PCDAS Version 2. 2: Remote network control and data acquisition

This manual is intended for both technical and non-technical people who want to use the PCDAS remote network control and data acquisition software. If you are unfamiliar with remote data collection hardware systems designed at Pacific Northwest Laboratory (PNL), this introduction should answer your basic questions. Even if you have some experience with the PNL-designed Field Data Acquisition Systems (FDAS), it would be wise to review this material before attempting to set up a network. This manual was written based on the assumption that you have a rudimentary understanding of personal computer (PC) operations using Disk Operating System (DOS) version 2.0 or greater (IBM 1984). You should know how to create subdirectories and get around the subdirectory tree

Impact evaluation of an energy savings plan project at Bellingham Cold Storage

This impact evaluation of an energy conservation measure (ECM) that was recently installed at Bellingham Cold Storage (BCS) was conducted for the Bonneville Power Administration (Bonneville) as part of an evaluation of its Energy Savings Plan (ESP) Program. The Program awards cash incentives to firms that install energy conservation measures in their industrial processes. The objective of this impact evaluation was to assess how much electrical energy is being saved at BCS as a result of the ESP and to determine how much savings cost Bonneville and the region. The impact of the ECM was evaluated with a combination of engineering analysis, financial analysis, interviews, submittal reviews (BCS's Completion Report, Proposal, and Abstract), and process evaluation reviews. The ECM itself consists of an energy management and control system that is used to manage energy consumption by a large refrigeration system at BCS's Orchard Drive facility in Bellingham, Washington. At this facility, BCS freezes and stores fruits, berries, and fish products, while two tenants process frozen fish products. Energy savings resulting from this ECM are expected to be at least 1,094, 402 kWh during the first year (a savings of 23%) with greater savings in subsequent years. The ECM cost BCS $169,300 to install, Bonneville paid an incentive to BCS of$65,100, and the local utility paid an additional incentive of $21,700. The levelized cost of these savings to Bonneville will be no greater than 5.0 mills/kWh over the ECM's expected 15-year life, and the cost to the region will be no greater than 12.5 mills/kWh. It is expected that this ECM would have been installed within 3 years even without the incentive from Bonneville, and the levelized cost to Bonneville for just the first three years' savings is 21.03 mills/kWh or less. 3 tabs

Humoral protection against mosquito bite-transmitted Plasmodium falciparum infection in humanized mice

Contains fulltext : 189906.pdf (publisher's version ) (Open Access

Correlative light-electron microscopy methods to characterize the ultrastructural features of the replicative and dormant liver stages of Plasmodium parasites

Abstract Background The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. Results The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples’ features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. Conclusions A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi. Graphical Abstrac

A rapid and scalable density gradient purification method for <it>Plasmodium</it> sporozoites

Abstract Background Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes. Methods Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants. Results This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates. Conclusions This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.</p

PfCap380 as a marker for Plasmodium falciparum oocyst development in vivo and in vitro

Abstract Background Despite the importance of the Plasmodium berghei oocyst capsule protein (PbCap380) in parasite survival, very little is known about the orthologous Plasmodium falciparum capsule protein (PfCap380). The goal of this work was to study the growth of P. falciparum oocysts using PfCap380 as a developmental marker. Methods To study P. falciparum oocyst development using both in vivo (mosquito-derived) and in vitro (culture-derived) growth conditions, antibodies (polyclonal antisera) were raised against PfCap380. For studies on in vivo oocysts, mature P. falciparum gametocytes were fed to Anopheles stephensi mosquitoes. For studies on in vitro parasites, P. falciparum gametocytes were induced and matured for subsequent ookinete production. Ookinetes were purified and then tested for binding affinity to basal lamina components and transformation into early oocysts, which were grown on reconstituted basal lamia coated wells with novel oocyst media. To monitor in vivo oocyst development, immunofluorescence assays (IFA) were performed using anti-PfCap380 antisera on Pf-infected mosquito midguts. IFA were also performed on culture-derived oocysts to follow in vitro oocyst development. Results The anti-PfCap380 antisera allowed detection of early midgut oocysts starting at 2 days after gametocyte infection, while circumsporozoite protein was definitively observed on day 6. For in vitro culture, significant transformation of gametocytes to ookinetes (24%) and of ookinetes to early oocysts (85%) was observed. After screening several basal lamina components, collagen IV provided greatest binding of ookinetes and transformation into early oocysts. Finally, PfCap380 expression was observed on the surface of culture-derived oocysts but not on gametocytes or ookinetes. Conclusions This study presents developmental monitoring of P. falciparum oocysts produced in vivo and in vitro. The anti-PfCap380 antisera serves as an important reagent for developmental studies of oocysts from the mosquito midgut and also from oocyst culture using in vitro methodology. The present data demonstrate that PfCap380 is a useful marker to follow the development and maturation of in vivo and in vitro produced oocysts as early as 2 days after zygote formation. Further in vitro studies focused on oocyst and sporozoite maturation will support the manufacturing of whole sporozoites for malaria vaccines

Plasmodium falciparum liver stage infection to stable blood stage infection in liver-humanized and blood-humanized FRGN KO mice enables testing of blood stage inhibitory antibodies (reticulocyte-binding protein homolog 5) in vivo

The invention of liver-humanized mouse models has made it possible to directly study the preerythrocytic stages ofPlasmodium falciparum. In contrast, the current models to directly study blood stage infectionin vivoare extremely limited. Humanization of the mouse blood stream is achievable by frequent injections of human red blood cells (hRBCs) and is currently the only system with which to study human malaria blood stage infections in a small animal model. Infections have been primarily achieved by direct injection ofP. falciparum-infected RBCs but as such, this modality of infection does not model the natural route of infection by mosquito bite and lacks the transition of parasites from liver stage infection to blood stage infection. Including these life cycle transition points in a small animal model is of relevance for testing therapeutic interventions. To this end, we used FRGN KO mice that were engrafted with human hepatocytes and performed a blood exchange under immune modulation to engraft the animals with more than 50% hRBCs. These mice were infected by mosquito bite with sporozoite stages of a luciferase-expressingP. falciparumparasite, resulting in noninvasively measurable liver stage burden byin vivobioluminescent imaging (IVIS) at days 5-7 postinfection. Transition to blood stage infection was observed by IVIS from day 8 onward and then blood stage parasitemia increased with a kinetic similar to that observed in controlled human malaria infection. To assess the utility of this model, we tested whether a monoclonal antibody targeting the erythrocyte invasion ligand reticulocyte-binding protein homolog 5 (with known growth inhibitory activityin vitro) was capable of blocking blood stage infectionin vivowhen parasites emerge from the liver and found it highly effective. Together, these results show that a combined liver-humanized and blood-humanized FRGN mouse model infected with luciferase-expressingP. falciparumwill be a useful tool to studyP. falciparumpreerythrocytic and erythrocytic stages and enables the testing of interventions that target either one or both stages of parasite infection

Plasmodium falciparum liver stage infection to stable blood stage infection in liver-humanized and blood-humanized FRGN KO mice enables testing of blood stage inhibitory antibodies (reticulocyte-binding protein homolog 5) in vivo

The invention of liver-humanized mouse models has made it possible to directly study the preerythrocytic stages ofPlasmodium falciparum. In contrast, the current models to directly study blood stage infectionin vivoare extremely limited. Humanization of the mouse blood stream is achievable by frequent injections of human red blood cells (hRBCs) and is currently the only system with which to study human malaria blood stage infections in a small animal model. Infections have been primarily achieved by direct injection ofP. falciparum-infected RBCs but as such, this modality of infection does not model the natural route of infection by mosquito bite and lacks the transition of parasites from liver stage infection to blood stage infection. Including these life cycle transition points in a small animal model is of relevance for testing therapeutic interventions. To this end, we used FRGN KO mice that were engrafted with human hepatocytes and performed a blood exchange under immune modulation to engraft the animals with more than 50% hRBCs. These mice were infected by mosquito bite with sporozoite stages of a luciferase-expressingP. falciparumparasite, resulting in noninvasively measurable liver stage burden byin vivobioluminescent imaging (IVIS) at days 5-7 postinfection. Transition to blood stage infection was observed by IVIS from day 8 onward and then blood stage parasitemia increased with a kinetic similar to that observed in controlled human malaria infection. To assess the utility of this model, we tested whether a monoclonal antibody targeting the erythrocyte invasion ligand reticulocyte-binding protein homolog 5 (with known growth inhibitory activityin vitro) was capable of blocking blood stage infectionin vivowhen parasites emerge from the liver and found it highly effective. Together, these results show that a combined liver-humanized and blood-humanized FRGN mouse model infected with luciferase-expressingP. falciparumwill be a useful tool to studyP. falciparumpreerythrocytic and erythrocytic stages and enables the testing of interventions that target either one or both stages of parasite infection

Multiplex, DNase-free one-step reverse transcription PCR for Plasmodium 18S rRNA and spliced gametocyte-specific mRNAs

Plasmodium gametocytes are sexual stages transmitted to female Anopheles mosquitoes. While Plasmodium parasites can be differentiated microscopically on Giemsa-stained blood smears, molecular methods are increasingly used because of their increased sensitivity. Molecular detection of gametocytes requires methods that discriminate between asexual and sexual stage parasites. Commonly tested gametocyte-specific mRNAs are pfs25 and pfs230 detected by reverse transcription polymerase chain reaction (RT-PCR). However, detection of these unspliced mRNA targets requires preceding DNase treatment of nucleic acids to eliminate co-purified genomic DNA. If gametocyte-specific, spliced mRNAs could be identified, DNase treatment could be eliminated and one-step multiplexed molecular methods utilized. Expression data was used to identify highly-expressed mRNAs in mature gametocytes that were also low in antisense RNA expression in non-gametocyte stages. After testing numerous candidate mRNAs, the spliced female Pf3D7_0630000 mRNA was selected as a Plasmodium falciparum gametocyte-specific biomarker compatible with Plasmodium 18S rRNA RT-PCR. This mRNA was only detected in samples containing mature gametocytes and was absent in those containing only asexual stage parasites or uninfected human blood. PF3D7_0630000 RT-PCR detected gametocytes across a wide range of parasite densities in both spiked and clinical samples and agreed with pfs25 RT-PCR, the gold standard for RT-PCR-based gametocyte detection. PF3D7_0630000 multiplexed with Plasmodium 18S rRNA RT-PCR was more sensitive than other spliced mRNA targets for one-step RT-PCR gametocyte detection. Because the spliced target does not require DNase treatment, the PF3D7_0630000 assay can be multiplexed with Plasmodium 18S rRNA for direct one-step detection of gametocytes from whole human blood