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At least 145 records · Page 8

Hybridization of downregulated-COMT transgenic switchgrass lines with field-selected switchgrass for improved biomass traits

Transgenic switchgrass (Panicum virgatum L.) has been produced for improved cell walls for biofuels. For instance, downregulated caffeic acid 3-O-methyltransferase (COMT) switchgrass produced significantly more biomass and biofuel than the non-transgenic progenitor line. In this present study we sought to further improve biomass characteristics by crossing the downregulated COMT T 1 lines with high-yielding switchgrass accessions in two genetic backgrounds ('Alamo' and 'Kanlow'). Crosses and T 2 progeny analyses were made under greenhouse conditions to assess maternal effects, plant morphology and yield, and cell wall traits. Female parent type influenced morphology, but had no effect on cell wall traits. T 2 hybrids produced with T 1 COMT-downregulated switchgrass as the female parent were taller, produced more tillers, and produced 63% more biomass compared with those produced using the field selected accession as the female parent. Transgene status (presence or absence of transgene) influenced both growth and cell wall traits. T 2 transgenic hybrids were 7% shorter 80 days after sowing and produced 43% less biomass than non-transgenic null-segregant hybrids. Cell wall-related differences included lower lignin content, reduced syringyl-to-guaiacyl (S/G) lignin monomer ratio, and a 12% increase in total sugar release in the T 2 transgenic hybrids compared to non-transgenic null segregants. This is the first study to evaluate the feasibility of transferring the low-recalcitrance traits associated with a transgenic switchgrass line into high-yielding field varieties in an attempt to improve growth-related traits. Lastly, our results provide insights into the possible improvement of switchgrass productivity via biotechnology paired with plant breeding.

09 BIOMASS FUELS↗

Quality assurance of temporal variability of natural decay chain and neutron induced background for low-level NORM analysis

In this study, twenty-one high purity germanium (HPGe) background spectra were collected over 2 years at Los Alamos National Laboratory. A quality assurance methodology was developed to monitor spectral background levels from thermal and fast neutron flux levels and naturally occurring radioactive material decay series radionuclides. 238 U decay products above 222 Rn demonstrated minimal temporal variability beyond that expected from counting statistics. 238 U and 232 Th progeny below Rn gas displayed at most twice the expected variability. Further, an analysis of the 139 keV 74 Ge(n, γ) and 691 keV 72 Ge(n, n') spectral features demonstrated temporal stability for both thermal and fast neutron fluxes.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

Time sequence determination of parent–daughter radionuclides using gamma-spectrometry

The acquisition of time-stamped list (TLIST) data provides additional information useful to gamma-spectrometry analysis. A novel technique is described that uses non-linear least-squares fitting and the Levenberg-Marquardt algorithm to simultaneously determine parent-daughter atoms from time sequence measurements of only the daughter radionuclide. This has been demonstrated for the radioactive decay of short-lived radon progeny (214Pb/214Bi, 212Pb/212Bi) described using the Bateman first-order differential equation. The calculated atoms are in excellent agreement with measured atoms, with a difference of 1.3 – 4.8% for parent atoms and 2.4% - 10.4% for daughter atoms. Measurements are also reported with reduced uncertainty. The technique has potential to redefine gamma-spectrometry analysis.

Burnett, J. L. (ORCID:0000000322586252)↗

Results from a controlled depleted uranium metal casting experiment designed to investigate nuclear Forensic Radiochronometry Signatures

The accurate interpretation of uranium metal 230 Th/ 234 U and 231 Pa/ 235 U radiochronometry model ages requires an understanding of how uranium parent nuclides and decay progeny ( 230 Th and 231 Pa) behave during uranium metal casting. In order to directly measure the spatial distribution of 230 Th and 231 Pa in uranium metal before and after vacuum induction melting (VIM), Los Alamos National Laboratory identified uranium metal feedstock, characterized the metal feedstock, conducted a controlled casting experiment of an approximately 120 kg uranium metal rod, and characterized the cast metal. Herein this study presents radiochronometry results and quantified 230 Th and 231 Pa VIM separation factors from bulk uranium.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

Genetic architecture of leaf morphological and physiological traits in a Populus deltoides ‘Danhong’ × P. simonii ‘Tongliao1’ pedigree revealed by quantitative trait locus analysis

Understanding the genetic architecture of leaf morphological and physiological traits will help plant breeders develop high biomass poplar genotypes. Quantitative trait locus (QTL) studies combining next-generation sequencing techniques can advance our understanding of the genetic basis of complex traits. In this study, we measured 13 leaf morphological and physiological traits and identified quantitative trait loci (QTLs) in a Populus deltoides ‘Danhong’ × P. simonii ‘Tongliao1’ F1 population (500 progenies) using a high-density genetic map constructed by whole genome re-sequencing. This linkage map consisted of 5796 single nucleotide polymorphism (SNP) markers assigned to 19 linkage groups (LGs), spanning 2683.80 centimorgans (cM) of genetic length, with an average marker density of 0.46 cM. We identified 109 QTLs on 18 LGs for leaf morphological traits and 55 QTLs on 14 LGs for leaf physiological traits. One-hundred eight putative candidate genes were identified within the candidate genomic region. Co-expression network and gene ontology enrichment analyses suggested that these candidate genes were involved in the photosynthetic process. The differential expression patterns of the CYCLIN (Potri.015G112200) and RED CHLOROPHYLL REDUCTASE (Potri.007G043600) genes between two parents indicated their potential roles in leaf morphological and physiological traits. These findings decipher the genetic architecture of leaf morphological and physiological traits in the P. deltoides ‘Danhong’ × P. simonii ‘Tongliao1’ pedigree and provide candidate genes for future poplar genetic improvement.

59 BASIC BIOLOGICAL SCIENCES↗

Nuclear genome shuffling significantly increases production of chloroplast-based recombinant protein in Chlamydomonas reinhardtii

Algae biotechnology holds great promise for the production of sustainable and environmentally friendly products, however more robust strains of microalgae must be developed before algae can be used as a commercially relevant production platform. Here we demonstrate a timely and effective approach to significantly improve a desired phenotype in algae through a combination of mutagenesis and genome shuffling. In this study, we engineered a strain of Chlamydomonas reinhardtii to produce recombinant green fluorescent protein in the chloroplast and then subjected the nuclear genome to various rounds of UV-induced mutagenesis and genome shuffling via sexual recombination, followed by isolation of strains with enhanced recombinant protein expression using fluorescence-activated cell sorting (FACS). Here, the greatest success came from first UV-mutagenizing the host strain to create mutations in the nuclear genome, followed by FACS to isolate the best GFP-expressing mutant, then shuffling the nuclear genome of that mutant by mating it with selected wild-type strains, and then again isolating the best progeny via FACS. Using this strategy, a novel strain was evolved in <3 months to express GFP over 2% total soluble protein (TSP), a 15-fold increase from the initial recombinant strain which expressed GFP at 0.14% TSP. Furthermore, this significant increase in GFP expression from the chloroplast was accomplished without altering the GFP gene itself or reducing the growth rate of the strain, demonstrating the effectiveness of this strategy to enhance a specific phenotype and significantly improve product production within an algal species in a commercially relevant timeframe.

59 BASIC BIOLOGICAL SCIENCES↗

Selection-enriched genomic loci (SEGL) reveals genetic loci for environmental adaptation and photosynthetic productivity in Chlamydomonas reinhardtii

This work demonstrates an approach to produce and select hybrid algal strains exhibiting increased photosynthetic productivity under multiple environmental conditions. This simultaneously addresses two major impediments to improving algal bioenergy production: 1) generating new genetic variants with improved performance; and 2) disentangling complex interactions between genetic and physiological factors contributing to these improvements. We pooled progeny generated from mating two environmental isolates of the green alga Chlamydomonas reinhardtii and cultured the pools under multiple environmental conditions. Strains from the outcompeting populations showed substantial (in some cases over 3 fold) increases in productivity over the parental lines under certain environments related to biomass production, including laboratory conditions as well as hyperoxia, fluctuating light, high salinity and high temperature. The results indicate that C. reinhardtii has remarkable, untapped, directed evolution capacity that may be harnessed using breeding and competition approaches. The populations were deep sequenced at multiple time points to identify “Selection-Enriched Genomic Loci” (SEGL) that accumulated in the populations, and thus likely confer increased fitness under the respective environmental conditions. With improved resolution, SEGL mapping can identify allelic combinations used for targeted breeding approaches, generating elite algal lines with multiple desirable traits, as well as to further understand the genetic and mechanistic bases of photosynthetic productivity. Increasing the photosynthetic efficiency of algae during biomass production is perhaps the most critical hurdle for economically sustainable algal based biofuels. This presents unique challenges because modifications designed to increase photosynthesis often result in decreased fitness, due to production of toxic reactive oxygen species when photosynthesis is not adequately regulated. These problems are exacerbated under natural and outdoor production environments because of the complex nature of photosynthesis and the multifaceted interactions between genetic, environmental and physiological factors. Here, we demonstrate a high throughput biotechnological screening approach that simultaneously produces algal strains with highly increased autotrophic productivity and identifies genomic loci contributing to these improvements. Our results demonstrate that Chlamydomonas reinhardtii exhibits high directed evolutionary capacity readily accessed through breeding and selection.

59 BASIC BIOLOGICAL SCIENCES↗

Comparative analysis of thorium and uranium fuel for transuranic recycle in a sodium cooled Fast Reactor

The present paper compares the reactor physics and transmutation performance of sodium-cooled Fast Reactors (FRs) for TRansUranic (TRU) burning with thorium (Th) or uranium (U) as fertile materials. The 1000 MWt Toshiba-Westinghouse Advanced Recycling Reactor (ARR) conceptual core has been used as benchmark for the comparison. Both burner and breakeven configurations sustained or started with a TRU supply, and assuming full actinide homogeneous recycle strategy, have been developed. State-of-the-art core physics tools have been employed to establish fuel inventory and reactor physics performances for equilibrium and transition cycles. Results show that Th fosters large improvements in the reactivity coefficients associated with coolant expansion and voiding, which enhances safety margins and, for a burner design, can be traded for maximizing the TRU burning rate. A trade-off of Th compared to U is the significantly larger fuel inventory required to achieve a breakeven design, which entails additional blankets at the detriment of core compactness as well as fuel manufacturing and separation requirements. The gamma field generated by the progeny of U-232 in the U bred from Th challenges fuel handling and manufacturing, but in case of full recycle, the high contents of Am and Cm in the transmutation fuel impose remote fuel operations regardless of the presence of U-232.

99 GENERAL AND MISCELLANEOUS↗

Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.

60 APPLIED LIFE SCIENCES↗

Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles

A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials. - Highlights: • Cell penetrable human scFvs (transbodies) to Ebolavirus (EBOV) VP40 were produced. • The transbodies inhibited egress of EBOV-like particles (VLPs) from human hepatocytes. • They interacted with VP40 CTD basic residues important for plasma membrane binding. • And hence interfere with viral matrix assembly and viral progeny budding. • This is the first report on human antibodies that target intracellular EBOV VP40.

60 APPLIED LIFE SCIENCES↗

Molecular pathways for learning in the single-cell Stentor coeruleus

The single-cell Stentor coeruleus contracts in response to mechanical taps but habituates and learns to ignore the taps after repeated stimulation. Here, we explored the molecular changes that occur during the formation of this cellular memory in order to improve our understanding of non-synaptic learning. We impaired cellular protein synthesis with cycloheximide and puromycin and found that, contrary to the effects of such treatments on metazoa, these drugs accelerate habituation and prolong memory retention in Stentor . Exploratory proteomic and transcriptomic analyses identified candidate proteins and genes that changed over the course of habituation and response recovery, pointing toward the regulation of Stentor learning by calcium signaling and protein phosphorylation. Building on these results, we found that using RNA interference to knock down the calcium-binding, EF-hand domain-containing protein SteCoe_6763 accelerated habituation. Furthermore, increased extracellular calcium improved Stentor learning, while treatment with kinase and phosphatase inhibitors impaired learning. In particular, KN-93, a drug known to inhibit calcium/calmodulin-dependent kinase II and voltage-gated calcium channels, decreased both the rate and extent of habituation in Stentor , similar to its effects on learning in metazoa. We also discovered that habituation memory can be maintained in progeny following cell division. Taken together, these results suggest that response recovery in Stentor requires new protein synthesis and that memory formation involves the modification of delocalized mechanoreceptors by phosphorylation and calcium signaling. This is consistent with our previous model of Stentor learning, in which habituation occurs through the inactivation of cell-surface receptors.

CaMKII↗

Influence of chronic low-dose/dose-rate high-LET irradiation from radium-226 in a human colorectal carcinoma cell line

Purpose: To evaluate potential damages of chronic environmentally relevant low-dose/dose-rate high-LET irradiation from a naturally occurring alpha-emitting radionuclide (radium-226, {sup 226}Ra) on a human colorectal carcinoma HCT116 p53{sup +/+} cell line. Methods: Clonogenic survival assays and mitochondrial membrane potential (MMP) measurement with a sensitive fluorescent MMP probe JC-1 were performed in HCT116 p53{sup +/+} cells chronically exposure to low doses/dose rates of {sup 226}Ra with high-LET. Comparisons were made with the human non-transformed keratinocyte HaCaT cell line and acute low-dose direct low-LET gamma radiation. Results and conclusion: The chronic low-dose/dose-rate alpha radiation (CLD/DRAR) did not reduce the clonogenic survival of HCT116 p53{sup +/+} cells over the period of 70 days of exposure. Only one significant reduction in the HCT116 p53{sup +/+} cells’ clonogenic survival was when cells were grown with 10,000 mBq/mL {sup 226}Ra for 40 days and progeny cells were clonogenically assessed in the presence of 10,000 mBq/mL {sup 226}Ra. The cumulative doses that cells received during this period ranged from 0.05 to 46.2 mGy. The mitochondrial membrane potential (MMP) dropped initially in both HCT116 p53{sup +/+} and HaCaT cells in response to CLD/DRAR. The MMP in HCT116 p53{sup +/+} cells recovered more quickly at all dose points than and that in HaCaT cells until the end of the exposure period. The highest dose rate of 0.66 mGy/day depolarized the HaCaT's mitochondria more consistently during the exposure period. The faster recovery status of the MMP in HCT116 p53{sup +/+} cells than that in HaCaT cells was also observed after exposure to acute low-dose gamma rays. Overall, it was found that CLD/DRAR had little impact on the MMP of human colorectal cancer and keratinocyte cell lines. - Highlights: • Human colorectal carcinoma HCT116 p53{sup +/+} and keratinocyte HaCaT cells were exposed to chronic low-dose/dose-rate alpha radiation (CLD/DRAR). • Unlike the HaCaT cells, HCT116 p53{sup +/+} cells’ clonogenic survival and mitochondrial membrane potential (MMP) changed little upon CLD/DRAR. • Upon the mitochondrial membrane depolarization response induced by CLD/DRAR, HaCaT cells were more sensitive than HCT116 p53{sup +/+}. • Gamma radiation depolarized the mitochondria of both HaCaT and HCT116 p53{sup +/+} in a dose-dependent manner. • The MMP in HCT116 p53{sup +/+} could recover more quickly than that in HaCaT cells after an acute low-dose gamma irradiation challenge.

54 ENVIRONMENTAL SCIENCES↗

Low doses and non-targeted effects in environmental radiation protection; where are we now and where should we go?

The field of low dose radiobiology has advanced considerably in the last 30 years from small indications in the 1980's that all was not simple, to a paradigm shift which occurred during the 1990's, which severely dented the dose-driven models and DNA centric theories which had dominated until then. However while the science has evolved, the application of that science in environmental health protection has not. A reason for this appears to be the uncertainties regarding the shape of the low dose response curve, which lead regulators to adopt a precautionary approach to radiation protection. Radiation protection models assume a linear relationship between dose (i.e. energy deposition) and effect (in this case probability of an adverse DNA interaction leading to a mutation). This model does not consider non-targeted effects (NTE) such as bystander effects or delayed effects, which occur in progeny cells or offspring not directly receiving energy deposition from the dose. There is huge controversy concerning the role of NTE with some saying they reflect “biology” and that repair and homeostatic mechanisms sort out the apparent damage while others consider them to be a class of damage which increases the size of the target. One thing which has recently become apparent is that NTE may be very critical for modelling long-term effects at the level of the population rather than the individual. The issue is that NTE resulting from an acute high dose such as occurred after the A-bomb or Chernobyl occur in parallel with chronic effects induced by the continuing residual effects due to radiation dose decay. This means that if ambient radiation doses are measured for example 25 years after the Chernobyl accident, they only represent a portion of the dose effect because the contribution of NTE is not included. - Highlights: • This paper presents a new concept in Environmental Radiobiology, which could explain why effects are detected in Chernobyl and Fukushima wildlife at ambient doses which are very low. • We suggest that non-targeted effects such as genomic instability, lethal mutations and bystander signalling, contribute a persistent legacy dimension to the total measured effect. • This “historic dose” component needs to be incorporated into dose effect models in order to predict true levels of risk.

54 ENVIRONMENTAL SCIENCES↗

The 2014 Integrated Field Exercise of the Comprehensive Nuclear-Test-Ban Treaty revisited: The case for data fusion

The International Monitoring System of the Preparatory Commission for the Comprehensive Nuclear-Test-Ban Treaty Organization (CTBTO) uses a global network of radionuclide monitoring stations to detect evidence of a nuclear explosion. The two radionuclide technologies employed—particulate and noble gas (radioxenon) detection—have applications for data fusion to improve detection of a nuclear explosion. Using the hypothetical 0.5 kT nuclear explosive test scenario of the CTBTO 2014 Integrated Field Exercise, the intrinsic relationship between particulate and noble gas signatures has been examined. Here this study shows that, depending upon the time of the radioxenon release, the particulate progeny can produce the more detectable signature. Thus, as both particulate and noble gas signatures are inherently coupled, the authors recommend that the sample categorization schemes should be linked.

98 NUCLEAR DISARMAMENT, SAFEGUARDS, AND PHYSICAL P↗

Development and testing of a continuous maritime monitor for radionuclide aerosols

Monitoring airborne concentrations of radionuclide activity may provide a timely warning to sea-based assets to avoid contamination from a radioactive plume. The development and testing of an automated aerosol monitoring system that can capture and detect radioactive particulate from marine air is presented. A custom electrostatic precipitator (ESP) was designed to capture particulate onto a reusable collection media. The collection efficiency of the ESP system for radon progeny was determined to be ~23%. A conservative calculation of the minimum detectable concentration of 214 Bi was estimated as 0.3-8 Bq/m 3 . The system was demonstrated in continuous operation, without consumables and limited maintenance, in a marine environment at the PNNL campus in Sequim, Washington. In conclusion, a successful 2-month deployment indicates the feasibility of the approach for continuous maritime monitoring for radionuclide aerosols.

Moore, Michael E. [Pacific Northwest National Labo↗