Solubilization of EGF receptor with Triton X-100 alters stimulation of tyrosine residue phosphorylation by EGF and dimethyl sulfoxide.

In isolated hepatic membranes, epidermal growth factor (EGF) and the polar solvent dimethyl sulfoxide (Me2SO) selectively stimulated the phosphorylation of the 170,000-dalton EGF receptor (p170) by 13.6 +/- 2.0- and 10.9 +/- 1.1-fold, respectively. The stimulation by maximally effective concentrations of the two substances was similar in rapidity of onset (less than 30 s at 0 degree C), time course of phosphorylation, and tyrosine residue specificity. These maximal effects were not additive when the substances were combined, indicating that the same kinase/substrate combination is activated by each. The lectin concanavalin A, which inhibits EGF receptor binding, blocked the effect of EGF but not Me2SO. In membranes solubilized with Triton X-100, EGF stimulated p170 phosphorylation by 40- to 55-fold. Me2SO also stimulated phosphorylation, indicating that it acts directly on the protein. However, the effect of the solvent was reduced by half. Additionally, Me2SO blocks the effect of EGF in the solubilized preparation. A room temperature preincubation after addition of either substance was necessary for maximal stimulation of p170 phosphorylation in solubilized membranes. With EGF, 30-40 min was necessary; with Me2SO, only 10 min was required. Thus, a secondary process appears to be involved in EGF receptor/kinase activation

Genome-wide allele- and strand-specific expression profiling

Recent reports have shown that most of the genome is transcribed and that transcription frequently occurs concurrently on both DNA strands. In diploid genomes, the expression level of each allele conditions the degree to which sequence polymorphisms affect the phenotype. It is thus essential to quantify expression in an allele- and strand-specific manner. Using a custom-designed tiling array and a new computational approach, we piloted measuring allele- and strand-specific expression in yeast. Confident quantitative estimates of allele-specific expression were obtained for about half of the coding and non-coding transcripts of a heterozygous yeast strain, of which 371 transcripts (13%) showed significant allelic differential expression greater than 1.5-fold. The data revealed complex allelic differential expression on opposite strands. Furthermore, combining allele-specific expression with linkage mapping enabled identifying allelic variants that act in cis and in trans to regulate allelic expression in the heterozygous strain. Our results provide the first high-resolution analysis of differential expression on all four strands of an eukaryotic genome

Influence of real-world characteristics on outcomes for patients with methicillin-resistant Staphylococcal skin and soft tissue infections:a multi-country medical chart review in Europe

BACKGROUND: Patient-related (demographic/disease) and treatment-related (drug/clinician/hospital) characteristics were evaluated as potential predictors of healthcare resource use and opportunities for early switch (ES) from intravenous (IV)-to-oral methicillin-resistant Staphylococcus aureus (MRSA)-active antibiotic therapy and early hospital discharge (ED). METHODS: This retrospective observational medical chart study analyzed patients (across 12 European countries) with microbiologically confirmed MRSA complicated skin and soft tissue infections (cSSTI), ≥3 days of IV anti-MRSA antibiotics during hospitalization (July 1, 2010-June 30, 2011), and discharged alive by July 31, 2011. Logistic/linear regression models evaluated characteristics potentially associated with actual resource use (length of IV therapy, length of hospital stay [LOS], IV-to-oral antibiotic switch), and ES and ED (using literature-based and expert-verified criteria) outcomes. RESULTS: 1542 patients (mean ± SD age 60.8 ± 16.5 years; 61.5% males) were assessed with 81.0% hospitalized for MRSA cSSTI as the primary reason. Several patient demographic, infection, complication, treatment, and hospital characteristics were predictive of length of IV therapy, LOS, IV-to-oral antibiotic switch, or ES and ED opportunities. Outcomes and ES and ED opportunities varied across countries. Length of IV therapy and LOS (r = 0.66, p < 0.0001) and eligibilities for ES and ED (r = 0.44, p < 0.0001) showed relatively strong correlations. IV-to-oral antibiotic switch patients had significantly shorter length of IV therapy (−5.19 days, p < 0.001) and non-significantly shorter LOS (−1.86 days, p > 0.05). Certain patient and treatment characteristics were associated with increased odds of ES (healthcare-associated/ hospital-acquired infection) and ED (patient living arrangements, healthcare-associated/ hospital-acquired infection, initiating MRSA-active treatment 1–2 days post cSSTI index date, existing ED protocol), while other factors decreased the odds of ES (no documented MRSA culture, ≥4 days from admission to cSSTI index date, IV-to-oral switch, IV line infection) and ED (dementia, no documented MRSA culture, initiating MRSA-active treatment ≥3 days post cSSTI index date, existing ES protocol). CONCLUSIONS: Practice patterns and opportunity for further ES and ED were affected by several infection, treatment, hospital, and geographical characteristics, which should be considered in identifying ES and ED opportunities and designing interventions for MRSA cSSTI to reduce IV days and LOS while maintaining the quality of care. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2334-14-476) contains supplementary material, which is available to authorized users

Computational modelling of cancerous mutations in the EGFR/ERK signalling pathway

This article has been made available through the Brunel Open Access Publishing Fund - Copyright @ 2009 Orton et al.BACKGROUND: The Epidermal Growth Factor Receptor (EGFR) activated Extracellular-signal Regulated Kinase (ERK) pathway is a critical cell signalling pathway that relays the signal for a cell to proliferate from the plasma membrane to the nucleus. Deregulation of the EGFR/ERK pathway due to alterations affecting the expression or function of a number of pathway components has long been associated with numerous forms of cancer. Under normal conditions, Epidermal Growth Factor (EGF) stimulates a rapid but transient activation of ERK as the signal is rapidly shutdown. Whereas, under cancerous mutation conditions the ERK signal cannot be shutdown and is sustained resulting in the constitutive activation of ERK and continual cell proliferation. In this study, we have used computational modelling techniques to investigate what effects various cancerous alterations have on the signalling flow through the ERK pathway. RESULTS: We have generated a new model of the EGFR activated ERK pathway, which was verified by our own experimental data. We then altered our model to represent various cancerous situations such as Ras, B-Raf and EGFR mutations, as well as EGFR overexpression. Analysis of the models showed that different cancerous situations resulted in different signalling patterns through the ERK pathway, especially when compared to the normal EGF signal pattern. Our model predicts that cancerous EGFR mutation and overexpression signals almost exclusively via the Rap1 pathway, predicting that this pathway is the best target for drugs. Furthermore, our model also highlights the importance of receptor degradation in normal and cancerous EGFR signalling, and suggests that receptor degradation is a key difference between the signalling from the EGF and Nerve Growth Factor (NGF) receptors. CONCLUSION: Our results suggest that different routes to ERK activation are being utilised in different cancerous situations which therefore has interesting implications for drug selection strategies. We also conducted a comparison of the critical differences between signalling from different growth factor receptors (namely EGFR, mutated EGFR, NGF, and Insulin) with our results suggesting the difference between the systems are large scale and can be attributed to the presence/absence of entire pathways rather than subtle difference in individual rate constants between the systems.This work was funded by the Department of Trade and Industry (DTI), under their Bioscience Beacon project programme. AG was funded by an industrial PhD studentship from Scottish Enterprise and Cyclacel

Pilot, randomized, placebo-controlled clinical field study to evaluate the effectiveness of bupivacaine liposome injectable suspension for the provision of post-surgical analgesia in dogs undergoing stifle surgery

Abstract Background Local anesthetics are an important component of perioperative pain management, but the duration of action of available products is limited. We hypothesized that a single local infiltration of a novel bupivacaine liposome injectable suspension (AT-003) would provide clinically effective analgesia over a 72-h period. In a masked, randomized, placebo-controlled, multi-center pilot field study, dogs undergoing lateral retinacular suture placement for cranial cruciate insufficiency were randomly assigned to surgical site infiltration with AT-003 (5.3 mg/kg) or an equivalent volume of saline. Infiltration of the surgical site was done prior to closure. Primary outcome measure was the Glasgow Composite Measure Pain Scale (CMPS-SF) assessed prior to surgery and at 2, 4, 8, 12, 24, 30, 36, 48, 54, 60 and 72 h following surgery by trained individuals. Provision for rescue analgesia was employed. Repeated measures analysis of variance were utilized to test for possible differences between treatment groups and a success/failure analysis was also employed, based on the need for rescue analgesia. Results Forty-six dogs were enrolled and evaluated. For CMPS-SF scores there was a significant overall treatment effect (p = 0.0027) in favor of AT-003. There were significantly more successes in the AT-003 group compared to placebo over each time period (p = 0.0001 for 0–24 h, p = 0.0349 for 0–48 h, and p = 0.0240 for 0-72 h). No significant adverse events were seen. Conclusions AT-003 (bupivacaine liposome injectable suspension) provided measurable local analgesia over a 72-h period following post-stifle surgery surgical site tissue infiltration. Further work is indicated to develop this product for clinical use

Identification of losses to follow-up in a community-based antiretroviral therapy clinic in South Africa using a computerized pharmacy tracking system

BACKGROUND: High rates of loss to follow-up (LTFU) are undermining rapidly expanding antiretroviral treatment (ART) services in sub-Saharan Africa. The intelligent dispensing of ART (iDART) is an open-source electronic pharmacy system that provides an efficient means of generating lists of patients who have failed to pick-up medication. We determined the duration of pharmacy delay that optimally identified true LTFU. METHODS: We conducted a retrospective cross-sectional study of a community-based ART cohort in Cape Town, South Africa. We used iDART to identify groups of patients known to be still enrolled in the cohort on the 1st of April 2008 that had failed to pick-up medication for periods of ≥ 6, ≥ 12, ≥ 18 and ≥ 24 weeks. We defined true LTFU as confirmed failure to pick up medication for 3 months since last attendance. We then assessed short-term and long-term outcomes using a prospectively maintained database and patient records. RESULTS: On the date of the survey, 2548 patients were registered as receiving ART but of these 85 patients (3.3%) were found to be true LTFU. The numbers of individuals (proportion of the cohort) identified by iDART as having failed to collect medication for periods of ≥ 6, ≥ 12, ≥ 18 and ≥ 24 weeks were 560 (22%), 194 (8%), 117 (5%) and 80 (3%), respectively. The sensitivities of these pharmacy delays for detecting true LTFU were 100%, 100%, 62.4% and 47.1%, respectively. The corresponding specificities were 80.7%, 95.6%, 97.4% and 98.4%. Thus, the optimal delay was ≥ 12 weeks since last attendance at this clinic (equivalent to 8 weeks since medication ran out). Pharmacy delays were also found to be significantly associated with LTFU and death one year later. CONCLUSIONS: The iDART electronic pharmacy system can be used to detect patients potentially LTFU and who require recall. Using a short a cut-off period was too non-specific for LTFU and would require the tracing of very large numbers of patients. Conversely prolonged delays were too insensitive. Of the periods assessed, a ≥ 12 weeks delay appeared optimal. This system requires prospective evaluation to further refine its utility

Teaching politics after the practice turn

The ‘practice turn’ and its associated ontology, epistemology and methodology are now well established in political research. In this article, we identify and explore a corollary pedagogy. After outlining the principal components of practice theory, we compare case- and placement-based approaches to learning, designed to develop skills for use in practice. We introduce and describe our own rather different course, which we designed to develop an understanding of the nature of practice as such. We discuss its scope and dynamics, particularly with regard to power in the classroom, and identify broader opportunities and challenges for the development of practice-based pedagogy

Traditional electrosurgery and a low thermal injury dissection device yield different outcomes following bilateral skin-sparing mastectomy: a case report

<p>Abstract</p> <p>Introduction</p> <p>Although a skin- and nipple-sparing mastectomy technique offers distinct cosmetic and reconstructive advantages over traditional methods, partial skin flap and nipple necrosis remain a significant source of post-operative morbidity. Prior work has suggested that collateral thermal damage resulting from electrocautery use during skin flap development is a potential source of this complication. This report describes the case of a smoker with recurrent ductal carcinoma <it>in situ </it>(DCIS) who experienced significant unilateral skin necrosis following bilateral skin-sparing mastectomy while participating in a clinical trial examining mastectomy outcomes with two different surgical devices. This unexpected complication has implications for the choice of dissection devices in procedures requiring skin flap preservation.</p> <p>Case presentation</p> <p>The patient was a 61-year-old Caucasian woman who was a smoker with recurrent DCIS of her right breast. As part of the clinical trial, each breast was randomized to either the standard of care treatment group (a scalpel and a traditional electrosurgical device) or treatment with a novel, low thermal injury dissection device, allowing for a direct, internally controlled comparison of surgical outcomes. Post-operative follow-up at six days was unremarkable for both operative sites. At 16 days post-surgery, the patient presented with a significant wound necrosis in the mastectomy site randomized to the control study group. Following debridement and closure, this site progressively healed over 10 weeks. The contralateral mastectomy, randomized to the alternative device, healed normally.</p> <p>Conclusion</p> <p>We hypothesize that thermal damage to the subcutaneous microvasculature during flap dissection may have contributed to this complication and that the use of a low thermal injury dissection device may be advantageous in select patients undergoing skin- and nipple-sparing mastectomy.</p

Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage

© 2012 Millet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.This work was supported by the Research Grant Council of Hong Kong (RGC#760208)and the RESPARI project of the International Network of Pasteur Institutes