Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality

A Plasmodium falciparum hexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevis oocytes and the use of a glucose analogue inhibitor permitted chemical validation of PfHT as a novel drug target. Following recent re-annotations of the P. falciparum genome, other putative sugar transporters have been identified. To investigate further if PfHT is the key supplier of hexose to P. falciparum and to extend studies to different stages of Plasmodium spp., we functionally analysed the hexose transporters of both the human parasite P. falciparum and the rodent parasite Plasmodium berghei using gene targeting strategies. We show here the essential function of pfht for the erythrocytic parasite growth as it was not possible to knockout pfht unless the gene was complemented by an episomal construct. Also, we show that parasites are rescued from the toxic effect of a glucose analogue inhibitor when pfht is overexpressed in these transfectants. We found that the rodent malaria parasite orthologue, P. berghei hexose transporter (PbHT) gene, was similarly refractory to knockout attempts. However, using a single cross-over transfection strategy, we generated transgenic P. berghei parasites expressing a PbHT–GFP fusion protein suggesting that locus is amenable for gene targeting. Analysis of pbht-gfp transgenic parasites showed that PbHT is constitutively expressed through all the stages in the mosquito host in addition to asexual stages. These results provide genetic support for prioritizing PfHT as a target for novel antimalarials that can inhibit glucose uptake and kill parasites, as well as unveiling the expression of this hexose transporter in mosquito stages of the parasite, where it is also likely to be critical for survival

The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation

<b>Background</b> Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. <b>Methods:</b> The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. <b>Results:</b> The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. <b>Conclusions:</b> The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation

Impact of Beta-Amyloid-Specific Florbetaben PET Imaging on Confidence in Early Diagnosis of Alzheimer’s Disease

BACKGROUND: Early diagnosis of Alzheimer's disease (AD) may be corroborated by imaging of beta-amyloid plaques using positron emission tomography (PET). Here, we performed an add-on questionnaire study to evaluate the relevance of florbetaben imaging (BAY 949172) in diagnosis and consecutive management of probable AD patients. METHODS: AD patients with a clinical diagnosis in accordance with the NINCDS-ADRDA criteria or controls were imaged using florbetaben. Referring physicians were asked on a voluntary basis about their confidence in initial diagnosis, significance of PET imaging results, and their anticipated consequences for future patient care. RESULTS: 121 questionnaires for probable AD patients and 80 questionnaires for controls were evaluated. In 18% of patients who had initially received the diagnosis of probable AD, PET scans were rated negative, whereas in controls 18% of scans were positive. An increase in confidence in the initial diagnosis was frequently reported (80%). Imaging results had a significant impact on the intended patient care, as judged by the referring physicians; this was most prominent in those patients with a contradicting scan and/or a low confidence in the initial diagnosis. CONCLUSION: Florbetaben amyloid imaging increases the overall confidence in diagnosis of AD and may frequently influence clinical decisions and patient management

Toxoplasma and Plasmodium protein kinases: roles in invasion and host cell remodelling

Some apicomplexan parasites have evolved distinct protein kinase families to modulate host cell structure and function. Toxoplasma gondii rhoptry protein kinases and pseudokinases are involved in virulence and modulation of host cell signalling. The proteome of Plasmodium falciparum contains a family of putative kinases called FIKKs, some of which are exported to the host red blood cell and might play a role in erythrocyte remodelling. In this review we will discuss kinases known to be critical for host cell invasion, intracellular growth and egress, focusing on (i) calcium-dependent protein kinases and (ii) the secreted kinases that are unique to Toxoplasma (rhoptry protein kinases and pseudokinases) and Plasmodium (FIKKs)

MERLIN - A meV resolution beamline at the ALS

An ultra-high resolution beamline is being constructed at the Advanced Light Source (ALS) for the study of low energy excitations in strongly correlated systems with the use of high-resolution inelastic scattering and angle-resolved photoemission. This new beamline, given the acronym Merlin (for meV resolution line), will cover the energy range 10-150 eV. The monochromator has fixed entrance and exit slits and a plane mirror that can illuminate a spherical grating at the required angle of incidence (as in the SX-700 mechanism). The monochromator can be operated in two different modes. In the highest resolution mode, the energy scanning requires translating the monochromator chamber (total travel 1.1 m) as well as rotating the grating and the plane mirror in front of the grating. The resolution in this mode is practically determined by the slits width. In the second mode, the scanning requires rotating the grating and the plane mirror. This mode can be used to scan a few eV without a significant resolution loss. The source for the beamline is a 1.9 m long, 90 mm period quasi periodic EPU. The expected flux at the sample is higher than 10 photons/s at a resolving power of 5 × 10 in the energy range 16-130 eV. A second set of gratings can be used to obtain higher flux at the expense of resolution. © 2007 American Institute of Physics. 11

Scaling a Community-Wide Campaign Intervention to Manage Hypertension and Weight Loss

Public health impacts can be achieved when evidence-based interventions are implemented to those most in need. Too often implementation never or slowly occurs. The community-wide campaign intervention Tu Salud ¡Si Cuenta! has evidence of improving health outcomes related to chronic disease among low-income, Latinos. Using the RE-AIM Framework, this study examined if the scaled-up version of the intervention is associated with improvements in hypertension and obesity in 12 locations. Each element of the RE-AIM framework was examined. For Effectiveness, we examined outcomes overall and by implementing location. We used linear and logistic regression to assess if exposure in the intervention was associated with improvement in hypertension and weight loss. Participants were stratified into low exposure (2-3 outreach visits) vs. high exposure (4-5 outreach visits). Based on the RE-AIM Framework, the intervention reached its intended population of low-income Latinos, demonstrated effectiveness in improving hypertension and obesity, was adopted at a high level in all but one site, was implemented with fidelity to the intervention model with moderate success across locations, and showed high maintenance over time. For effectiveness specifically, we found that out of 5,019 participants, 2,508 (50%) had a baseline hypertensive blood pressure (BP) reading. Of the 2,508, 1,245 (49.9%) recovered to normal blood pressure or pre-hypertension stage by last follow-up. After adjusting for baseline BP and potential confounders in multivariable linear regression models, the high exposure group had significantly more reduction in systolic BP (adjusted mean difference in % change = -0.96; p = 0.002) and diastolic BP (adjusted mean difference in % change = -1.61; p \u3c 0.0001) compared to the low exposure group. After controlling for baseline weight and other confounders, the high exposure group had significantly greater decrease in weight compared to the low exposure group (adjusted mean difference in % change = -1.28; p \u3c 0.0001). Results from the multivariable logistic regression models indicated that compared to the low exposure group the high exposure group was more likely to achieve a clinically significant minimum 5% weight loss [adjusted odds ratio (OR) = 2.97; p \u3c 0.0001). This study contributes evidence that a Community-Wide Campaign model holds promise for addressing hypertension and obesity among low-income Latinos

Evaluating the Dissemination and Implementation of a Community Health Worker-Based Community Wide Campaign to Improve Fruit and Vegetable Intake and Physical Activity among Latinos along the U.S.-Mexico Border

This study evaluated the dissemination and implementation of a culturally tailored community-wide campaign (CWC), Tu Salud ¡Si Cuenta! (TSSC), to augment fruit and vegetable (FV) consumption and physical activity (PA) engagement among low-income Latinos of Mexican descent living along the U.S.-Mexico Border in Texas. TSSC used longitudinal community health worker (CHW) home visits as a core vehicle to enact positive change across all socioecological levels to induce behavioral change. TSSC’s reach, effectiveness, adoption, implementation, and maintenance (RE-AIM) was examined. A dietary questionnaire and the Godin-Shepherd Exercise Questionnaire measured program effectiveness on mean daily FV consumption and weekly PA engagement, respectively. Participants were classified based on CHW home visits into “low exposure” (2–3 visits) and “high exposure” (4–5 visits) groups. The TSSC program reached low-income Latinos (n = 5686) across twelve locations. TSSC demonstrated effectiveness as, compared to the low exposure group, the high exposure group had a greater FV intake (mean difference = +0.65 FV servings daily, 95% CI: 0.53–0.77) and an increased PA (mean difference = +185.6 MET-minutes weekly, 95% CI: 105.9–265.4) from baseline to the last follow-up on a multivariable linear regression analysis. Multivariable logistic regression revealed that the high exposure group had higher odds of meeting both FV guidelines (adjusted odds ratio (AOR) = 2.03, 95% CI: 1.65–2.47) and PA guidelines (AOR = 1.36, 95% CI: 1.10–1.68) at the last follow-up. The program had a 92.3% adoption rate, with 58.3% of adopting communities meeting implementation fidelity, and 91.7% of communities maintaining TSSC. TSSC improved FV consumption and PA engagement behaviors among low-income Latinos region wide. CHW delivery and implementation funding positively influenced reach, effectiveness, adoption, and maintenance, while lack of qualified CHWs negatively impacted fidelit

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Background: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. Results: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. Conclusions: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2. © 2016 The Author(s)

Measurement of single electron emission in two-phase xenon

We present the first measurements of the electroluminescence response to the emission of single electrons in a two-phase noble gas detector. Single ionization electrons generated in liquid xenon are detected in a thin gas layer during the 31-day background run of the ZEPLIN-II experiment, a two-phase xenon detector for WIMP dark matter searches. Both the pressure dependence and magnitude of the single-electron response are in agreement with previous measurements of electroluminescence yield in xenon. We discuss different photoionization processes as possible cause for the sample of single electrons studied in this work. This observation may have implications for the design and operation of future large-scale two-phase systems.Comment: 11 pages, 6 figure

The Emergence of Population Health in US Academic Medicine

Importance In response to rapidly growing interest in population health, academic medical centers are launching department-level initiatives that focus on this evolving discipline. This trend, with its potential to extend the scope of academic medicine, has not been well characterized. Objective To describe the emergence of departments of population health at academic medical centers in the United States, including shared areas of focus, opportunities, and challenges. Design, Setting, and Participants This qualitative study was based on a structured in-person convening of a working group of chairs of population health–oriented departments on November 13 and 14, 2017, complemented by a survey of core characteristics of these and additional departments identified through web-based review of US academic medical centers. United States medical school departments with the word population in their name were included. Centers, institutes, and schools were not included. Main Outcomes and Measures Departments were characterized by year of origin, areas of focus, organizational structure, faculty size, teaching programs, and service engagement. Opportunities and challenges faced by these emerging departments were grouped thematically and described. Results Eight of 9 population health–oriented departments in the working group were launched in the last 6 years. The 9 departments had 5 to 97 full-time faculty. Despite varied organizational structures, all addressed essential areas of focus spanning the missions of research, education, and service. Departments varied significantly in their relationships with the delivery of clinical care, but all engaged in practice-based and/or community collaboration. Common attributes include core attention to population health–oriented research methods across disciplines, emphasis on applied research in frontline settings, strong commitment to partnership, interest in engaging other sectors, and focus on improving health equity. Tensions included defining boundaries with other academic units with overlapping areas of focus, identifying sources of sustainable extramural funding, and facilitating the interface between research and health system operations. Conclusions and Relevance Departments addressing population health are emerging rapidly in academic medical centers. In supporting this new framing, academic medicine affirms and strengthens its commitment to advancing population health and health equity, to improving the quality and effectiveness of care, and to upholding the social mission of medicine