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At least 1,369 records · Page 76

System and method for measuring fluorescence of a sample

The present disclosure provides a system and a method for measuring fluorescence of a sample. The sample may be a polymerase-chain-reaction (PCR) array, a loop-mediated-isothermal amplification array, etc. LEDs are used to excite the sample, and a photodiode is used to collect the sample's fluorescence. An electronic offset signal is used to reduce the effects of background fluorescence and the noises from the measurement system. An integrator integrates the difference between the output of the photodiode and the electronic offset signal over a given period of time. The resulting integral is then converted into digital domain for further processing and storage.

47 OTHER INSTRUMENTATION↗

Clinical features and risk factors for adverse outcome in Ebola virus disease in Moyamba District Sierra Leone.

BACKGROUND The current outbreak of Ebola virus disease (EVD) in West Africa has attacked 24000 people, killed more than 10000 and disrupted social life. METHODS We studied retrospectively the clinical presentation and risk factors for fatal outcome in EVD among all patients admitted to the Ebola Treatment Center in Moyamba District, Sierra Leone. RESULTS Among a total of 88 admitted patients, eighty-two were tested by PCR and 31 (38%) were positive for Ebola virus. Ninety percent reported previous contact with EVD patients and 35% had participated in burials of EVD suspect deceased. No healthworkers were admitted. The most common symptoms on admission were weakness (97%), diarrhea (68%), fever (62%), loss of appetite (62%), vomiting (58%), pain in muscles (62%) and joints (55%), headache (55%), abdominal pain (45%) and conjunctivitis (42%). On admission, bleeding was present in one-third (11/31), while more than half (17/31) bled during the hospital stay. Fifty-eight percent (18/31) died, most within 4-11 days of onset. Significant predictors for fatal outcome were shorter time from onset to admission (P=0.02), high initial viral load (P<0.001), bleeding (P=0.004), and severe pain (P=0.001). The only two patients with hiccups died. CONCLUSIONS Bleeding was more common in our cohort than reported elsewhere during this epidemic, and predicted poor prognosis. Severe pain was common, particularly in fatal cases, and calls for improved and safe palliation, for instance with transdermal opiates. The lack of fever in one third of EBV cases may have implications for screening procedures and case definitions.

Haaskjold, Yngvar Lunde↗

Integrated solid-state rapid thermo-cycling system

Techniques, systems, and devices are disclosed for implementing a portable lab system for PCR testing. The portable lab system comprises a thermal cycling device including a first well and a second well to receive samples to be thermally cycled, a thermoelectric cooling (TEC) element coupled to the first well and the second well, and a controller to control operation of the TEC element. The portable lab system further includes an electronic interface including a power interface to supply power to the TEC element and the controller of the thermal cycling device to allow the TEC element to transfer energy from the first well to the second well when current flows through the TEC element in a first direction, and from the second well to the first well when current flows through the TEC element in a second direction, and a data interface to collect data from the controller.

59 BASIC BIOLOGICAL SCIENCES↗

In-Situ Testing and Performance Assessment of a Redesigned WIPP Panel Closure - 13192

There are two primary regulatory requirements for Panel Closures at the Waste Isolation Pilot Plant (WIPP), the nation's only deep geologic repository for defense related Transuranic (TRU) and Mixed TRU waste. The Federal requirement is through 40 CFR 191 and 194, promulgated by the U.S. Environmental Protection Agency (EPA). The state requirement is regulated through the authority of the Secretary of the New Mexico Environment Department (NMED) under the New Mexico Hazardous Waste Act (HWA), New Mexico Statutes Annotated (NMSA) 1978, chap. 74-4-1 through 74-4-14, in accordance with the New Mexico Hazardous Waste Management Regulations (HWMR), 20.4.1 New Mexico Annotated Code (NMAC). The state regulations are implemented for the operational period of waste emplacement plus 30 years whereas the federal requirements are implemented from the operational period through 10,000 years. The 10,000 year federal requirement is related to the adequate representation of the panel closures in determining long-term performance of the repository. In Condition 1 of the Final Certification Rulemaking for 40 CFR Part 194, the EPA required a specific design for the panel closure system. The U.S. Department of Energy (DOE) Carlsbad Field Office (CBFO) has requested, through the Planned Change Request (PCR) process, that the EPA modify Condition 1 via its rulemaking process. The DOE has also requested, through the Permit Modification Request (PMR) process, that the NMED modify the approved panel closure system specified in Permit Attachment G1. The WIPP facility is carved out of a bedded salt formation 655 meters below the surface of southeast New Mexico. Condition 1 of the Final Certification Rulemaking specifies that the waste panels be closed using Option D which is a combination of a Salado mass concrete (SMC) monolith and an isolation/explosion block wall. The Option D design was also accepted as the panel closure of choice by the NMED. After twelve years of waste handling operations and a greater understanding of the waste and the behavior of the underground salt formation, the DOE has established a revised panel closure design. This revised design meets both the short-term NMED Permit requirements for the operational period, and also the Federal requirements for long-term repository performance. This new design is simpler, easier to construct and has less of an adverse impact on waste disposal operations than the originally approved Option D design. The Panel Closure Redesign is based on: (1) the results of in-situ constructability testing performed to determine run-of-mine salt reconsolidation parameters and how the characteristics of the bedded salt formation affect these parameters and, (2) the results of air flow analysis of the new design to determine that the limit for the migration of Volatile Organic Compounds (VOCs) will be met at the compliance point. Waste panel closures comprise a repository feature that has been represented in WIPP performance assessment (PA) since the original Compliance Certification Application of 1996. Panel closures are included in WIPP PA models principally because they are a part of the disposal system, not because they play a substantive role in inhibiting the release of radionuclides to the outside environment. The 1998 rulemaking that certified WIPP to receive transuranic waste placed conditions on the panel closure design to be implemented in the repository. The revised panel closure design, termed the Run-of-Mine (ROM) Panel Closure System (ROMPCS), is comprised of 30.48 meters of ROM salt with barriers at each end. The ROM salt is generated from ongoing mining operations at the WIPP and may be compacted and/or moistened as it is emplaced in a panel entry. The barriers consist of bulkheads, similar to those currently used in the panels as room closures. A WIPP performance assessment has been completed that incorporates the ROMPCS design into the representation of the repository, and compares repository performance to that achieved with the approved Option D design. Several key physical processes and rock mechanics principles are incorporated into the performance assessment. First, creep closure of the salt rock surrounding a panel entry results in consolidation of the ROM salt emplaced in the entry. Eventually, the ROM salt comprising the ROMPCS will approach a condition similar to intact salt. As the ROM salt reaches higher fractional densities during consolidation, back stress will be imposed on the surrounding rock mass leading to eventual healing of the disturbed rock zone above and below the panel closure. Healing of the disturbed rock zone above and below the ROMPCS reduces the porosity and permeability in those areas. Analysis of the new design demonstrates that: (1) the WIPP continues to meet regulatory compliance requirements when the ROMPCS design is implemented instead of Option D, and (2) there is no impact on the short-term effectiveness of the panel closure to limit the concentration of VOCs at the WIPP site boundary to a fraction of the health-based exposure limits (HBLs) during the operational period. (authors)

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Integrated Systems-Based Approach for Reaching Acceptable End Points for Groundwater - 13629

The sheer mass and nature of contaminated materials at DOE and DoD sites, makes it impractical to completely restore these sites to pre-disposal conditions. DOE faces long-term challenges, particularly with developing monitoring and end state approaches for clean-up that are protective of the environment, technically based and documented, sustainable, and most importantly cost effective. Integrated systems-based monitoring approaches (e.g., tools for characterization and monitoring, multi-component strategies, geophysical modeling) could provide novel approaches and a framework to (a) define risk-informed endpoints and/or conditions that constitute completion of cleanup and (b) provide the understanding for implementation of advanced scientific approaches to meet cleanup goals. Multi-component strategies which combine site conceptual models, biological, chemical, and physical remediation strategies, as well as iterative review and optimization have proven successful at several DOE sites. Novel tools such as enzyme probes and quantitative PCR for DNA and RNA, and innovative modeling approaches for complex subsurface environments, have been successful at facilitating the reduced operation or shutdown of pump and treat facilities and transition of clean-up activities into monitored natural attenuation remedies. Integrating novel tools with site conceptual models and other lines of evidence to characterize, optimize, and monitor long term remedial approaches for complex contaminant plumes are critical for transitioning active remediation into cost effective, yet technically defensible endpoint strategies. (authors)

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Integrated solid-state rapid thermo-cycling system

Techniques, systems, and devices are disclosed for implementing a portable lab system for PCR testing. An example method for operating an integrated thermal cycling system includes depositing samples into the integrated thermal cycling system that includes a thermal cycling device and an electronic interface. The thermal cycling device includes multiple wells to receive the samples to be thermally cycled, a thermoelectric cooling (TEC) element connected to the multiple wells, a substrate on which the TEC element is positioned, and a controller coupled to the TEC element. The multiple wells are positioned within the substrate that includes a thermally conductive ground positioned between adjacent wells. Supplying power to the integrated thermal cycling system, via the electronic interface, allows the multiple wells to exchange heat with the substrate and for each well to operate independently from other wells.

Beer, N. Reginald↗

Detection of phosphate transporter genes from arbuscular mycorrhizal fungi in mature tree roots under experimental soil pH manipulation

We present the majority of terrestrial plant roots are colonized by arbuscular mycorrhizal (AM) fungi that, in exchange for carbon, provide plants with enhanced nutrient uptake — most notably inorganic phosphate (P i ). To mediate the uptake of Pi from the soil, AM fungi possess high affinity inorganic phosphate transporters (PTs). Under laboratory conditions, P i concentrations have been shown to regulate AM fungal-specific PT gene expression. The relationship between PT expression and P i in the field remains unexplored. Here we quantify AM fungal-specific PTs from maple tree roots in situ. In an effort to limit edaphic parameters, root samples were collected from manipulated forested plots that had elevated soil P i availability, either through direct P i application or elevating pH to lower exchangeable aluminum. The aim of the study was to examine AM fungal-specific PT gene expression both prior to and following soil P i amendment; however, a direct correlation between soil P i concentration and PT gene expression was not observed. PT transcripts were detected to a greater extent under elevated pH and, while our results are confounded by an overall low detection of PT genes (23 % of all samples collected), our findings raise interesting questions regarding the role of soil pH on PT function. In conclusion, our study is a first step in understanding how edaphic properties influence PT expression and plant P acquisition in mature tree roots.

54 ENVIRONMENTAL SCIENCES↗

Comparison of pectin-degrading fungal communities in temperate forests using glycosyl hydrolase family 28 pectinase primers targeting Ascomycete fungi

Here, fungi have developed a wide assortment of enzymes to break down pectin, a prevalent polymer in plant cell walls that is important in plant defense and structure. One enzyme family used to degrade pectin is the glycosyl hydrolase family 28 (GH28). In this studywe developed primers for the amplification of GH28 coding genes from a database of 293 GH28 sequences from40 fungal genomes. The primerswere used to successfully amplify GH28 pectinases from all Ascomycota cultures tested, but only three out of seven Basidiomycota cultures. In addition, we further tested the primers in PCRs on metagenomic DNA extracted from senesced tree leaves from different forest ecosystems, followed by cloning and sequencing. Taxonomic specificity for Ascomycota GH28 genes was tested by comparing GH28 composition in leaves to internal transcribed spacer (ITS) amplicon composition using pyrosequencing. All sequences obtained from GH28 primers were classified as Ascomycota; in contrast, ITS sequences indicated that fungal communitieswere up to 39% Basidiomycetes. Analysis of leaf samples indicated that both forest stand and ecosystemtype were important in structuring fungal communities. However, site played the prominent role in explaining GH28 composition, whereas ecosystem type was more important for ITS composition, indicating possible genetic drift between populations of fungi. Overall, these primers will have utility in understanding relationships between fungal community composition and ecosystem processes, as well as detection of potentially pathogenic Ascomycetes.

59 BASIC BIOLOGICAL SCIENCES↗

Differential structure and functional gene response to geochemistry associated with the suspended and attached shallow aquifer microbiomes from the Illinois Basin, IL

Despite the clear ecological significance of the microbiomes inhabiting groundwater and connected ecosystems, our current understanding of their habitats, functionality, and the ecological processes controlling their assembly have been limited. In this study, an efficient pipeline combining geochemistry, high-throughput Fluidigm TM functional gene amplification and sequencing was developed to analyze the suspended and attached microbial communities inhabiting five groundwater monitoring wells in the Illinois Basin, USA. The dominant taxa in the suspended and the attached microbial communities exhibited significantly different spatial and temporal changes in both alpha- and beta-diversity. Further analyses of representative functional genes affiliated with N 2 fixation (nifH), methane oxidation (pmoA), and sulfate reduction (dsrB, and aprA), suggested functional redundancy within the shallow aquifer microbiomes. While more diversified functional gene taxa were observed for the suspended microbial communities than the attached ones except for pmoA, different levels of changes over time and space were observed between these functional genes. Notably, deterministic and stochastic ecological processes shaped the assembly of microbial communities and functional gene reservoirs differently. While homogenous selection was the prevailing process controlling assembly of microbial communities, the neutral processes (e.g., dispersal limitation, drift and others) were more important for the functional genes. The results suggest complex and changing shallow aquifer microbiomes, whose functionality and assembly vary even between the spatially proximate habitats and fractions. As a result, this research underscored the importance to include all the interface components for a more holistic understanding of the biogeochemical processes in aquifer ecosystems, which is also instructive for practical applications.

54 ENVIRONMENTAL SCIENCES↗

Symptomatic Plasmodium vivax Infection in Rwanda

Here, we report a Plasmodium vivax infection in a Rwandan child misdiagnosed with Plasmodium falciparum and administered artemether-lumefantrine. Antigen detection revealed an absence of P falciparum histidine-rich protein 2 (HRP2) and presence of Plasmodium vivax lactate dehydrogenase. Nested and real-time polymerase chain reactions verified that the sample only contained P vivax deoxyribonucleic acid.

60 APPLIED LIFE SCIENCES↗

Transcriptomic analysis of metabolic function in the giant kelp, Macrocystis pyrifera , across depth and season

To increase knowledge of transcript diversity for the giant kelp, Macrocystis pyrifera, and assess gene expression across naturally occurring depth gradients in light, temperature and nutrients, we sequenced four cDNA libraries created from blades collected at the sea surface and at 18 m depth during the winter and summer. Comparative genomics cluster analyses revealed novel gene families (clusters) in existing brown alga expressed sequence tag data compared with other related algal groups, a pattern also seen with the addition of M. pyrifera sequences. Assembly of 228 Mbp of sequence generated c. 9000 isotigs and c. 12 000 open reading frames. Annotations were assigned using families of hidden Markov models for c. 11% of open reading frames; M. pyrifera had highest similarity to other members of the Phaeophyceae, namely Ectocarpus siliculosus and Laminaria digitata. Quantitative polymerase chain reaction of transcript targets verified depth-related differences in gene expression; stress response and light-harvesting transcripts, especially members of the LI818 (also known as LHCSR) family, showed high expression in the surface compared with 18 m depth, while some nitrogen acquisition transcripts (e.g. nitrite reductase) were upregulated at depth compared with the surface, supporting a conceptual biological model of depth-dependent physiology.

54 ENVIRONMENTAL SCIENCES↗

Agricultural management and plant selection interactively affect rhizosphere microbial community structure and nitrogen cycling

BACKGROUND: Rhizosphere microbial communities are key regulators of plant performance, yet few studies have assessed the impact of different management approaches on the rhizosphere microbiomes of major crops. Rhizosphere microbial communities are shaped by interactions between agricultural management and host selection processes, but studies often consider these factors individually rather than in combination. We tested the impacts of management (M) and rhizosphere effects (R) on microbial community structure and co-occurrence networks of maize roots collected from long-term conventionally and organically managed maize-tomato agroecosystems. We also explored the interaction between these factors (M × R) and how it impacts rhizosphere microbial diversity and composition, differential abundance, indicator taxa, co-occurrence network structure, and microbial nitrogen-cycling processes. RESULTS: Host selection processes moderate the influence of agricultural management on rhizosphere microbial communities, although bacteria and fungi respond differently to plant selection and agricultural management. We found that plants recruit management-system-specific taxa and shift N-cycling pathways in the rhizosphere, distinguishing this soil compartment from bulk soil. Rhizosphere microbiomes from conventional and organic systems were more similar in diversity and network structure than communities from their respective bulk soils, and community composition was affected by both M and R effects. In contrast, fungal community composition was affected only by management, and network structure only by plant selection. Quantification of six nitrogen-cycling genes (nifH, amoA [bacterial and archaeal], nirK, nrfA, and nosZ) revealed that only nosZ abundance was affected by management and was higher in the organic system. CONCLUSIONS: Plant selection interacts with conventional and organic management practices to shape rhizosphere microbial community composition, co-occurrence patterns, and at least one nitrogen-cycling process. Reframing research priorities to better understand adaptive plant-microbe feedbacks and include roots as a significant moderating influence of management outcomes could help guide plant-oriented strategies to improve productivity and agroecosystem sustainability.

59 BASIC BIOLOGICAL SCIENCES↗

Differentiating Vaccine-Related Fowl Cholera from Naturally Occurring Disease

Vaccine-related fowl cholera must be considered when flock mortality increases after use of a live Pasteurella multocida vaccine product. All registered live vaccines serotype as Heddleston 3,4; however, in some regions this is also the most common serotype of outbreak isolates in broiler breeders and turkeys. Therefore, serotyping may not be useful for diagnosing vaccine-related fowl cholera. This project sought to apply a vaccine-specific test to differentiate vaccine-related disease from naturally occurring outbreaks. Results indicate that vaccine strains were commonly isolated from broiler breeders exhibiting signs of fowl cholera post vaccination, but some of these isolates exhibited only serotype 4 antigenicity. The isolates’ lipopolysaccharides, the target antigen for serotyping, contained compositional changes that may explain the varying serotype results and virulence of the commercial preparations. Furthermore, these results suggest that vaccine-related disease may be common in broiler breeders, and live commercial vaccine preparations need to be assessed for serotype and titer prior to use in order to reduce vaccine-related fowl cholera.

60 APPLIED LIFE SCIENCES↗

Post-Closure Report for Closed Resource Conservation and Recovery Act Corrective Action Units, Nevada National Security Site, Nevada for fiscal year 2013 (October 2012 - September 2013)

This report serves as the combined annual report for post-closure activities for the following closed Corrective Action Units (CAUs): CAU 90, Area 2 Bitcutter Containment; CAU 91, Area 3 U-3fi Injection Well; CAU 92, Area 6 Decon Pond Facility; CAU 110, Area 3 WMD U-3ax/bl Crater; CAU 111, Area 5 WMD Retired Mixed Waste Pits; and, CAU 112, Area 23 Hazardous Waste Trenches.

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Post-Closure Inspection Report for the Tonopah Test Range, Nevada, for Calendar Year 2013

This report provides the results of the annual post-closure inspections conducted at the closed Corrective Action Units (CAUs) located on the Tonopah Test Range (TTR), Nevada. This report covers calendar year 2013 and includes inspection and repair activities completed at the following CAUs: • CAU 400: Bomblet Pit and Five Points Landfill (TTR) • CAU 407: Roller Coaster RadSafe Area (TTR) • CAU 424: Area 3 Landfill Complexes (TTR) • CAU 453: Area 9 UXO Landfill (TTR) • CAU 487: Thunderwell Site (TTR) Inspections were conducted according to the post-closure plans in the approved Closure Reports and subsequent correspondence with the Nevada Division of Environmental Protection. The post-closure inspection plans and subsequent correspondence modifying the requirements for each CAU are included in Appendix B. The inspection checklists are included in Appendix C. Field notes are included in Appendix D. Photographs taken during inspections are included in Appendix E. The annual post-closure inspections were conducted on May 14, 2013. Maintenance was performed at CAU 400, CAU 424, and CAU 453. At CAU 400, animal burrows were backfilled. At CAU 424, erosion repairs were completed at Landfill Cell A3-3, subsidence was repaired at Landfill Cell A3-4, and additional lava rock was placed in high-traffic areas to mark the locations of the surface grade monuments at Landfill Cell A3-3 and Landfill Cell A3-8. At CAU 453, two areas of subsidence were repaired and animal burrows were backfilled. Vegetation monitoring was performed at the CAU 400 Five Points Landfill and CAU 407 in June 2013. The vegetation monitoring report is included in Appendix F.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.

59 BASIC BIOLOGICAL SCIENCES↗