The isolated perfused mouse uterus as a model for the study of implantation in vitro. Methodology and morphology

In order to facilitate investigations of mammalian blastocyst implantation in the endometrium, an in-vitro organ perfusion technique was developed. This technique was designed to avoid the drawbacks of inVivo and cell culture investigations, while retaining physiological resolution of the endo- and paracrinology and specifically a normal epithelium to stroma relationship. The ovary, oviduct and uterine horn from 21 mice were perfused in-vitro for 10 hours. The surgical techniques for isolation of the organs as well as the perfusion procedure are described. The resultant morphology of the perfused tissue, including implantations is described and illustrated by light and transmission electron microscopy. The model seems to be useful for studying the mammalian implantation as implantation takes place and decidua is formed during perfusion

Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in <i>Pectobacterium spp</i>

In order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;Pectobacterium atrosepticum&lt;/i&gt; with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that &lt;i&gt;Pectobacterium spp.&lt;/i&gt; carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of &lt;i&gt;Pectobacterium carotovorum&lt;/i&gt; and &lt;i&gt;atrosepticum&lt;/i&gt; that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells

CFD Investigation on Fluid Flow Analysis in Fluid Separator

The analysis of fully developed flow in the two fluid separators is an important issue in the industry such as production, processing, and petrochemical. The role of the two fluid separator is to separate two different fluid by using an appropriate mechanism without changing the quality. In this study, we have reviewed different mechanisms of two fluid separations such as gravity sedimentation, centrifugation, and electro kinetics, etc. The current work focuses on the design aspect of a fluid separator with respect to geometry and thermal design. CFD has been used to simulate flow in a fluid separator and its results have been verified experimentally. Flow rates used in the simulation have different values in interval 0.1 LPM. The study shows the best performance of fluid separator with respect to shape and flow rates. The given work helps to co-relate various design of separator in the industry with laboratory separators

Localism in Finland : The changing role and current crisis of the Finnish municipal system

Peer reviewe

The Structure and Regulation of Human Muscle α-Actinin

SummaryThe spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins

Novel computational methods for increasing PCR primer design effectiveness in directed sequencing

<p>Abstract</p> <p>Background</p> <p>Polymerase chain reaction (PCR) is used in directed sequencing for the discovery of novel polymorphisms. As the first step in PCR directed sequencing, effective PCR primer design is crucial for obtaining high-quality sequence data for target regions. Since current computational primer design tools are not fully tuned with stable underlying laboratory protocols, researchers may still be forced to iteratively optimize protocols for failed amplifications after the primers have been ordered. Furthermore, potentially identifiable factors which contribute to PCR failures have yet to be elucidated. This inefficient approach to primer design is further intensified in a high-throughput laboratory, where hundreds of genes may be targeted in one experiment.</p> <p>Results</p> <p>We have developed a fully integrated computational PCR primer design pipeline that plays a key role in our high-throughput directed sequencing pipeline. Investigators may specify target regions defined through a rich set of descriptors, such as Ensembl accessions and arbitrary genomic coordinates. Primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the specified target regions. As part of the tiling process, primer pairs are computationally screened to meet the criteria for success with one of two PCR amplification protocols. In the process of improving our sequencing success rate, which currently exceeds 95% for exons, we have discovered novel and accurate computational methods capable of identifying primers that may lead to PCR failures. We reveal the laboratory protocols and their associated, empirically determined computational parameters, as well as describe the novel computational methods which may benefit others in future primer design research.</p> <p>Conclusion</p> <p>The high-throughput PCR primer design pipeline has been very successful in providing the basis for high-quality directed sequencing results and for minimizing costs associated with labor and reprocessing. The modular architecture of the primer design software has made it possible to readily integrate additional primer critique tests based on iterative feedback from the laboratory. As a result, the primer design software, coupled with the laboratory protocols, serves as a powerful tool for low and high-throughput primer design to enable successful directed sequencing.</p

Evaluation of the current knowledge limitations in breast cancer research: a gap analysis

BACKGROUND A gap analysis was conducted to determine which areas of breast cancer research, if targeted by researchers and funding bodies, could produce the greatest impact on patients. METHODS Fifty-six Breast Cancer Campaign grant holders and prominent UK breast cancer researchers participated in a gap analysis of current breast cancer research. Before, during and following the meeting, groups in seven key research areas participated in cycles of presentation, literature review and discussion. Summary papers were prepared by each group and collated into this position paper highlighting the research gaps, with recommendations for action. RESULTS Gaps were identified in all seven themes. General barriers to progress were lack of financial and practical resources, and poor collaboration between disciplines. Critical gaps in each theme included: (1) genetics (knowledge of genetic changes, their effects and interactions); (2) initiation of breast cancer (how developmental signalling pathways cause ductal elongation and branching at the cellular level and influence stem cell dynamics, and how their disruption initiates tumour formation); (3) progression of breast cancer (deciphering the intracellular and extracellular regulators of early progression, tumour growth, angiogenesis and metastasis); (4) therapies and targets (understanding who develops advanced disease); (5) disease markers (incorporating intelligent trial design into all studies to ensure new treatments are tested in patient groups stratified using biomarkers); (6) prevention (strategies to prevent oestrogen-receptor negative tumours and the long-term effects of chemoprevention for oestrogen-receptor positive tumours); (7) psychosocial aspects of cancer (the use of appropriate psychosocial interventions, and the personal impact of all stages of the disease among patients from a range of ethnic and demographic backgrounds). CONCLUSION Through recommendations to address these gaps with future research, the long-term benefits to patients will include: better estimation of risk in families with breast cancer and strategies to reduce risk; better prediction of drug response and patient prognosis; improved tailoring of treatments to patient subgroups and development of new therapeutic approaches; earlier initiation of treatment; more effective use of resources for screening populations; and an enhanced experience for people with or at risk of breast cancer and their families. The challenge to funding bodies and researchers in all disciplines is to focus on these gaps and to drive advances in knowledge into improvements in patient care

PETALS: Proteomic Evaluation and Topological Analysis of a mutated Locus' Signaling

<p>Abstract</p> <p>Background</p> <p>Colon cancer is driven by mutations in a number of genes, the most notorious of which is <it>Apc</it>. Though much of <it>Apc</it>'s signaling has been mechanistically identified over the years, it is not always clear which functions or interactions are operative in a particular tumor. This is confounded by the presence of mutations in a number of other putative cancer driver (CAN) genes, which often synergize with mutations in <it>Apc</it>.</p> <p>Computational methods are, thus, required to predict which pathways are likely to be operative when a particular mutation in <it>Apc </it>is observed.</p> <p>Results</p> <p>We developed a pipeline, PETALS, to predict and test likely signaling pathways connecting <it>Apc </it>to other CAN-genes, where the interaction network originating at <it>Apc </it>is defined as a "blossom," with each <it>Apc</it>-CAN-gene subnetwork referred to as a "petal." Known and predicted protein interactions are used to identify an Apc blossom with 24 petals. Then, using a novel measure of bimodality, the coexpression of each petal is evaluated against proteomic (2 D differential In Gel Electrophoresis, 2D-DIGE) measurements from the <it>Apc</it>^{<it>1638N</it>+/-}mouse to test the network-based hypotheses.</p> <p>Conclusions</p> <p>The predicted pathways linking <it>Apc </it>and <it>Hapln1 </it>exhibited the highest amount of bimodal coexpression with the proteomic targets, prioritizing the <it>Apc-Hapln1 </it>petal over other CAN-gene pairs and suggesting that this petal may be involved in regulating the observed proteome-level effects. These results not only demonstrate how functional 'omics data can be employed to test in <it>silico </it>predictions of CAN-gene pathways, but also reveal an approach to integrate models of upstream genetic interference with measured, downstream effects.</p

wKinMut: An integrated tool for the analysis and interpretation of mutations in human protein kinases

BACKGROUND: Protein kinases are involved in relevant physiological functions and a broad number of mutations in this superfamily have been reported in the literature to affect protein function and stability. Unfortunately, the exploration of the consequences on the phenotypes of each individual mutation remains a considerable challenge. RESULTS: The wKinMut web-server offers direct prediction of the potential pathogenicity of the mutations from a number of methods, including our recently developed prediction method based on the combination of information from a range of diverse sources, including physicochemical properties and functional annotations from FireDB and Swissprot and kinase-specific characteristics such as the membership to specific kinase groups, the annotation with disease-associated GO terms or the occurrence of the mutation in PFAM domains, and the relevance of the residues in determining kinase subfamily specificity from S3Det. This predictor yields interesting results that compare favourably with other methods in the field when applied to protein kinases. Together with the predictions, wKinMut offers a number of integrated services for the analysis of mutations. These include: the classification of the kinase, information about associations of the kinase with other proteins extracted from iHop, the mapping of the mutations onto PDB structures, pathogenicity records from a number of databases and the classification of mutations in large-scale cancer studies. Importantly, wKinMut is connected with the SNP2L system that extracts mentions of mutations directly from the literature, and therefore increases the possibilities of finding interesting functional information associated to the studied mutations. CONCLUSIONS: wKinMut facilitates the exploration of the information available about individual mutations by integrating prediction approaches with the automatic extraction of information from the literature (text mining) and several state-of-the-art databases. wKinMut has been used during the last year for the analysis of the consequences of mutations in the context of a number of cancer genome projects, including the recent analysis of Chronic Lymphocytic Leukemia cases and is publicly available at http://wkinmut.bioinfo.cnio.es