Paromomycin Affects Translation and Vesicle-Mediated Trafficking as Revealed by Proteomics of Paromomycin –Susceptible –Resistant Leishmania donovani

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL) and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr) strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC) followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr) strain identified a total of 226 proteins at ≥95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani

Increase in Near-Surface Atmospheric Moisture Content due to Land Use Changes: Evidence from the Observed Dewpoint Temperature Data

Land use change can significantly affect root zone soil moisture, surface energy balance, and near-surface atmospheric temperature and moisture content. During the second half of the twentieth century, portions of the North American Great Plains have experienced extensive introduction of irrigated agriculture. It is expected that land use change from natural grass to irrigated land use would significantly increase nearsurface atmospheric moisture content. Modeling studies have already shown an enhanced rate of evapotranspiration from the irrigated areas. The present study analyzes observed dewpoint temperature (Td) to assess the affect of irrigated land use on near-surface atmospheric moisture content. This investigation provides a unique opportunity to use long-term (1982–2003) mesoscale Td data from the Automated Weather Data Network of the high plains. Long-term daily Td data from 6 nonirrigated and 11 irrigated locations have been analyzed. Daily time series were developed from the hourly data. The length of time series was the primary factor in selection of these stations. Results suggest increase in growing-season Td over irrigated areas. For example, average growing-season Td due to irrigation can be up to 1.56°C higher relative to nonirrigated land uses. It is also found that Td for individual growing-season month at irrigated locations can be increased up to 2.17°C by irrigation. Based on the results, it is concluded that the land use change in the Great Plains has modified near-surface moistness

Expression of biopterin transporter (BT1) protein in Leishmania

The present work focuses on the growth phase regulated expression of biopterin transporter gene (BT1) from the LD1 locus on chromosome 35 of Leishmania donovani. Antiserum against recombinant BT1 detected a polypeptide of 45 kDa of equal intensity at lag, log and stationary phases of promastigote growth, both in L. donovani strain LSB-7.1 (MHOM/BL/67/ITMAP263), and strain LSB-146.1 (HOM/IR/95/X81), a natural isolate from Isfehan, Iran that caused cutaneous leishmaniasis. However, in both these strains an additional polypeptide of higher molecular mass (50 kDa) was also observed during lag phase only. In addition, polypeptides of 40, 20, 18 and 16 kDa were seen only during the lag and log phases of both strains. Analysis of L. donovani single, double and triple (null) BT1 knockout mutants confirmed that the 45-kDa polypeptide was the BT1 gene product, as it was absent in the null mutant. These results indicate that 45-kDa BT1 protein in Leishmania is consistently and constitutively expressed in all the growth stages of the parasite

Role of biopterin transporter (BT1) gene on growth and infectivity of Leishmania

Leishmania are known to be auxotrophic for pteridines that are known to play a critical role in the parasites survival. In the present work the role of biopterin transporter in the growth of the parasite and infectivity in to macrophages has been worked out. The role of biopterin transporter in the susceptibility of Leishmania to antimonial compounds has also been demonstrated. This role has been verified by using attenuated strains of Leishmania with single, double, and triple (null) biopterin (BT1) mutants made by targeted gene replacement with specific antibiotic markers. Growth analysis of these mutants revealed that wild type, single and double knock out cell lines maintained high growth rates in the medium supplemented with biopterin and folate, whereas the triple knock out or null BT1 mutants were unable to grow in the absence of supplemental biopterin. Using wild type and null BT1 mutants, we examined the role of BT1 gene in infectivity and parasite survival. The cell lines with amplified BT1 gene showed increased infectivity and survival in the macrophages where as the cell lines with disrupted BT1 gene showed reduced infectivity and survival in the macrophages. We also examined the interaction between pteridine and antimonial compounds using recombinant Leishmania strains with reduced or absent biopterin transporter gene (BT1) alleles. No difference in susceptibility to Pentostam or Glucantime was observed in both wild type and BT1-knock out strains. However, pterin or folate supplementation resulted in reversal of Glucantime but not Pentostam susceptibility in both wild type and BT1-knock out strains. The reversal of Glucantime susceptibility by pterins in BT1-knock out strains suggests that the effect may be exerted independently of biopterin transporter, possibly by blocking Glucantime uptake

Tissue Doppler imaging following paediatric cardiac surgery : early patterns of change and relationship to outcome

In this study, tissue Doppler imaging (TDI) was used to assess changes in ventricular function following repair of congenital heart defects. The relationship between TDI indices, myocardial injury and clinical outcome was explored. Forty-five children were studied; 35 withcardiac lesions and 10 controls. TDI was performed preoperatively, on admission to paediatric intensive care unit (PICU) and day 1. Regional myocardial Doppler signals were acquired from the right ventricle (RV), left ventricle (LV) and septum. TDI indices included: peak systolicvelocities, isovolumetric velocities (IVV) and isovolumetric acceleration (IVA). Preoperatively, bi-ventricular TDI velocities in the study groupwere reduced compared with normal controls. Postoperatively, RV velocities were significantly reduced and this persisted to day-1 (PreOp vs. PICU and day-1: 7.7+2.2 vs. 3.4+1.0, P < 0.0001 and 3.55+1.29, P < 0.0001). LV velocities initially declined but recovered towards baseline by day-1 (PreOp vs. PICU: 5.31+1.50 vs. 3.51+1.23, P < 0.0001). Isovolumetric parameters in all regions were reduced throughout the postoperative period. Troponin-I release correlated with longer X-clamp times (r=0.82, P < 0.0001) and reduced RV velocities (r=0.42, P=0.028). Reduced pre- and postoperative LV velocities correlated with longer ventilation (PreOp: r=0.54, P=0.002; PostOp: r=0.42, P=0.026). This study identified reduced postoperative RV velocities correlated with myocardial injury while reduced LV TDI correlated with longer postoperative ventilation

The Implementation of a Terminal Master’s Program in Applied Economics

This paper examines the impacts of transforming a traditional master’s program into a professionally-oriented, terminal degree program in applied economics. Alumni surveys were conducted before and after the curricular innovation and the results indicate that the change in program provided high quality training that alumni judged to be more relevant to the post-graduate work environment. This case study illustrates that master’s programs in economics can benefit by implementing curricular structures that explicitly serve terminal degree-seeking students while, at the same time, provide elements of training that students destined for the Ph. D. are unlikely to receive in a doctoral program

Russian Missile Technology and Nuclear Reactor Transfers to Iran

Many in Congress and the Clinton Administration charge that Russian entities are assisting Iran in developing ballistic missiles. Russia is also building a nuclear power station and is finishing other nuclear services to, Iran. Congress has passed legislation requiring the President to impose sanctions for missile technology transfers, arms sales, nuclear technology transfers, and large-scale investments in Iran. H.R 2709, which includes the "Iran Missile Proliferation Sanctions Act of 1997," is one of several bills designed to tighten existing sanctions law. It was amended and passed by the Senate on May 22, 1998, and by the House on June 9 by very large bipartisan majorities. Nevertheless, President Clinton vetoed the bill on June 23 and said he would work to sustain the veto

Russian Missile Technology and Nuclear Reactor Transfers to Iran

Many in Congress and the Clinton Administration charge that Russian entities are assisting Iran in developing ballistic missiles. Russia is also building a nuclear power station and is finishing other nuclear services to, Iran. Congress has passed legislation requiring the President to impose sanctions for missile technology transfers, arms sales, nuclear technology transfers, and large-scale investments in Iran. H.R 2709, which includes the "Iran Missile Proliferation Sanctions Act of 1997," is one of several bills designed to tighten existing sanctions law. It was amended and passed by the Senate on May 22, 1998, and by the House on June 9 by very large bipartisan majorities. Nevertheless, President Clinton vetoed the bill on June 23 and said he would work to sustain the veto