EVOLUTION OF THE KINETICS AND DYNAMICS OF HEME-CREVICE LOOP REGULATING CHEMISTRY IN HUMAN CYTOCHROME C

Cytochrome c, cytc, is a metalloprotein that plays primary roles in electron transport and intrinsic apoptotic pathways. Much of the chemistry that cytc is involved with is regulated by a highly conserved region known as the heme crevice loop, consisting of residues 70-85. Only three of these residues (those at positions 81, 83 and 85) are not universally conserved within the evolutionary timeline. Here I look to elucidate possible evolutionary roles for several of the key residues known to be important in regulating heme chemistry of cytc. I first address the role that lysine 72 plays in cytc folding and chemistry. Here I provide evidence that K72 alters the alkaline conformational transition of cytc. Trimethylated lysine 72, tmK72, was previously investigated and shows similar trends in peroxidase activity (McClelland et al 2013). Lastly I address I81 which is not only within the heme crevice loop and not universally conserved, but is also a hydrophobic surface residue. Here I make a Hu I81A variant, mutating to the alanine seen in yeast cytc. Our hypothesis was that this mutation would show a destabilization of the heme crevice loop region when monitoring the charge transfer band (695 nm), and more importantly observing an increase in peroxidase activity when monitoring for tetraguaiacol (470 nm) in our enzymatic assay. This signifies this mutation could have evolved to lock down that heme crevice loop in order to decrease peroxidase activity when intrinsic apoptosis pathways evolved in mammals. pH titration data showed a decrease in stability of the alkaline conformational transition in our I81A variant when compared to Hu WT. When looking at peroxidase activity we see a significant increase in kcat(s-1) values of I81A compared to Hu WT. The Hu I81A indeed shows what we would expect of a mutation which evolved to decrease peroxidase activity. Analysis of pH jump data in the Soret region of cytc shows that there is an effect on lysine 73 or 79 bound alkaline ligands (unable to be determined) by a decrease in amplitude, however there is no effect on the lysine 72 bound alkaline ligand

Failure of Interferon γ to Induce the Anti-Inflammatory Interleukin 18 Binding Protein in Familial Hemophagocytosis

Background: Familial hemophagocytosis (FHL) is a rare disease associated with defects in proteins involved in CD8+ T-cell cytotoxicity. Hyperactivation of immune cells results in a perilous, Th1-driven cytokine storm. We set out to explore the regulation of cytokines in an FHL patient who was clinically stable on low-dose immunosuppressive therapy after bone marrow transplantation over a six-month period. During this period, chimerism analyses showed that the fraction of host cells was between 1 and 10%. Both parents of the patient as well as healthy volunteers were studied for comparison. Methods/Principal Findings: Using ELISA, quantitative real-time PCR, and clinical laboratory methods, we investigated constitutive and inducible cytokines, polymorphisms, and clinical parameters in whole blood and whole blood cultures. Although routine laboratory tests were within the normal range, the chemokines IP-10 and IL-8 as well as the cytokine IL-27p28 were increased up to 10-fold under constitutive and stimulated conditions compared to healthy controls. Moreover, high levels of IFNgamma and TNFalpha were produced upon stimulation. Unexpectedly, IFNgamma induction of IL-18 binding protein (IL-18BP) was markedly reduced (1.6-fold vs 5-fold in controls). The patient's mother featured intermediately increased cytokine levels, whereas levels in the father were similar to those in the controls. Conclusions/Significance: Since IL-18 plays a major role in perpetuating hemophagocytosis, the failure of IFNgamma to induce IL-18BP may constitute a fundamental pathogenetic mechanism. Furthermore, increased production of IL-8 and IL-27 appears to be associated with this disease. Such dysregulation of cytokines was also found in the heterozygous parents, providing a novel insight into genotype-phenotype correlation of FHL which may encourage future research of this rare disease

Wetting on a spherical wall: influence of liquid-gas interfacial properties

We study the equilibrium of a liquid film on an attractive spherical substrate for an intermolecular interaction model exhibiting both fluid-fluid and fluid-wall long-range forces. We first reexamine the wetting properties of the model in the zero-curvature limit, i.e., for a planar wall, using an effective interfacial Hamiltonian approach in the framework of the well known sharp-kink approximation (SKA). We obtain very good agreement with a mean-field density functional theory (DFT), fully justifying the use of SKA in this limit. We then turn our attention to substrates of finite curvature and appropriately modify the so-called soft-interface approximation (SIA) originally formulated by Napi\'orkowski and Dietrich [Phys. Rev. B 34, 6469 (1986)] for critical wetting on a planar wall. A detailed asymptotic analysis of SIA confirms the SKA functional form for the film growth. However, it turns out that the agreement between SKA and our DFT is only qualitative. We then show that the quantitative discrepancy between the two is due to the overestimation of the liquid-gas surface tension within SKA. On the other hand, by relaxing the assumption of a sharp interface, with, e.g., a simple smoothing of the density profile there, markedly improves the predictive capability of the theory, making it quantitative and showing that the liquid-gas surface tension plays a crucial role when describing wetting on a curved substrate. In addition, we show that in contrast to SKA, SIA predicts the expected mean-field critical exponent of the liquid-gas surface tension

Influence of temperature fluctuations on plasma turbulence investigations with Langmuir probes

The reliability of Langmuir probe measurements for plasma-turbulence investigations is studied on GEMR gyro-fluid simulations and compared with results from conditionally sampled I-V characteristics as well as self-emitting probe measurements in the near scrape-off layer of the tokamak ASDEX Upgrade. In this region, simulation and experiment consistently show coherent in-phase fluctuations in density, plasma potential and also in electron temperature. Ion-saturation current measurements turn out to reproduce density fluctuations quite well. Fluctuations in the floating potential, however, are strongly influenced by temperature fluctuations and, hence, are strongly distorted compared to the actual plasma potential. These results suggest that interpreting floating as plasma-potential fluctuations while disregarding temperature effects is not justified near the separatrix of hot fusion plasmas. Here, floating potential measurements lead to corrupted results on the ExB dynamics of turbulent structures in the context of, e.g., turbulent particle and momentum transport or instability identification on the basis of density-potential phase relations

Data Platforms and Cities

This section offers a series of joint reflections on (open) data platform from a variety of cases, from cycling, traffic and mapping to activism, environment and data brokering. Data platforms play a key role in contemporary urban governance. Linked to open data initiatives, such platforms are often proposed as both mechanisms for enhancing the accountability of administrations and performing as sites for 'bottom-up' digital invention. Such promises of smooth flows of data, however, rarely materialise unproblematically. The development of data platforms is always situated in legal and administrative cultures, databases are often built according to the standards of existing digital ecologies, access always involves processes of social negotiation, and interfaces (such as sensors) may become objects of public contestation. The following contributions explore the contested and mutable character of open data platforms as part of heterogeneous publics and trace the pathways of data through different knowledge, skills, public and private configurations. They also reflect on the value of STS approaches to highlight issues and tensions as well as to shape design and governance

Spatial awareness comparisons between large-screen, integrated pictorial displays and conventional EFIS displays during simulated landing approaches

An extensive simulation study was performed to determine and compare the spatial awareness of commercial airline pilots on simulated landing approaches using conventional flight displays with their awareness using advanced pictorial 'pathway in the sky' displays. Sixteen commercial airline pilots repeatedly made simulated complex microwave landing system approaches to closely spaced parallel runways with an extremely short final segment. Scenarios involving conflicting traffic situation assessments and recoveries from flight path offset conditions were used to assess spatial awareness (own ship position relative the the desired flight route, the runway, and other traffic) with the various display formats. The situation assessment tools are presented, as well as the experimental designs and the results. The results demonstrate that the integrated pictorial displays substantially increase spatial awareness over conventional electronic flight information systems display formats

Comparisons of Medical Student Knowledge Regarding Life-Threatening CT Images Before and After Clinical Experience

Introduction. Currently, no national standard exists for educatingmedical students regarding radiography or formal research indicatingthe level of improvement regarding computed tomography(CT) interpretation of medical students during clinical rotations. Methods. Students were evaluated based on their response totwenty-two open-ended questions regarding diagnosis and treatmentof eleven de-identified CT images of life-threatening injuries.The number of incorrect answers was compared withcorrect or partially correct answers between students startingthird-year clinical rotations and those starting their fourth year. Results. Survey results were collected from 65 of 65 (100%) beginningthird-year students and 9 of 60 (15%) beginning fourthyearstudents. Students in their fourth-year had less incorrectanswers compared to third-year students, with five questionsreflecting a statistically significant reduction in incorrect responses.The image with the least incorrect for both groups wasepidural hemorrhage, 33.9% and 18.5% incorrect for third-yearstudents for diagnosis and treatment, respectively, and 11.1%and 0% incorrect for fourth-year students. Outside of this image,the range of incorrect answers for third-year students was75.4% to 100% and 44.4% to 100% for fourth-year students. Conclusion. Baseline CT knowledge of medical students,regardless of clinical experience, indicated a strong deficit,as more students were incorrect than correct for themajority of CT images. KS J Med 2017;10(3):55-58