Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Chronic OVA allergen challenged Siglec-F deficient mice have increased mucus, remodeling, and epithelial Siglec-F ligands which are up-regulated by IL-4 and IL-13

Abstract Background In this study we examined the role of Siglec-F, a receptor highly expressed on eosinophils, in contributing to mucus expression, airway remodeling, and Siglec-F ligand expression utilizing Siglec-F deficient mice exposed to chronic allergen challenge. Methods Wild type (WT) and Siglec-F deficient mice were sensitized and challenged chronically with OVA for one month. Levels of airway inflammation (eosinophils), Siglec-F ligand expresion and remodeling (mucus, fibrosis, smooth muscle thickness, extracellular matrix protein deposition) were assessed in lung sections by image analysis and immunohistology. Airway hyperreactivity to methacholine was assessed in intubated and ventilated mice. Results Siglec-F deficient mice challenged with OVA for one month had significantly increased numbers of BAL and peribronchial eosinophils compared to WT mice which was associated with a significant increase in mucus expression as assessed by the number of periodic acid Schiff positive airway epithelial cells. In addition, OVA challenged Siglec-F deficient mice had significantly increased levels of peribronchial fibrosis (total lung collagen, area of peribronchial trichrome staining), as well as increased numbers of peribronchial TGF-β1+ cells, and increased levels of expression of the extracellular matrix protein fibronectin compared to OVA challenged WT mice. Lung sections immunostained with a Siglec-Fc to detect Siglec-F ligand expression demonstrated higher levels of expression of the Siglec-F ligand in the peribronchial region in OVA challenged Siglec-F deficient mice compared to WT mice. WT and Siglec-F deficient mice challenged intranasally with IL-4 or IL-13 had significantly increased levels of airway epithelial Siglec-F ligand expression, whereas this was not observed in WT or Siglec-F deficient mice challenged with TNF-α. There was a significant increase in the thickness of the peribronchial smooth muscle layer in OVA challenged Siglec-F deficient mice, but this was not associated with significant increased airway hyperreactivity compared to WT mice. Conclusions Overall, this study demonstrates an important role for Siglec-F in modulating levels of chronic eosinophilic airway inflammation, peribronchial fibrosis, thickness of the smooth muscle layer, mucus expression, fibronectin, and levels of peribronchial Siglec-F ligands suggesting that Siglec-F may normally function to limit levels of chronic eosinophilic inflammation and remodeling. In addition, IL-4 and IL-13 are important regulators of Siglec-F ligand expression by airway epithelium

Determining the Repertoire of Immunodominant Proteins via Whole-Genome Amplification of Intracellular Pathogens

Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

The kaleidoscopic midwife: A conceptual metaphor illustrating first-time mothers' perspectives of a good midwife during childbirth. A grounded theory study

Background: The literature review reveals general information about a good midwife from a range of perspectives and what childbearing women generally value in a midwife, but there is a lack of information around mothers’ perspectives of what makes a good midwife specifically during labour and birth, and even less in the context of different places of birth. Aim: To conceptualise first-time mothers’ expectations and experiences of a good midwife during childbirth in the context of different birthplaces. Design: Qualitative Straussian grounded theory methodology. Setting: Three National Health Service Trusts in England providing maternity care that offered women the possibility of giving birth in different settings (home, freestanding midwifery unit and obstetric unit). Participants: Fourteen first-time mothers in good general health with a straightforward singleton pregnancy anticipating a normal birth. Methods: Ethical approval was gained. Data were collected through two semi-structured interviews for each participant (before and after birth). Data analysis included the processes of coding and conceptualising data, with constant comparison between data, literature and memos. Findings: The model named ‘The kaleidoscopic midwife: a conceptual metaphor illustrating first-time mothers’ perspectives of a good midwife during childbirth’ was developed. The model is dynamic and woman-centred, and is operationalised as the midwife adapts to each woman's individual needs in the context of each specific labour. Four pillars of intrapartum care were identified for a good midwife in the labour continuum: promoting individuality; supporting embodied limbo; helping to go with the flow; providing information and guidance. The metaphor of a kaleidoscopic figure is used to describe a midwife who is ‘multi-coloured’ and ever changing in the light of the woman's individual needs, expectations and labour journey, in order to create an environment that enables her to move forward despite the uncertainty and the expectations-experiences gap. The following elements are harmonised by the kaleidoscopic midwife: relationship-mediated being; knowledgeable doing; physical presence; immediately available presence. Conclusion: The model presented has relevance to contemporary debates about quality of care and place of birth and can be used by midwives to pursue excellence in caring for labouring mothers. Independently from the place of birth, when the woman is cared for by a midwife demonstrating the above characteristics, she is likely to have an optimum experience of birth. Future research is necessary to tease out individual components of the model in a variety of practice settings

The Drosophila melanogaster PeptideAtlas facilitates the use of peptide data for improved fly proteomics and genome annotation

<p>Abstract</p> <p>Background</p> <p>Crucial foundations of any quantitative systems biology experiment are correct genome and proteome annotations. Protein databases compiled from high quality empirical protein identifications that are in turn based on correct gene models increase the correctness, sensitivity, and quantitative accuracy of systems biology genome-scale experiments.</p> <p>Results</p> <p>In this manuscript, we present the <it>Drosophila melanogaster </it>PeptideAtlas, a fly proteomics and genomics resource of unsurpassed depth. Based on peptide mass spectrometry data collected in our laboratory the portal <url>http://www.drosophila-peptideatlas.org</url> allows querying fly protein data observed with respect to gene model confirmation and splice site verification as well as for the identification of proteotypic peptides suited for targeted proteomics studies. Additionally, the database provides consensus mass spectra for observed peptides along with qualitative and quantitative information about the number of observations of a particular peptide and the sample(s) in which it was observed.</p> <p>Conclusion</p> <p>PeptideAtlas is an open access database for the <it>Drosophila </it>community that has several features and applications that support (1) reduction of the complexity inherently associated with performing targeted proteomic studies, (2) designing and accelerating shotgun proteomics experiments, (3) confirming or questioning gene models, and (4) adjusting gene models such that they are in line with observed <it>Drosophila </it>peptides. While the database consists of proteomic data it is not required that the user is a proteomics expert.</p

Effect of an education programme for patients with osteoarthritis in primary care - a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Osteoarthritis (OA) is a degenerative disease, considered to be one of the major public health problems. Research suggests that patient education is feasible and valuable for achieving improvements in quality of life, in function, well-being and improved coping. Since 1994, Primary Health Care in Malmö has used a patient education programme directed towards OA. The aim of this study was to evaluate the effects of this education programme for patients with OA in primary health care in terms of self-efficacy, function and self-perceived health.</p> <p>Method</p> <p>The study was a single-blind, randomized controlled trial (RCT) in which the EuroQol-5D and Arthritis self-efficacy scale were used to measure self-perceived health and self-efficacy and function was measured with Grip Ability Test for the upper extremity and five different functional tests for the lower extremity.</p> <p>Results</p> <p>We found differences between the intervention group and the control group, comparing the results at baseline and after 6 months in EuroQol-5D (p < 0.001) and in standing one leg eyes closed (p = 0.02) in favour of the intervention group. No other differences between the groups were found.</p> <p>Conclusion</p> <p>This study has shown that patient education for patients with osteoarthritis is feasible in a primary health care setting and can improve self-perceived health as well as function in some degree, but not self-efficacy. Further research to investigate the effect of exercise performance on function, as well as self-efficacy is warranted.</p> <p>Trial registration</p> <p>The trial is registered with ClinicalTrials.gov. Registration number: NCT00979914</p

BRCA1 tumours correlate with a HIF-1α phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression

BACKGROUND: There are limited data regarding the hypoxia pathway in familial breast cancers. We therefore performed a study of hypoxic factors in BRCA1, BRCA2 and BRCAX breast cancers. METHODS: Immunoperoxidase staining for HIF-1alpha, PHD1, PHD2, PHD3, VEGF and FIH was carried out in 125 (38 BRCA1, 33 BRCA2 and 54 BRCAX) breast carcinomas. These were correlated with clinicopathological parameters and the intrinsic breast cancer phenotypes. RESULTS: BRCA1 tumours correlated with positivity for HIF-1alpha (P=0.008) and negativity for PHD3 (P=0.037). HIF-1alpha positivity (P=0.001), PHD3 negativity (P=0.037) and nuclear FIH negativity (P=0.011) was associated with basal phenotype. HIF-1alpha expression correlated with high tumour grade (P=0.009), negative oestrogen receptor (ER) status (P=0.001) and the absence of lymph node metastasis (P=0.028). Nuclear FIH expression and PHD3 correlated with positive ER expression (P=0.024 and P=0.035, respectively). BRCA1 cancers with positive HIF-1alpha or cytoplasmic FIH had a significantly shorter relapse-free survival (P=0.007 and P=0.049, respectively). CONCLUSIONS: The aggressive nature of BRCA1 and basal-type tumours may be partly explained by an enhanced hypoxic drive and hypoxia driven ER degradation because of suppressed PHD and aberrantly located FIH expression. This may have important implications, as these tumours may respond to compounds directed against HIF-1alpha or its downstream targets

Direct Recognition of Fusobacterium nucleatum by the NK Cell Natural Cytotoxicity Receptor NKp46 Aggravates Periodontal Disease

Periodontitis is a common human chronic inflammatory disease that results in the destruction of the tooth attachment apparatus and tooth loss. Although infections with periopathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are essential for inducing periodontitis, the nature and magnitude of the disease is determined by the host's immune response. Here, we investigate the role played by the NK killer receptor NKp46 (NCR1 in mice), in the pathogenesis of periodontitis. Using an oral infection periodontitis model we demonstrate that following F. nucleatum infection no alveolar bone loss is observed in mice deficient for NCR1 expression, whereas around 20% bone loss is observed in wild type mice and in mice infected with P. gingivalis. By using subcutaneous chambers inoculated with F. nucleatum we demonstrate that immune cells, including NK cells, rapidly accumulate in the chambers and that this leads to a fast and transient, NCR1-dependant TNF-α secretion. We further show that both the mouse NCR1 and the human NKp46 bind directly to F. nucleatum and we demonstrate that this binding is sensitive to heat, to proteinase K and to pronase treatments. Finally, we show in vitro that the interaction of NK cells with F. nucleatum leads to an NCR1-dependent secretion of TNF-α. Thus, the present study provides the first evidence that NCR1 and NKp46 directly recognize a periodontal pathogen and that this interaction influences the outcome of F. nucleatum-mediated periodontitis