Fusion of green fluorescent protein to the C-terminus of granulysin alters its intracellular localization in comparison to the native molecule

The engineering of green fluorescent protein (GFP) fusion constructs in order to visibly tag a protein of interest has become a commonly used cell biology technique. Although caveats to this approach are obvious, literature reports in which the chimeric molecule behaves differently than the native molecule are scant. This brief report describes one such case. Granulysin, a small lytic and antimicrobial protein produced by cytotoxic lymphocytes, traffics to the regulated secretory system and is subsequently released from cells upon proper stimulus. In an attempt to elucidate mechanisms by which it accumulates in and is released from cytolytic granules, GFP was fused to the C-terminus of granulysin and expressed in an NK cell line. A control construct expressing the native protein was similarly expressed. The data demonstrate that, while the fusion protein is expressed and secreted, its subcellular localization is altered in comparison to native granulysin. Thus, the addition of GFP to the C-terminus of granulysin obscures the signal(s) that cytotoxic lymphocytes use to sort it to the regulated secretory pathway despite its normal biosynthesis and secretion. This example is offered as a cautionary account for other researchers who contemplate using this technology

Are Commonly Used Resident Measurements Associated with Procedural Skills in Internal Medicine Residency Training?

BACKGROUND: Acquisition of competence in performing a variety of procedures is essential during Internal Medicine (IM) residency training. PURPOSES: Determine the rate of procedural complications by IM residents; determine whether there was a correlation between having 1 or more complications and institutional procedural certification status or attending ratings of resident procedural skill competence on the American Board of Internal Medicine (ABIM) monthly evaluation form (ABIM-MEF). Assess if an association exists between procedural complications and in-training examination and ABIM board certification scores. METHODS: We retrospectively reviewed all procedure log sheets, procedural certification status, ABIM-MEF procedural skills ratings, in-training exam and certifying examination (ABIM-CE) scores from the period 1990–1999 for IM residency program graduates from a training program. RESULTS: Among 69 graduates, 2,212 monthly procedure log sheets and 2,475 ABIM-MEFs were reviewed. The overall complication rate was 2.3/1,000 procedures (95% CI: 1.4–3.1/1,000 procedure). With the exception of procedural certification status as judged by institutional faculty, there was no association between our resident measurements and procedural complications. CONCLUSIONS: Our findings support the need for a resident procedural competence certification system based on direct observation. Our data support the ABIM’s action to remove resident procedural competence from the monthly ABIM-MEF ratings

An Approach to Evaluation of the Effect of Bioremediation on Biological Activity of Environmental Contaminants: Dechlorination of Polychlorinated Biphenyls

The effectiveness of bioremediation efforts is assessed traditionally from the loss of the chemical of interest. In some cases, analytical techniques are coupled with evaluation of toxicity to organisms representative of those found in the affected environment or surrogate organisms. Little is known, however, about the effect of remediation of environmental chemicals on potential toxicity to mammalian organisms. We discuss both an approach that employs mammalian cell system bioassays and the criteria for selection of the assays. This approach has been used to evaluate the biological response to mixtures of polychlorinated biphenyls (PCBs) before and after remediation by reductive dechlorination. The dechlorination process used results in accumulation of congeners substituted in only the ortho and para positions and containing fewer chlorines than the starting mixtures. Evaluation of the dechlorinated mixture reveals a loss of biological activity that could be ascribed to coplanar PCBs not containing chlorine in the ortho positions. Conversely, biological activity associated with ortho-substituted PCB congeners is unaffected or increased by remediation. Thus, the results of the bioassays are consistent with the remediation-induced change in the profile of PCB congeners and the known mechanisms of action of PCBs. The results emphasize a need for evaluation of the products of remediation for biological activity in mammalian systems. Furthermore, the approach outlined demonstrates the potential to assess the impact of remediation on a range of biological activities in mammalian cells and thus to estimate positive and negative effects of remediation strategies on toxicity. Future needs in this area of research include assays to evaluate biological effects under conditions of exposure that mimic those found in the environment and models to extrapolate effects to assess risk to people and wildlife

Raising argument strength using negative evidence: A constraint on models of induction

Both intuitively, and according to similarity-based theories of induction, relevant evidence raises argument strength when it is positive and lowers it when it is negative. In three experiments, we tested the hypothesis that argument strength can actually increase when negative evidence is introduced. Two kinds of argument were compared through forced choice or sequential evaluation: single positive arguments (e.g., “Shostakovich’s music causes alpha waves in the brain; therefore, Bach’s music causes alpha waves in the brain”) and double mixed arguments (e.g., “Shostakovich’s music causes alpha waves in the brain, X’s music DOES NOT; therefore, Bach’s music causes alpha waves in the brain”). Negative evidence in the second premise lowered credence when it applied to an item X from the same subcategory (e.g., Haydn) and raised it when it applied to a different subcategory (e.g., AC/DC). The results constitute a new constraint on models of induction

Absence of a Fermi surface in classical minimal four-dimensional gauged supergravity

We demonstrate that the two point function of the supercurrent dual to the gravitino in the four-dimensional extremal anti-de Sitter Reissner-Nordstrom black hole does not exhibit a Fermi surface singularity. In our analysis, we utilize the ingoing Eddington-Finkelstein coordinate system, which enables us to bypass certain complications in the determination of the allowed near horizon behavior of the gravitino field at zero frequency. We check that our method agrees with previous results for the massless charged Dirac field.Comment: 12 pages, 1 figur

Patient-centric trials for therapeutic development in precision oncology

An enhanced understanding of the molecular pathology of disease gained from genomic studies is facilitating the development of treatments that target discrete molecular subclasses of tumours. Considerable associated challenges include how to advance and implement targeted drug-development strategies. Precision medicine centres on delivering the most appropriate therapy to a patient on the basis of clinical and molecular features of their disease. The development of therapeutic agents that target molecular mechanisms is driving innovation in clinical-trial strategies. Although progress has been made, modifications to existing core paradigms in oncology drug development will be required to realize fully the promise of precision medicine

Ozone and PM(2.5) Exposure and Acute Pulmonary Health Effects: A Study of Hikers in the Great Smoky Mountains National Park

To address the lack of research on the pulmonary health effects of ozone and fine particulate matter (≤ 2.5 μm in aerodynamic diameter; PM(2.5)) on individuals who recreate in the Great Smoky Mountains National Park (USA) and to replicate a study performed at Mt. Washington, New Hampshire (USA), we conducted an observational study of adult (18–82 years of age) day hikers of the Charlies Bunion trail during 71 days of fall 2002 and summer 2003. Volunteer hikers performed pre- and posthike pulmonary function tests (spirometry), and we continuously monitored ambient O(3), PM(2.5), temperature, and relative humidity at the trailhead. Of the 817 hikers who participated, 354 (43%) met inclusion criteria (nonsmokers and no use of bronchodilators within 48 hr) and gave acceptable and reproducible spirometry. For these 354 hikers, we calculated the posthike percentage change in forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV(1)), FVC/FEV(1), peak expiratory flow, and mean flow rate between 25 and 75% of the FVC and regressed each separately against pollutant (O(3) or PM(2.5)) concentration, adjusting for age, sex, hours hiked, smoking status (former vs. never), history of asthma or wheeze symptoms, hike load, reaching the summit, and mean daily temperature. O(3) and PM(2.5) concentrations measured during the study were below the current federal standards, and we found no significant associations of acute changes in pulmonary function with either pollutant. These findings are contrasted with those in the Mt. Washington study to examine the hypothesis that pulmonary health effects are associated with exposure to O(3) and PM(2.5) in healthy adults engaged in moderate exercise

Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding

Background: Atherosclerosis is an inflammatory process that develops in individuals with known risk factors that include hypertension and hyperlipidaemia, influenced by diet. However, the interplay between diet, inflammatory mechanisms and vascular risk factors requires further research. We hypothesised that interleukin-1 (IL-1) signaling in the vessel wall would raise arterial blood pressure and promote atheroma. Methodology/Principal Findings: Apoe(-/-) and Apoe(-/-)/IL-1R1(-/-) mice were fed high fat diets for 8 weeks, and their blood pressure and atherosclerosis development measured. Apoe(-/-)/IL-R1(-/-) mice had a reduced blood pressure and significantly less atheroma than Apoe(-/-) mice. Selective loss of IL-1 signaling in the vessel wall by bone marrow transplantation also reduced plaque burden (p<0.05). This was associated with an IL-1 mediated loss of endothelium-dependent relaxation and an increase in vessel wall Nox 4. Inhibition of IL-1 restored endothelium-dependent vasodilatation and reduced levels of arterial oxidative stress. Conclusions/Significance: The IL-1 cytokine system links atherogenic environmental stimuli with arterial inflammation, oxidative stress, increased blood pressure and atherosclerosis. This is the first demonstration that inhibition of a single cytokine can block the rise in blood pressure in response to an environmental stimulus. IL-1 inhibition may have profound beneficial effects on atherogenesis in man

Endothelin-1 Predicts Hemodynamically Assessed Pulmonary Arterial Hypertension in HIV Infection.

BackgroundHIV infection is an independent risk factor for PAH, but the underlying pathogenesis remains unclear. ET-1 is a robust vasoconstrictor and key mediator of pulmonary vascular homeostasis. Higher levels of ET-1 predict disease severity and mortality in other forms of PAH, and endothelin receptor antagonists are central to treatment, including in HIV-associated PAH. The direct relationship between ET-1 and PAH in HIV-infected individuals is not well described.MethodsWe measured ET-1 and estimated pulmonary artery systolic pressure (PASP) with transthoracic echocardiography (TTE) in 106 HIV-infected individuals. Participants with a PASP ≥ 30 mmHg (n = 65) underwent right heart catheterization (RHC) to definitively diagnose PAH. We conducted multivariable analysis to identify factors associated with PAH.ResultsAmong 106 HIV-infected participants, 80% were male, the median age was 52 years and 77% were on antiretroviral therapy. ET-1 was significantly associated with higher values of PASP [14% per 0.1 pg/mL increase in ET-1, p = 0.05] and PASP ≥ 30 mmHg [PR (prevalence ratio) = 1.24, p = 0.012] on TTE after multivariable adjustment for PAH risk factors. Similarly, among the 65 individuals who underwent RHC, ET-1 was significantly associated with higher values of mean pulmonary artery pressure and PAH (34%, p = 0.003 and PR = 2.43, p = 0.032, respectively) in the multivariable analyses.ConclusionsHigher levels of ET-1 are independently associated with HIV-associated PAH as hemodynamically assessed by RHC. Our findings suggest that excessive ET-1 production in the setting of HIV infection impairs pulmonary endothelial function and contributes to the development of PAH