accessibility (DNasel sensitivity) data from the NHGRI ENCODE project. We show that DNaseI sensitivity is broadly correlated (though to differing degrees) with a number of different activating histone modifications. We examine the continuous relationship between the repressive histone modification H3K27me3 and the activating mark H3K4me2, and find these modifications to display significant duality, with both significant positively and negatively correlated genomic territories. While the former appear to recapitulate in definitive cells the so-called "bi-valent" pattern originally proposed as a signature of pluripotency, the presence of negatively correlated regions suggests that the regulatory events that underlie the observed modification patterns are complex and highly regionalized in the genome.

RNA and protein-dependent mechanisms in tauopathies: consequences for therapeutic strategies.

Tauopathies are a group of neurodegenerative diseases characterised by intracellular deposits of the microtubule-associated protein tau. The most typical example of a tauopathy is Alzheimer's disease. The importance of tau in neuronal dysfunction and degeneration has been demonstrated by the discovery of dominant mutations in the MAPT gene, encoding tau, in some rare dementias. Recent developments have shed light on the significance of tau phosphorylation and aggregation in pathogenesis. Furthermore, emerging evidence reveals the central role played by tau pre-mRNA processing in tauopathies. The present review focuses on the current understanding of tau-dependent pathogenic mechanisms and how realistic therapies for tauopathies can be developed.

Kernel-based data fusion and its application to protein function prediction in yeast.

Kernel methods provide a principled framework in which to represent many types of data, including vectors, strings, trees and graphs. As such, these methods are useful for drawing inferences about biological phenomena. We describe a method for combining multiple kernel representations in an optimal fashion, by formulating the problem as a convex optimization problem that can be solved using semidefinite programming techniques. The method is applied to the problem of predicting yeast protein functional classifications using a support vector machine (SVM) trained on five types of data. For this problem, the new method performs better than a previously-described Markov random field method, and better than the SVM trained on any single type of data.

Analysis of strain and regional variation in gene expression in mouse brain.

BACKGROUND: We performed a statistical analysis of a previously published set of gene expression microarray data from six different brain regions in two mouse strains. In the previous analysis, 24 genes showing expression differences between the strains and about 240 genes with regional differences in expression were identified. Like many gene expression studies, that analysis relied primarily on ad hoc 'fold change' and 'absent/present' criteria to select genes. To determine whether statistically motivated methods would give a more sensitive and selective analysis of gene expression patterns in the brain, we decided to use analysis of variance (ANOVA) and feature selection methods designed to select genes showing strain- or region-dependent patterns of expression. RESULTS: Our analysis revealed many additional genes that might be involved in behavioral differences between the two mouse strains and functional differences between the six brain regions. Using conservative statistical criteria, we identified at least 63 genes showing strain variation and approximately 600 genes showing regional variation. Unlike ad hoc methods, ours have the additional benefit of ranking the genes by statistical score, permitting further analysis to focus on the most significant. Comparison of our results to the previous studies and to published reports on individual genes show that we achieved high sensitivity while preserving selectivity. CONCLUSIONS: Our results indicate that molecular differences between the strains and regions studied are larger than indicated previously. We conclude that for large complex datasets, ANOVA and feature selection, alone or in combination, are more powerful than methods based on fold-change thresholds and other ad hoc selection criteria.

Long-term sexual co-habitation offers no protection from hypertensive disease of pregnancy.

Long-term sexual co-habitation and previous pregnancies are thought to protect against the development of hypertensive disease of pregnancy. In order to test the hypothesis that pregnancies conceived after prolonged exposure to the partner's spermatozoa have reduced rates of hypertensive disease this study examined the outcomes of pregnancies of women who conceived by donor insemination as compared with women who conceived after IVF with partner's spermatozoa. This was a retrospective cohort study of 218 women attending an IVF clinic, 45 of whom conceived by donor insemination and 173 of whom conceived by partner's spermatozoa. Cases were identified from the IVF unit and data were extracted from patients' notes by blinded observers. Results showed no difference between the groups, with 22% of women who conceived with donor spermatozoa and 24% who conceived with partner spermatozoa developing some form of hypertensive disease of pregnancy. Insemination by partner's spermatozoa was not associated with a reduction of hypertensive disease and neither was donor spermatozoa associated with an increased incidence.

Co-transfer of plasmids in association with conjugative transfer of mupirocin or mupirocin and penicillin resistance in methicillin-resistant Staphylococcus aureus.

Two distinct strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients in a dermatology ward were also resistant to mupirocin. The mupirocin resistance plasmids from both strains were indistinguishable by EcoRI and HindIII restriction digest analysis, except for the presence of genes apparently mediating penicillinase production in some transconjugants. Conjugative transfer of the plasmid mediating mupirocin resistance from one of these strains to a recipient S. aureus was accompanied in some cases by co-transfer of plasmids mediating resistance to tetracycline or erythromycin; in some instances a plasmid which possessed no apparent resistance markers was also transferred. The second strain demonstrated conjugative transfer of penicillin and mupirocin resistance as well as transfer of a plasmid mediating gentamicin resistance, but transfer of erythromycin resistance was not apparently plasmid-mediated.

Cytokine expression during allergen-induced late nasal responses: IL-4 and IL-5 mRNA is expressed early (at 6 h) predominantly by eosinophils.

The production of TH2-type cytokines [interleukin-4 (IL-4) and IL-5] and tissue eosinophilia are characteristic features of allergic diseases. It was previously reported that at 24 h after allergen provocation, CD3+ T-lymphocytes were the principal cell source of IL-4 and IL-5 mRNA transcripts in both atopic asthma and rhinitis. To investigate whether IL-4 and IL-5 mRNA are expressed earlier during late nasal responses and if so, which cell(s) are responsible. Nasal biopsies were obtained at 6 h after nasal allergen challenge and following a control challenge with the allergen diluent. Sections were immunostained for T-lymphocytes (CD3+, CD4+) and eosinophils (EG2+). In situ hybridization was used to detect the number of cells expressing messenger RNA (mRNA) for IL-4 and IL-5. In patients with allergic rhinitis, eosinophils (EG2+ cells P = 0. 006) but not T- cells (CD3+ cells) increased in the nasal mucosa at 6 h after allergen challenge. The number of cells expressing IL-4 mRNA (P = 0.01) and IL-5 mRNA (P = 0.05) also increased at 6 h. Co-localization studies showed that 76% of IL-4 mRNA+ cells and 77% of IL-5 mRNA+ cells were eosinophils, whereas at this time point, T-cells and mast cells accounted for

Self-reports about tinnitus and about cochlear implants.

OBJECTIVE: Analyze literature on self-report outcomes in two areas of audiological rehabilitation: 1) tinnitus and 2) cochlear implant hearing aids. DESIGN: 1) Tinnitus: survey of features in the development of self-report approaches and of formal scales used in assessment of tinnitus disability and handicaps. 2) Cochlear implants: summary of the literature using self-report approaches to cochlear implant experience that indicates points of theoretical significance. RESULTS: 1) Major features of tinnitus are: a) disabilities such as interference with and distortion of normal auditory perception; b) handicaps such as emotional distress, interference with sleep, and with personal and social life. Nonauditory factors-chronic depression, high self-focused attention-mediate the degree of experienced tinnitus handicap. 2) People with prelingual loss of hearing report that a cochlear implant primarily enables improved detection and discrimination of environmental sound; those with postlingual loss find that an implant in addition provides improved speech recognition. CONCLUSIONS: 1) Coping with tinnitus is influenced by the personal resources that can be brought to bear on the experience, highlighting a general point that any rehabilitation outcome is not only a matter of acoustical solutions. By the same token, tinnitus can be easier to cope with if its "psychoacoustic presence" can be diminished by some form of masking. 2) Cochlear implants fitted in childhood that do not provide meaningful input signals in real-world settings may be rejected in adolescence. 3) "Hearing," as a capacity, does not have a fixed worth. Different circumstances mean it will be taken as desirable or as delivering torment (extreme tinnitus, e.g.). Its value will also vary depending on the extent of a person's access to spoken language (aiding in very early childhood, e.g.).

Optimal outcome measures, research priorities, and international cooperation.

The participants in the Eriksholm Workshop on "Measuring Outcomes in Audiological Rehabilitation Using Hearing Aids" debated three issues that are reported in this article. First, it was agreed that the characteristics of an optimal outcome measure vary as a function of the purpose of the measurement. Potential characteristics of outcome self-report tools for four common goals of outcome measurement are briefly presented to illustrate this point. Second, 10 important research priorities in outcome measurement were identified and ranked. They are presented with brief discussion of the top five. Third, the concept of generating a brief universally applicable outcome measure was endorsed. This brief data set is intended to supplement existing outcome measures and to promote data combination and comparison across different social, cultural, and health-care delivery systems. A set of seven core items is proposed for further study.

Glandular abnormalities on cervical smear: a study to compare the accuracy of cytological diagnosis with underlying pathology.

The interpretation of glandular abnormalities detected by cervical smear provides a diagnostic dilemma. This study aims to compare the accuracy of cytological diagnosis with underlying pathology so that guidelines for the investigation and management of abnormal glandular smears may be formulated. A retrospective review of 150 women with glandular abnormalities reported on cervical smear collected over 12 months from 1996 in a University hospital was performed. Smears were graded by the initial report into 3 groups, dependent on the severity of abnormality. Investigation, treatment and subsequent 3-year follow-up were recorded. The accuracy of prediction for a significant neoplastic or preneoplastic glandular pathology only was 0% with mild, 9% (3/35) with moderate, and 24% (9/38) with severe abnormalities. When squamous lesions were included, the chance of finding any dysplastic squamous or glandular abnormality was 16% (12/77), 51% (18/35) and 82% (31/38), respectively, following a smear showing a suspected glandular abnormality only. Our results highlight the poor specificity of predicting glandular neoplasia or preneoplasia from cervical smears, with a final diagnosis of high grade CIN in 35% (17/49) of patients with dyskaryotic glandular cytological changes only and 83% (20/24) where concomitant squamous dyskaryosis was reported. The reporting of reactive or minor changes in endocervical cells was of no diagnostic value. Management protocols for moderate and severe glandular abnormalities should include visualisation and biopsy of the uterine cavity to exclude endometrial neoplasia.

Long-term clinical efficacy of grass-pollen immunotherapy.

BACKGROUND: Pollen immunotherapy is effective in selected patients with IgE-mediated seasonal allergic rhinitis, although it is questionable whether there is long-term benefit after the discontinuation of treatment. METHODS: We conducted a randomized, double-blind, placebo-controlled trial of the discontinuation of immunotherapy for grass-pollen allergy in patients in whom three to four years of this treatment had previously been shown to be effective. During the three years of this trial, primary outcome measures were scores for seasonal symptoms and the use of rescue medication. Objective measures included the immediate conjunctival response and the immediate and late skin responses to allergen challenge. Cutaneous-biopsy specimens obtained 24 hours after intradermal allergen challenge were examined for T-cell infiltration and the presence of cytokine-producing T helper cells (TH2 cells) (as evidenced by the presence of interleukin-4 messenger RNA). A matched group of patients with hay fever who had not received immunotherapy was followed as a control for the natural course of the disease. RESULTS: Scores for seasonal symptoms and the use of rescue antiallergic medication, which included short courses of prednisolone, remained low after the discontinuation of immunotherapy, and there was no significant difference between patients who continued immunotherapy and those who discontinued it. Symptom scores in both treatment groups (median areas under the curve in 1995, 921 for continuation of immunotherapy and 504 for discontinuation of immunotherapy; P=0.60) were markedly lower than those in the group that had not received immunotherapy (median value in 1995, 2863). Although there was a tendency for immediate sensitivity to allergen to return late after discontinuation, there was a sustained reduction in the late skin response and associated CD3+ T-cell infiltration and interleukin-4 messenger RNA expression. CONCLUSIONS: Immunotherapy for grass-pollen allergy for three to four years induces prolonged clinical remission accompanied by a persistent alteration in immunologic reactivity.

Fluoroquinolone resistance in Staphylococcus intermedius.

Four canine isolates of S. intermedius resistant to enrofloxacin were isolated amongst a total of 429 screened. Two of these were shown to exhibit resistance also to marbofloxacin and ciprofloxacin. Whilst molecular studies have shown the mechanism of resistance to these quinolone antibiotics to be similar in a number of staphylococcal species, it was not possible to confirm this mechanism in Staphylococcus intermedius.