Characterization of two putative potassium channels in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Potassium channels are essential for cell survival and participate in the regulation of cell membrane potential and electrochemical gradients. During its lifecycle, <it>Plasmodium falciparum </it>parasites must successfully traverse widely diverse environmental milieus, in which K⁺channel function is likely to be critical. Dramatically differing conditions will be presented to the parasite in the mosquito mid-gut, red blood cell (RBC) cytosol and the human circulatory system.</p> <p>Methods</p> <p><it>In silico </it>sequence analyses identified two open-reading frames in the <it>P. falciparum </it>genome that are predicted to encode for proteins with high homology to K⁺channels. To further analyse these putative channels, specific antisera were generated and used in immunoblot and immunofluorescence analyses of <it>P. falciparum</it>-infected RBCs. Recombinant genome methods in cultured <it>P. falciparum </it>were used to create genetic knock outs of each K⁺channel gene to asses the importance of their expression.</p> <p>Results</p> <p>Immunoblot and IFA analyses confirmed the expression of the two putative <it>P. falciparum </it>K⁺channels (PfK1 and PfK2). PfK1 is expressed in all asexual stage parasites, predominantly in late stages and localizes to the RBC membrane. Conversely, PfK2 is predominantly expressed in late schizont and merozoite stage parasites and remains primarily localized to the parasite. Repeated attempts to knockout PfK1 and PfK2 expression by targeted gene disruption proved unsuccessful despite evidence of recombinant gene integration, indicating that <it>pfk1 </it>and <it>pfk2 </it>are apparently refractory to genetic disruption.</p> <p>Conclusion</p> <p>Putative K⁺channel proteins PfK1 and PfK2 are expressed in cultured <it>P. falciparum </it>parasites with differing spatial and temporal patterns. Eventual functional characterization of these channels may reveal future pharmacological targets.</p

A Novel Transcription Factor, T-bet, Directs Th1 Lineage Commitment

AbstractNaive T helper cells differentiate into two subsets, Th1 and Th2, each with distinct functions and cytokine profiles. Here, we report the isolation of T-bet, a Th1-specific T box transcription factor that controls the expression of the hallmark Th1 cytokine, IFNγ. T-bet expression correlates with IFNγ expression in Th1 and NK cells. Ectopic expression of T-bet both transactivates the IFNγ gene and induces endogenous IFNγ production. Remarkably, retroviral gene transduction of T-bet into polarized Th2 and Tc2 primary T cells redirects them into Th1 and Tc1 cells, respectively, as evidenced by the simultaneous induction of IFNγ and repression of IL-4 and IL-5. Thus, T-bet initiates Th1 lineage development from naive Thp cells both by activating Th1 genetic programs and by repressing the opposing Th2 programs

Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a “class I” fusogen) and West Nile virus envelope protein (“class II”). Our study of VSV now extends this description to “class III” viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion

Sulfate was a trace constituent of Archean seawater

In the low-oxygen Archean world (>2400 million years ago), seawater sulfate concentrations were much lower than today, yet open questions frustrate the translation of modern measurements of sulfur isotope fractionations into estimates of Archean seawater sulfate concentrations. In the water column of Lake Matano, Indonesia, a low-sulfate analog for the Archean ocean, we find large (>20 per mil) sulfur isotope fractionations between sulfate and sulfide, but the underlying sediment sulfides preserve a muted range of δ^(34)S values. Using models informed by sulfur cycling in Lake Matano, we infer Archean seawater sulfate concentrations of less than 2.5 micromolar. At these low concentrations, marine sulfate residence times were likely 10^3 to 10^4 years, and sulfate scarcity would have shaped early global biogeochemical cycles, possibly restricting biological productivity in Archean oceans

Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma in Patients With Child-Pugh B or C Cirrhosis

Purpose: Our purpose was to report outcomes in patients with Child-Pugh B or C (CP B/C) hepatocellular carcinoma (HCC) treated with stereotactic body radiation therapy (SBRT). Methods and Materials: Patients with HCC suitable for SBRT were prospectively enrolled in the study from 2012 to 2018. Outcomes in patients with CP B/C were analyzed. Cox proportional hazard models were used to compare survival outcomes between baseline CP score and post-SBRT CP score. Results: Twenty-three patients with CP B/C with a total of 29 HCC tumors were treated with SBRT. Eighty-seven percent of patients were CP B8-C10. Median tumor size was 3.1 cm (range, 1-10 cm). Median dose delivered was 40 Gy in a median of 5 fractions. Eighteen of 23 patients (78.3%) had been previously treated with transarterial chemoembolization. Median follow-up was 14.5 months. Rates of 6- and 12-month local control were 100% and 92.3%, respectively. Six- and 12-month survival rates were 73.9% and 56.5%, respectively. Median survival was 14.5 months overall and 9.2, 22.5, 14.5, and 14.4 months for patients with CP B7, B8, B9, and C10, respectively. No patients exhibited symptoms of classic radiation-induced liver disease. However, 10 patients had CP score progression, with 4 patients (17%) having a \u3e /=2-point increase in CP score by 6 months (or time of censor). There were 7 liver-related deaths, and based on independent review by a hepatologist, 1 of these deaths may have been attributable to SBRT-related liver injury. Fifteen of 23 patients were listed for liver transplant (LT) at the time of SBRT and 9 went on to receive LT with a pathologic complete response rate of 63.6%. Median survival, excluding patients who received LT, was 7.3 months. Conclusions: SBRT is a reasonable treatment option for carefully selected patients with CP B7-C10. In our small cohort, there was no detectable difference between local control or overall survival and baseline CP score

miR451 and AMPK Mutual Antagonism in Glioma Cell Migration and Proliferation: A Mathematical Model

Glioblastoma multiforme (GBM) is the most common and the most aggressive type of brain cancer; the median survival time from the time of diagnosis is approximately one year. GBM is characterized by the hallmarks of rapid proliferation and aggressive invasion. miR-451 is known to play a key role in glioblastoma by modulating the balance of active proliferation and invasion in response to metabolic stress in the microenvironment. The present paper develops a mathematical model of GBM evolution which focuses on the relative balance of growth and invasion. In the present work we represent the miR-451/AMPK pathway by a simple model and show how the effects of glucose on cells need to be “refined” by taking into account the recent history of glucose variations. The simulations show how variations in glucose significantly affect the level of miR-451 and, in turn, cell migration. The model predicts that oscillations in the levels of glucose increase the growth of the primary tumor. The model also suggests that drugs which upregulate miR-451, or block other components of the CAB39/AMPK pathway, will slow down glioma cell migration. The model provides an explanation for the growth-invasion cycling patterns of glioma cells in response to high/low glucose uptake in microenvironment in vitro, and suggests new targets for drugs, associated with miR-451 upregulation

Immune Activation Caused By Vascular Oxidation Promotes Fibrosis And Hypertension

Vascular oxidative injury accompanies many common conditions associated with hypertension. In the present study, we employed mouse models with excessive vascular production of ROS (tg(sm/p22phox) mice, which overexpress the NADPH oxidase subunit p22(phox) smooth muscle, and mice with vascular-specific deletion of extracellular SOD) and have shown that these animals develop vascular collagen deposition, aortic stiffening, renal dysfunction, and hypertension with age. T cells from tg(5m/p22phox) mice produced high levels of IL-17A and IFN-gamma. Crossing tg(sm/p22phox) mice with lymphocyte-deficient Rag1(-/-) mice eliminated vascular inflammation, aortic stiffening, renal dysfunction, and hypertension; however, adoptive transfer of T cells restored these processes. Isoketal-protein adducts, which are immunogenic, were increased in aortas, DCs, and macrophages of tg(sm/P22Phox) mice. Autologous pulsing with tg(sm/p22phox) aortic homogenates promoted DCs of tg(sm/p22phox) mice to stimulate T cell proliferation and production of IFN-gamma, IL-17A, and TNF-alpha. Treatment with the superoxide scavenger tempol or the isoketal scavenger 2-hydroxybenzylamine (2-HOBA) normalized blood pressure; prevented vascular inflammation, aortic stiffening, and hypertension; and prevented DC and T cell activation. Moreover, in human aortas, the aortic content of isoketal adducts correlated with fibrosis and inflammation severity. Together, these results define a pathway linking vascular oxidant stress to immune activation and aortic stiffening and provide insight into the systemic inflammation encountered in common vascular diseases.12615067NIH [DK059637, DK020593]NIH R01 [HL105294, HL039006, HL108701]VITA [HHSN268201400010C]American Heart Association predoctoral fellowship [13PRE14480008]NIH K08 [1K08HL121671]Vanderbilt Physician Scientist Development AwardNIH F32 [1F32HL124972-01][P01 HL58000

Statistical Model Checking for Stochastic Hybrid Systems

This paper presents novel extensions and applications of the UPPAAL-SMC model checker. The extensions allow for statistical model checking of stochastic hybrid systems. We show how our race-based stochastic semantics extends to networks of hybrid systems, and indicate the integration technique applied for implementing this semantics in the UPPAAL-SMC simulation engine. We report on two applications of the resulting tool-set coming from systems biology and energy aware buildings.Comment: In Proceedings HSB 2012, arXiv:1208.315

Observer Dependent Horizon Temperatures: a Coordinate-Free Formulation of Hawking Radiation as Tunneling

We reformulate the Hamilton-Jacobi tunneling method for calculating Hawking radiation in static, spherically-symmetric spacetimes by explicitly incorporating a preferred family of frames. These frames correspond to a family of observers tied to a locally static timelike Killing vector of the spacetime. This formulation separates the role of the coordinates from the choice of vacuum and thus provides a coordinate-independent formulation of the tunneling method. In addition, it clarifies the nature of certain constants and their relation to these preferred observers in the calculation of horizon temperatures. We first use this formalism to obtain the expected temperature for a static observer at finite radius in the Schwarzschild spacetime. We then apply this formalism to the Schwarzschild-de Sitter spacetime, where there is no static observer with 4-velocity equal to the static timelike Killing vector. It is shown that a preferred static observer, one whose trajectory is geodesic, measures the lowest temperature from each horizon. Furthermore, this observer measures horizon temperatures corresponding to the well-known Bousso-Hawking normalization.Comment: 11 pages, 1 2-part figure, references added, appendix added, discussion streamline