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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

PcsR2 Is a LuxR-Type Regulator That Is Upregulated on Wheat Roots and Is Unique to Pseudomonas chlororaphis

LuxR solos are common in plant-associated bacteria and increasingly recognized for playing important roles in plant-microbe interkingdom signaling. Unlike the LuxR-type transcriptional regulators of prototype LuxR/LuxI quorum sensing systems, luxR solos do not have a LuxI-type autoinducer synthase gene associated with them. LuxR solos in plant-pathogenic bacteria are important for virulence and in plant endosymbionts contribute to symbiosis. In the present study, we characterized an atypical LuxR solo, PcsR2, in the biological control species Pseudomonas chlororaphis 30–84 that is highly conserved among sequenced P. chlororaphis strains. Unlike most LuxR solos in the plant-associated bacteria characterized to date, pcsR2 is not associated with a proline iminopeptidase gene and the protein has an atypical N-terminal binding domain. We created a pcsR2 deletion mutant and used quantitative RT-PCR to show that the expression of pcsR2 and genes in the operon immediately downstream was upregulated ~10-fold when the wild type strain was grown on wheat roots compared to planktonic culture. PcsR2 was involved in upregulation. Using a GFP transcriptional reporter, we found that expression of pcsR2 responded specifically to root-derived substrates as compared to leaf-derived substrates but not to endogenous AHLs. Compared to the wild type, the mutant was impaired in the ability to utilize root carbon and nitrogen sources in wheat root macerate and to colonize wheat roots. Phenazine production and most biofilm traits previously shown to be correlated with phenazine production also were diminished in the mutant. Gene expression of several of the proteins in the phenazine regulatory network including PhzR, Pip (phenazine inducing protein) and RpeA/RpeB were reduced in the mutant, and overexpression of these genes in trans restored phenazine production in the mutant to wild-type levels, indicating PcsR2 affects the activity of the these regulatory genes upstream of RpeA/RpeB via an undetermined mechanism. Our results indicate PcsR2 upregulates the expression of the adjacent operon in response to unknown wheat root-derived signals and belongs to a novel subfamily of LuxR-type transcriptional regulators found in sequenced P. chlororaphis strains.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.

NASA Discipline Plant Biology↗
NASA NTRS2003

Inhibition of HSP70 and a Collagen-Specific Molecular Chaperone (HSP47) Expression in Rat Osteoblasts by Microgravity

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1alpha, 25 dihydroxyvitamin D(3) during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-beta1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1 x g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, approximately 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-beta1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-beta1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space.

Animals↗
NASA NTRS2005

Early immune response and regulation of IL-2 receptor subunits

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

Non-NASA Center↗
NASA NTRS2005

Microgravity and Signaling Molecules in Rat Osteoblasts: Downstream of Receptor Tyrosine Kinase, G-Protein-Coupled Receptor, and Small GTP-Binding Proteins

Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts.

Kumel, Yasuhiro↗
NASA NTRS2016

Skeletal Micro-RNA Responses to Simulated Weightlessness

Astronauts lose bone structure during long-duration spaceflight. These changes are due, in part, to insufficient bone formation by the osteoblast cells. Little is known about the role that small (approximately 22 nucleotides), non-coding micro-RNAs (miRNAs) play in the osteoblast response to microgravity. We hypothesize that osteoblast-lineage cells alter their miRNA status during microgravity exposure, contributing to impaired bone formation during weightlessness. To simulate weightlessness, female mice (C57BL/6, Charles River, 10 weeks of age, n = 7) were hindlimb unloaded up to 12 days. Age-matched and normally ambulating mice served as controls (n=7). To assess the expression of miRNAs in skeletal tissue, the tibia was collected ex vivo and cleaned of soft-tissue and marrow. Total RNA was collected from tibial bone and relative abundance was measured for miRNAs of interest using quantitative real time PCR array looking at 372 unique and well-characterized mature miRNAs using the delta-delta Ct method. Transcripts of interest were normalized to an average of 6 reference RNAs. Preliminary results show that hindlimb unloading decreased the expression of 14 miRNAs to less than 0.5 times that of the control levels and increased the expression of 5 miRNAs relative to the control mice between 1.2-1.5-fold (p less than 0.05, respectively). Using the miRSystem we assessed overlapping target genes predicted to be regulated by multiple members of the 19 differentially expressed miRNAs as well as in silico predicted targets of our individual miRNAs. Our miRsystem results indicated that a number of our differentially expressed miRNAs were regulators of genes related to the Wnt-Beta Catenin pathway-a known regulator of bone health-and, interestingly, the estrogen-mediated cell-cycle regulation pathway, which may indicate that simulated weightlessness modulated systemic hormonal levels or hormonal transduction that additionally contributed to bone loss. We plan to follow up these findings by measuring gene expression of miRNA-regulated genes within these two pathways with the aim of furthering our understanding of the function of miRNAs in the skeletal response to spaceflight.

musculoskeletal system↗
NASA NTRS2016

Regulation of Bone Formation During Disuse by miRNA

Astronauts lose bone structure during long-duration spaceflight. These changes are due, in part, to insufficient bone formation by the osteoblast cells. Little is known about the role that small (approximately 22 nucleotide), non-coding micro-RNAs (miRNAs) play in the osteoblast response to microgravity. We hypothesize that osteoblast-lineage cells alter their miRNA status during microgravity exposure, contributing to impaired bone formation during weightlessness. To simulate weightlessness, female mice (C57BL/6, Charles River, 10 weeks of age, n = 6) were hindlimb unloaded for 12 days. Age-matched and normally ambulating mice served as controls (n=6). To assess the expression of miRNAs in skeletal tissue, the right and left tibia of the mice were collected ex vivo and cleaned of soft-tissue and marrow. Total RNA was collected from tibial bone and relative abundance was measured for miRNAs of interest using quantitative real time PCR array looking at 372 unique and well-characterized mature miRNAs using the delta-delta Ct method. Transcripts of interest were normalized to an average of 6 reference RNAs. Preliminary results show that hindlimb unloading decreased the expression of 14 miRNAs to less than 1.4-2.9X control levels and increased the expression of 5 miRNAs relative to the control mice greater than 1-2-1.5X (p less than 0.05, respectively). Using the miRSystem we assessed overlapping target genes predicted to be regulated by multiple members of the 19 differentially expressed miRNAs as well as in silico predicted targets of our individual miRNAs. Our miRSystem results indicated that a number of our differentially expressed miRNAs were regulators of genes related to the Wnt-Beta Catenin pathway-a known regulator of bone health-and, interestingly, the estrogen-mediated cell-cycle regulation pathway, which may indicate that simulated weightlessness induced systemic hormonal changes that contributed to bone loss. We plan to follow up these findings by measuring gene expression of miRNA-regulated genes within these two pathways with the aim of furthering our understanding of the function of miRNAs in the skeletal response to spaceflight.

bone↗
DOE OSTI2021

Transcription factor FOXC1 positively regulates SFRP1 expression in androgenetic alopecia

Androgenetic alopecia (AGA) is the most common type of hair loss dysfunction. Secreted frizzled related protein 1 (SFRP1) is found to be associated with hair loss, but its role in AGA and the regulation mechanism of its transcription level is unclear. The aim of our study is to explore the expression of SFRP1 in AGA samples and its transcriptional mechanism. Male frontal and occipital scalp hair follicles from AGA patients were collected, and human dermal papilla cells (DPCs) were isolated and cultured. SFRP1 gene was cloned and constructed into recombinant plasmids to perform dual-luciferase reporter assay. Transcription factor binding sites were predicted through the Jaspar website and further confirmed by the chromatin immunoprecipitation (ChIP) assay. Expression of genes in DPCs was determined by immunofluorescence (IF) staining, quantitative real-time PCR (qRT-PCR) and western blotting. Our findings showed that SFRP1 was highly expressed in DPCs of AGA patients. The core promoter region of SFRP1 was from −100 to +50 bp and was found to be positively regulated by forkhead box C1 (FOXC1), a transcription factor related to hair growth, both at mRNA and protein level in DPCs. Our study suggests that FOXC1 plays an important role in regulating SFRP1 transcription, which may provide new insights into the development of therapeutic strategies for the treatment of AGA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

DYNC1H1 regulates NSCLC cell growth and metastasis by IFN-γ-JAK-STAT signaling and is associated with an aberrant immune response

Highlights: • DYNC1H1 is upregulated and its high expression predicted poor outcomes in NSCLC. • Loss of function of DYNC1H1 inhibits cell growth and metastasis of NSCLC cells in vitro. • DYNC1H1 knockdown inhibited metastasis of NSCLC cells in vivo. • DYNC1H1 loss of function suppresses IFN-γ-JAK-STAT signaling pathway in NSCLC. • DYNC1H1 expression is correlated with aberrant immune response in NSCLC indicated by analysis of TCGA immune data. It is urgent to identify new biomarkers and therapeutic targets to ameliorate the clinical prognosis of patients with lung cancer. The functional significance and molecular mechanism of dynein cytoplasmic 1 heavy chain 1 (DYNC1H1) in nonsmall cell lung cancer (NSCLC) progression is still elusive. In our current study, publicly available data and Western blotting experiments confirmed that DYNC1H1 expression was upregulated in lung cancer samples compared with noncancerous samples. Quantitative real-time PCR (qPCR) results indicated that high DYNC1H1 expression in lung cancer tissues was significantly associated with clinical tumor stage and distal metastasis; moreover, its high expression was negatively correlated with prognosis. Functional experiments demonstrated that DYNC1H1 loss of function caused a significant decrease in cell viability and cell proliferative ability, inhibition of the cell cycle, and promotion of both migration potential and invasion potential in vitro. Animal experiments by tail vein injection of lung cancer cells showed that DYNC1H1 knockdown significantly decreased lung cancer metastasis. Mechanistically, the results from a human protein array showed changes in the IFN-γ-JAK-STAT signaling pathway, and analysis of The Cancer Genome Atlas (TCGA) immune data demonstrated that disturbance of the immune microenvironment might be involved in the impaired growth and metastatic ability mediated by DYNC1H1 loss in NSCLC. DYNC1H1 might serve as a promising biological marker of prognosis and a potential clinical therapeutic target for patients with NSCLC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Molecular and Dual-Isotopic Profiling of the Microbial Controls on Nitrogen Leaching in Agricultural Soils under Managed Aquifer Recharge

Nitrate (NO 3 – ) leaching is a serious health and ecological concern in global agroecosystems, particularly those under the application of agricultural-managed aquifer recharge (Ag-MAR); however, there is an absence of information on microbial controls affecting NO 3 – leaching outcomes. We combine natural dual isotopes of NO 3 – ( 15 N/ 14 N and 18 O/ 16 O) with metagenomics, quantitative polymerase chain reaction (PCR), and a threshold indicator taxa analysis (TITAN) to investigate the activities, taxon profiles, and environmental controls of soil microbiome associated with NO 3 – leaching at different depths from Californian vineyards under Ag-MAR application. The isotopic signatures demonstrated a significant priming effect (P < 0.01) of Ag-MAR on denitrification activities in the topsoil (0–10 cm), with a 12–25-fold increase of 15 N–NO 3 – and 18 O–NO 3 – after the first 24 h of flooding, followed by a sharp decrease in the enrichment of both isotopes with ~80% decline in denitrification activities thereafter. In contrast, deeper soils (60–100 cm) showed minimal or no denitrification activities over the course of Ag-MAR application, thus resulting in 10–20-fold of residual NO 3 – being leached. Metagenomic profiling and laboratory microcosm demonstrated that both nitrifying and denitrifying groups, responsible for controlling NO 3 – leaching, decreased in abundance and potential activity rates with soil depth. TITAN suggested that Nitrosocosmicus and Bradyrhizobium, as the major nitrifier and denitrifier, had the highest and lowest tipping points with regard to the NO 3 – changes (P < 0.05), respectively. Overall, our study provides new insight into specific depth limitations of microbial controls on soil NO 3 – leaching in agroecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Dynamics and activity of an ammonia-oxidizing archaea bloom in South San Francisco Bay

Abstract Transient or recurring blooms of ammonia-oxidizing archaea (AOA) have been reported in several estuarine and coastal environments, including recent observations of AOA blooms in South San Francisco Bay. Here, we measured nitrification rates, quantified AOA abundance, and analyzed both metagenomic and metatranscriptomic data to examine the dynamics and activity of nitrifying microorganisms over the course of an AOA bloom in South San Francisco Bay during the autumn of 2018 and seasonally throughout 2019. Nitrification rates were correlated with AOA abundance in quantitative polymerase chain reaction (PCR) data, and both increased several orders of magnitude between the autumn AOA bloom and spring and summer seasons. From bloom samples, we recovered an extremely abundant, high-quality Candidatus Nitrosomarinus catalina-like AOA metagenome-assembled genome that had high transcript abundance during the bloom and expressed >80% of genes in its genome. We also recovered a putative nitrite-oxidizing bacteria metagenome-assembled genome from within the Nitrospinaceae that was of much lower abundance and had lower transcript abundance than AOA. During the AOA bloom, we observed increased transcript abundance for nitrogen uptake and oxidative stress genes in non-nitrifier metagenome-assembled genomes. This study confirms AOA are not only abundant but also highly active during blooms oxidizing large amounts of ammonia to nitrite—a key intermediate in the microbial nitrogen cycle—and producing reactive compounds that may impact other members of the microbial community.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Novel insights into the role of translesion synthesis polymerase in DNA incorporation and bypass of 5-fluorouracil in colorectal cancer

Abstract 5-Fluorouracil (5-FU) is the first-line chemotherapeutic agent in colorectal cancer, and resistance to 5-FU easily emerges. One of the mechanisms of drug action and resistance of 5-FU is through DNA incorporation. Our quantitative reverse-transcription PCR data showed that one of the translesion synthesis (TLS) DNA polymerases, DNA polymerase η (polη), was upregulated within 72 h upon 5-FU administration at 1 and 10 μM, indicating that polη is one of the first responding polymerases, and the only TLS polymerase, upon the 5-FU treatment to incorporate 5-FU into DNA. Our kinetic studies revealed that 5-fluoro-2′-deoxyuridine triphosphate (5FdUTP) was incorporated across dA 41 and 28 times more efficiently than across dG and across inosine, respectively, by polη indicating that the mutagenicity of 5-FU incorporation is higher in the presence of inosine and that DNA lesions could lead to more mutagenic incorporation of 5-FU. Our polη crystal structures complexed with DNA and 5FdUTP revealed that dA:5FdUTP base pair is like dA:dTTP in the active site of polη, while 5FdUTP adopted 4-enol tautomer in the base pairs with dG and HX increasing the insertion efficiency compared to dG:dTTP for the incorrect insertions. These studies confirm that polη engages in the DNA incorporation and bypass of 5-FU.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Comparative proteomics of a versatile, marine, iron-oxidizing chemolithoautotroph

This study conducted a comparative proteomic analysis to identify potential genetic markers for the biological function of chemolithoautotrophic iron oxidation in the marine bacterium Ghiorsea bivora. To date, this is the only characterized species in the class Zetaproteobacteria that is not an obligate iron-oxidizer, providing a unique opportunity to investigate differential protein expression to identify key genes involved in iron-oxidation at circumneutral pH. Over 1000 proteins were identified under both iron- and hydrogen-oxidizing conditions, with differentially expressed proteins found in both treatments. Notably, a gene cluster upregulated during iron oxidation was identified. This cluster contains genes encoding for cytochromes that share sequence similarity with the known iron-oxidase, Cyc2. Interestingly, these cytochromes, conserved in both Bacteria and Archaea, do not exhibit the typical β-barrel structure of Cyc2. This cluster potentially encodes a biological nanowire-like transmembrane complex containing multiple redox proteins spanning the inner membrane, periplasm, outer membrane, and extracellular space. The upregulation of key genes associated with this complex during iron-oxidizing conditions was confirmed by quantitative reverse transcription-PCR. These findings were further supported by electromicrobiological methods, which demonstrated negative current production by G. bivora in a three-electrode system poised at a cathodic potential. This research provides significant insights into the biological function of chemolithoautotrophic iron oxidation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Data for Characterization of the Ghd8 Flowering Time Gene in a Mini-Core Collection of Miscanthus sinensis

The optimal flowering time for bioenergy crop miscanthus is essential for environmental adaptability and biomass accumulation. However, little is known about how genes controlling flowering in other grasses contribute to flowering regulation in miscanthus. Here, we report on the sequence characterization and gene expression of Miscanthus sinensisGhd8 , a transcription factor encoding a HAP3/NF-YB DNA-binding domain, which has been identified as a major quantitative trait locus in rice, with pleiotropic effects on grain yield, heading date and plant height. In M. sinensis , we identified two homoeologous loci, MsiGhd8A located on chromosome 13 and MsiGhd8B on chromosome 7, with one on each of this paleo-allotetraploid species’ subgenomes. A total of 46 alleles and 28 predicted protein sequence types were identified in 12 wild-collected accessions. Several variants of MsiGhd8 showed a geographic and latitudinal distribution. Quantitative real-time PCR revealed that MsiGhd8 expressed under both long days and short days, and MsiGhd8B showed a significantly higher expression than MsiGhd8A . The comparison between flowering time and gene expression indicated that MsiGhd8B affected flowering time in response to day length for some accessions. This study provides insight into the conserved function of Ghd8 in the Poaceae, and is an important initial step in elucidating the flowering regulatory network of Miscanthus.

Feedstock Production↗
DOE OSTI2023

Development of a colloidal gold immunochromatographic assay strip using monoclonal antibody for rapid detection of porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) cause diarrhea and dehydration in newborn piglets and has the potential for cross-species transmission. Rapid and early diagnosis is important for preventing and controlling infectious disease. In this study, two monoclonal antibodies (mAbs) were generated, which could specifically recognize recombinant PDCoV nucleocapsid (rPDCoV-N) protein. A colloidal gold immunochromatographic assay (GICA) strip using these mAbs was developed to detect PDCoV antigens within 15 min. Results showed that the detection limit of the GICA strip developed in this study was 10 3 TCID 50 /ml for the suspension of virus-infected cell culture and 0.125 μg/ml for rPDCoV-N protein, respectively. Besides, the GICA strip showed high specificity with no cross-reactivity with other porcine pathogenic viruses. Three hundred and twenty-five fecal samples were detected for PDCoV using the GICA strip and reverse transcription-quantitative real-time PCR (RT-qPCR). The coincidence rate of the GICA strip and RT-qPCR was 96.9%. The GICA strip had a diagnostic sensitivity of 88.9% and diagnostic specificity of 98.5%. The specific and efficient detection by the strip provides a convenient, rapid, easy to use and valuable diagnostic tool for PDCoV under laboratory and field conditions.

Wang, Wei↗
DOE OSTI2022

Effect of Endosymbiotic Bacteria on Fungal Resistance Toward Heavy Metals

Most studies on metal removal or tolerance by fungi or bacteria focus on single isolates, without taking into consideration that some fungi in nature may be colonized by endobacteria. To address this knowledge gap, we investigated the tolerance and removal of diverse metals with two fungal species: Linnemannia elongata containing Burkholderia-related endobacteria and Benniella erionia containing Mollicute-related endobacteria. Isogenic lines of both species were generated with antibiotic treatments to remove their respective endobacteria. Experiments involved comparing the isogenic lines and wild type fungi in relation to the minimum inhibitory concentration for the metals, the fungal ability to remove these different metals via atomic adsorption spectroscopy, and the interaction of the metals with specific functional groups of the fungi and fungi-bacteria to determine the role of the bacteria via attenuated total reflection fourier transformed infrared (ATR-FTIR). Finally, we determined the influence of different metal concentrations, associated with moderate and high fungal growth inhibition, on the presence of the endobacteria inside the fungal mycelium via quantitative real-time PCR. Results showed that the presence of the endosymbiont increased B. erionia resistance to Mn 2+ and increased the removal of Fe 2+ compared to isogenic lines. The absence of the endosymbiont in L. elongata increased the fungal resistance toward Fe 2+ and improved the removal of Fe 2+ . Furthermore, when the bacterial endosymbiont was present in L. elongata , a decrease in the fungal resistance to Ca 2+ , Fe 2+ , and Cr 6+ was noticeable. In the ATR-FTIR analysis, we determined that C-H and C = O were the major functional groups affected by the presence of Cu 2+ , Mn 2+ , and Fe 2+ for L. elongata and in the presence of Cu 2+ and Ca 2+ for B. eronia . It is noteworthy that the highest concentration of Pb 2+ led to the loss of endobacteria in both L. elongata and B. eronia , while the other metals generally increased the concentration of endosymbionts inside the fungal mycelium. From these results, we concluded that bacterial endosymbionts of fungi can play a fundamental role in fungal resistance to metals. This study provides the first step toward a greater understanding of symbiotic interactions between bacteria and fungi in relation to metal tolerance and remediation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Characterization of the Ghd8 Flowering Time Gene in a Mini-Core Collection of Miscanthus sinensis

The optimal flowering time for bioenergy crop Miscanthus is essential for environmental adaptability and biomass accumulation. However, little is known about how genes controlling flowering in other grasses contribute to flowering regulation in Miscanthus. Here, we report on the sequence characterization and gene expression of Miscanthus sinensisGhd8, a transcription factor encoding a HAP3/NF-YB DNA-binding domain, which has been identified as a major quantitative trait locus in rice, with pleiotropic effects on grain yield, heading date and plant height. In M. sinensis, we identified two homoeologous loci, MsiGhd8A located on chromosome 13 and MsiGhd8B on chromosome 7, with one on each of this paleo-allotetraploid species’ subgenomes. A total of 46 alleles and 28 predicted protein sequence types were identified in 12 wild-collected accessions. Several variants of MsiGhd8 showed a geographic and latitudinal distribution. Quantitative real-time PCR revealed that MsiGhd8 expressed under both long days and short days, and MsiGhd8B showed a significantly higher expression than MsiGhd8A. The comparison between flowering time and gene expression indicated that MsiGhd8B affected flowering time in response to day length for some accessions. This study provides insight into the conserved function of Ghd8 in the Poaceae, and is an important initial step in elucidating the flowering regulatory network of Miscanthus.

geographic distribution↗