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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 847 records · Page 47
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Subtoxic dose of lithium cobalt oxide nanosheets impacts critical molecular pathways in trout gill epithelial cells

Increasing use of nanoscale lithium cobalt oxide (LCO) particles in nanotechnologies, including catalysis and energy storage, raises concerns over their release into the environment and subsequent biological impact. Here we study the impact of LCO nanosheets on trout gill epithelial cells – model cells for environmental exposures – by global transcriptomics using RNA-Seq. We identify molecular processes impacted by subtoxic and toxic nanoparticle (NP) doses as well as dissolved Li+ and Co2+ ions. We found that the ions, at concentrations released from the toxic NP dose, did not impact cell viability and downregulated the expression of few genes following 24 h exposure, which recovered to normal levels at 48 h. In contrast, the toxic NP dose upregulated the expression of over 1000 genes at each time point, indicating the intact NPs are responsible for perturbing gene expression and toxicity. Importantly, the subtoxic NP dose, despite having no impact on cell viability, upregulated the expression of over 500 genes at 24 h, and 150 genes at 48 h. Clustering analysis showed distinct gene expression profiles induced by the toxic and subtoxic NP doses, and functional enrichment identified pathways with distinct patterns of regulations. The impacted pathways fell into four main functional categories: metabolic and energy related processes, oxygen and hypoxia related processes, membrane binding and internalization processes, and developmental processes. Together, our observations indicate that LCO NP toxicity originates from the intact NP, not the dissolved ions, and even subtoxic NP dose impacts multiple pathways critical to the normal function of the cell.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Shade signals alter the expression of circadian clock genes in newly-formed bioenergy sorghum internodes

Stem internodes of bioenergy sorghum inbred R.07020 are longer at high plant density (shade) than at low plant density (control). Initially, the youngest newly-formed subapical stem internodes of shade-treated and control plants are comparable in length. However, full-length internodes of shade-treated plants are three times longer than the internodes of the control plants. To identify the early molecular events associated with internode elongation in response to shade, we analyzed the transcriptome of the newly-formed internodes of shade-treated and control plants sampled between 4 and 6 hr after the start of the light period (14 hr light/10 hr dark). Sorghum genes homologous to the Arabidopsis shade marker genes ATHB2 and PIL1 were not differentially expressed. The results indicate that shade signals promote internode elongation indirectly because sorghum internodes are not illuminated and grow while enclosed with leaf sheaths. Sorghum genes homologous to the Arabidopsis morning-phased circadian clock genes LHY, RVE, and LNK were downregulated and evening-phased genes such as TOC1, PRR5, and GI were upregulated in young internodes in response to shade. We hypothesize that a change in the function or patterns of expression of the circadian clock genes is the earliest molecular event associated with internode elongation in response to shade in bioenergy sorghum. Increased expression of CycD1, which promotes cell division, and decreased expression of cell wall-loosening and MBF1-like genes, which promote cell expansion, suggest that shade signals promote internode elongation in bioenergy sorghum in part through increasing cell number by delaying transition from cell division to cell expansion.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Transcriptomic Network Analysis of Cyanobacterial-Methylotroph Interactions in Coculture and Axenic Conditions

A previous study demonstrated the potential for Cyanobacterial-Methylotroph cocultures to facilitate biogas processing as well as to be used in other biotechnological applications. To advance this technology, we investigated potential interactions between Cyanobacterium stanieri HL-69 (HL69) and Methylotuvimicrobium alkaliphilum 20Z (20Z) by inferring and analyzing gene co-expression networks under co-culture and axenic conditions. Five different co-expression networks were examined. These networks were inferred using gene expression profiles for 20Z axenic condition, HL-69 axenic, HL-69, 20Z coculture, HL-69 coculture, and cross-species HL-69-20Z coculture. Through the analysis of node (gene) betweenness and node normalized degree values in all five network cases, we compared adjustments in gene expression between growth conditions (axenic vs co-culture) as well as identify biological functions relevant to interspecies interactions. This analysis was done to distinguish between gene interactions within an organism and gene interactions between two organisms. Moreover, for all five cases we investigated two different network cutoff levels of 3,000 and 10,000. By shedding light on inter- and intra- species interactions, we hope to gain a better understanding of how these two organisms interact. This research will allow the investigation of further biotechnological applications of coculture systems and optimization of such applications for biotechnological purposes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Metabolic and transcriptomic study of pennycress natural variation identifies targets for oil improvement

Pennycress (Thlaspi arvense L.), a member of the Brassicaceae family, produces seed oil high in erucic acid, suitable for biodiesel and aviation fuel. Although pennycress, a winter annual, could be grown as a dedicated bioenergy crop, an increase in its seed oil content is required to improve its economic competitiveness. The success of crop improvement relies upon finding the right combination of biomarkers and targets, and the best genetic engineering and/or breeding strategies. In this work, we combined biomass composition with metabolomic and transcriptomic studies of developing embryos from 22 pennycress natural variants to identify targets for oil improvement. The selected accession collection presented diverse levels of fatty acids at maturity ranging from 29% to 41%. Pearson correlation analyses, weighted gene co‐expression network analysis and biomarker identifications were used as complementary approaches to detect associations between metabolite level or gene expression and oil content at maturity. The results indicated that improving seed oil content can lead to a concomitant increase in the proportion of erucic acid without affecting the weight of embryos. Processes, such as carbon partitioning towards the chloroplast, lipid metabolism, photosynthesis, and a tight control of nitrogen availability, were found to be key for oil improvement in pennycress. Besides identifying specific targets, our results also provide guidance regarding the best timing for their modification, early or middle maturation. Thus, this work lays out promising strategies, specific for pennycress, to accelerate the successful development of lines with increased seed oil content for biofuel applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Developing Multi-Gene CRISPRa/I Programs to Accelerate DBTL Cycles in ABF Hosts Engineered for Chemical Production (CRADA 468)

Bacterial metabolism is comprised of large and complex gene networks that can produce valuable chemical products. Sophisticated organism engineering efforts are required to optimize production of high-value compounds from these networks. In principle, synthetic multi-gene transcriptional programs could be constructed to reengineer these networks for efficient industrial chemical production. In practice, however, our incomplete ability to understand and model the underlying networks, combined with our limited ability to predictably control the expression of multiple genes makes achieving this goal difficult. To overcome these challenges, we will combine new CRISPR-Cas multi-gene expression programs with computational modeling, machine learning, and multi-omics data to enhance the efficacy of design-build-test-learn (DBTL) cycles. For industrially promising microorganisms in early stages of development, creating technologies for rapidly engineering complex multi-gene programs could be transformative for accelerating data- and model-driven strain design. New CRISPR-Cas tools allow programmable gene activation (CRISPRa) or repression (CRISPRi) at multiple genes simultaneously, using the catalytically inactive Cas9 protein (dCas9) with guide RNAs that recognize DNA targets through predictable Watson-Crick base pairing. To enable accelerated DBTL cycles, we will combine these technologies with advanced Agile BioFoundry (ABF) capabilities for multi-omics data collection and machine learning. We will demonstrate the immediate applicability of these tools by rapidly improving the production of an industrial aromatic in multiple ABF organisms. We recently identified and optimized new transcriptional activators that can be linked to programmable CRISPR-Cas DNA binding domains to activate gene expression in E. coli. We can now use these CRISPRa tools as generalizable trans-acting regulators for combinatorial multi-gene expression tuning that can be easily transferred to new pathways and networks without additional genome engineering. We anticipate these tools will also transfer to new hosts. We have recently found that CRISPRa systems developed in E. coli can be readily ported to Pseudomonas putida, suggesting that multi-gene CRISPRa/i programs for diverse ABF organisms may be within reach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Data for A Role for Differential Rubisco Activase Isoform Expression in C4 Bioenergy Grasses at High Temperature

Rubisco activase (Rca) facilitates the release of sugar-phosphate inhibitors at Rubisco catalytic sites during CO2 fixation. Most plant species express two Rca isoforms, the larger Rca-α and the shorter Rca-β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C3 plants. However, the functional role of Rca in C4 plants where Rubisco and Rca are located in a much higher [CO2] compartment is less clear. In this study, we selected four C4 bioenergy grasses and the model C4 grass setaria ( Setaria viridis ) to investigate the role of Rca in C4 photosynthesis. All five C4 grass species contained two Rca genes, one encoding Rca-α and the other Rca-β, which were positioned closely together in the genomes. A variety of abiotic stress-related motifs were identified in the Rca-α promoter of each grass, and while the Rca-β gene was constantly highly expressed at ambient temperature, Rca-α isoforms were expressed only at high temperature but never surpassed 30% of Rca-β content. The pattern of Rca-α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C4 grasses. In sorghum ( Sorghum bicolor ), sugarcane ( Saccharum officinarum ), and setaria, the induction rate of Rca-α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca-α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca-α has a functional thermo-protective role in carbon fixation in C4 grasses by sustaining Rubisco activation at high temperature.

Genomics↗
DOE OSTI2022

Expression Patterns of Energy-Related Genes in Single Cells Uncover Key Isoforms and Enzymes That Gain Priority Under Nanoparticle-Induced Stress

Cellular responses to nanoparticles (NPs) have been largely studied in cell populations, providing averaged values that often misrepresent the true molecular processes that occur in the individual cell. To understand how a cell redistributes limited molecular resources to achieve optimal response and survival requires single-cell analysis. Here we applied multiplex single molecule-based fluorescence in situ hybridization (fliFISH) to quantify the expression of 10 genes simultaneously in individual intact cells, including glycolysis and glucose transporter genes, which are critical for restoring and maintaining energy balance. We focused on individual gill epithelial cell responses to lithium cobalt oxide (LCO) NPs, which are actively pursued as cathode materials in lithium-ion batteries, raising concerns about their impact on the environment and human health. We found large variabilities in the expression levels of all genes between neighboring cells under the same exposure conditions, from only a few transcripts to over 100 copies in individual cells. Gene expression ratios among the 10 genes in each cell uncovered shifts in favor of genes that play key roles in restoring and maintaining energy balance. Among these genes are isoforms that can secure and increase glycolysis rates more efficiently, as well as genes with multiple cellular functions, in addition to glycolysis, including DNA repair, regulation of gene expression, cell cycle progression, and proliferation. Our study uncovered prioritization of gene expression in individual cells for restoring energy balance under LCO NP exposures. Broadly, our study gained insight into single-cell strategies for redistributing limited resources to achieve optimal response and survival under stress.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

BdERECTA controls vasculature patterning and phloem-xylem organization in Brachypodium distachyon

Background: The vascular system of plants consists of two main tissue types, xylem and phloem. These tissues are organized into vascular bundles that are arranged into a complex network running through the plant that is essential for the viability of land plants. Despite their obvious importance, the genes involved in the organization of vascular tissues remain poorly understood in grasses. Results: We studied in detail the vascular network in stems from the model grass Brachypodium distachyon (Brachypodium) and identified a large set of genes differentially expressed in vascular bundles versus parenchyma tissues. To decipher the underlying molecular mechanisms of vascularization in grasses, we conducted a forward genetic screen for abnormal vasculature. We identified a mutation that severely affected the organization of vascular tissues. This mutant displayed defects in anastomosis of the vascular network and uncommon amphivasal vascular bundles. The causal mutation is a premature stop codon in ERECTA, a LRR receptor-like serine/threonine-protein kinase. Mutations in this gene are pleiotropic indicating that it serves multiple roles during plant development. This mutant also displayed changes in cell wall composition, gene expression and hormone homeostasis. Conclusion: In summary, ERECTA has a pleiotropic role in Brachypodium. We propose a major role of ERECTA in vasculature anastomosis and vascular tissue organization in Brachypodium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The Transcriptional Response of Soil Bacteria to Long-Term Warming and Short-Term Seasonal Fluctuations in a Terrestrial Forest

Terrestrial ecosystems are an important carbon store, and this carbon is vulnerable to microbial degradation with climate warming. After 30 years of experimental warming, carbon stocks in a temperate mixed deciduous forest were observed to be reduced by 30% in the heated plots relative to the controls. In addition, soil respiration was seasonal, as was the warming treatment effect. We therefore hypothesized that long-term warming will have higher expressions of genes related to carbohydrate and lipid metabolism due to increased utilization of recalcitrant carbon pools compared to controls. Because of the seasonal effect of soil respiration and the warming treatment, we further hypothesized that these patterns will be seasonal. We used RNA sequencing to show how the microbial community responds to long-term warming (~30 years) in Harvard Forest, MA. Total RNA was extracted from mineral and organic soil types from two treatment plots (+5°C heated and ambient control), at two time points (June and October) and sequenced using Illumina NextSeq technology. Treatment had a larger effect size on KEGG annotated transcripts than on CAZymes, while soil types more strongly affected CAZymes than KEGG annotated transcripts, though effect sizes overall were small. Although, warming showed a small effect on overall CAZymes expression, several carbohydrate-associated enzymes showed increased expression in heated soils (~68% of all differentially expressed transcripts). Further, exploratory analysis using an unconstrained method showed increased abundances of enzymes related to polysaccharide and lipid metabolism and decomposition in heated soils. Compared to long-term warming, we detected a relatively small effect of seasonal variation on community gene expression. Together, these results indicate that the higher carbohydrate degrading potential of bacteria in heated plots can possibly accelerate a self-reinforcing carbon cycle-temperature feedback in a warming climate.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Creating multifunctional synthetic lichen platforms for sustainable biosynthesis of biofuel precursors

In this project, we were creating a sustainable platform for biofuel production, utilizing carbon-fixing autotrophs to supply oxygen and organic substrates to heterotrophic partners, which in turn produce carbon dioxide to feed the autotrophs. This symbiotic lichen community could lower the input cost, optimize metabolic exchanges and improve the generation of biofuel precursors through multi-omics driven genetic engineering. The cyanobacteria Synechococcus elongatus (S. elongatus) was used as the primary autotroph to provide oxygen and organic substrates, especially sucrose, to a co-culture system. The strain with overexpression of sucrose transporter cscB demonstrated a significant increase in sucrose production under salt stress as what we expected. We also implemented 13C metabolic flux analysis on the sucrose secreting strain S. elongatus cscB-NaCl. Next, transporters proteins like glutamate exporter mscCG from Corynebacterium glutamicum was overexpressed in S. elongatus to improve metabolite exchange. We provided sucrose and glutamate to filamentous fungi to enable their growth and production of biochemicals using substrates from the cyanobacterium. Two fungi ( Aspergillus nidulans and Aspergillus niger ) and two yeast strains ( Rhodotorula toruloides and Lipomyces starkeyi ) served as heterotrophs in this system. They were co-cultured with S.elongatus under different pH condition, and growth on different carbon source to understand the symbiotic system and optimize the parameters for production. The two fungi strained grew much better at pH 7, 8 and 9 with Yeast Nitrogen Base (YNB ) supplementation. Two yeast strains grew well on pH 7 and 8 with YNB supplementation. All four species achieved the highest biomass level when utilizing sucrose as the main carbon sources, which demonstrates a great pairing with S.elongatus . In addition to investigate the metabolite exchange and growth condition of the co-culture system, we incorporated an Aspergillus strain expressing three genes (PAND, BAPAT and HPDH). Expression of these three genes enabled production of 3-hydroxypropionic acid (3HP) with a titer of 3-6 g/L regardless of pH and other nutrient conditions. Moreover, we developed a computational model with different heterotrophic fungi symbiosing with S.elongatus to evaluate the exchange of hundreds of metabolites. The accuracy and sensitivity had been optimized by high-throughput phenotyping assays. This project demonstrated ways to enhance a synthetic lichen platform’s efficiency and scalability to create a robust system for sustainable bioproduct synthesis. The project has enabled us to explore the technological and commercial potential of sustainable lichen co-culture. The project has also trained the next generation of scientists to address sustainability, carbon fixation, and design solutions to contemporary global challenges. The lichen platform represented a novel approach to sustainably producing bioproducts with a reduced carbon footprint and lower costs.

09 BIOMASS FUELS↗
DOE OSTI2022

Uptake of Phytoplankton-Derived Carbon and Cobalamins by Novel Acidobacteria Genera in Microcystis Blooms Inferred from Metagenomic and Metatranscriptomic Evidence

We report interactions between bacteria and phytoplankton can influence primary production, community composition, and algal bloom development. However, these interactions are poorly described for many consortia, particularly for freshwater bloom-forming cyanobacteria. Here, we assessed the gene content and expression of two uncultivated Acidobacteria from Lake Erie Microcystis blooms. These organisms were targeted because they were previously identified as important catalase producers in Microcystis blooms, suggesting that they protect Microcystis from H 2 O 2 . Metatranscriptomics revealed that both Acidobacteria transcribed genes for uptake of organic compounds that are known cyanobacterial products and exudates, including lactate, glycolate, amino acids, peptides, and cobalamins. Expressed genes for amino acid metabolism and peptide transport and degradation suggest that use of amino acids and peptides by Acidobacteria may regenerate nitrogen for cyanobacteria and other organisms. The Acidobacteria genomes lacked genes for biosynthesis of cobalamins but expressed genes for its transport and remodeling. This indicates that the Acidobacteria obtained cobalamins externally, potentially from Microcystis, which has a complete gene repertoire for pseudocobalamin biosynthesis; expressed them in field samples; and produced pseudocobalamin in axenic culture. Both Acidobacteria were detected in Microcystis blooms worldwide. Together, the data support the hypotheses that uncultured and previously unidentified Acidobacteria taxa exchange metabolites with phytoplankton during harmful cyanobacterial blooms and influence nitrogen available to phytoplankton. Thus, novel Acidobacteria may play a role in cyanobacterial physiology and bloom development.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Bioenergy sorghum stem growth regulation: intercalary meristem localization, development, and gene regulatory network analysis

SUMMARY Bioenergy sorghum is a highly productive drought tolerant C 4 grass that accumulates 80% of its harvestable biomass in approximately 4 m length stems. Stem internode growth is regulated by development, shading, and hormones that modulate cell proliferation in intercalary meristems (IMs). In this study, sorghum stem IMs were localized above the pulvinus at the base of elongating internodes using magnetic resonance imaging, microscopy, and transcriptome analysis. A change in cell morphology/organization occurred at the junction between the pulvinus and internode where LATERAL ORGAN BOUNDARIES ( SbLOB ), a boundary layer gene, was expressed. Inactivation of an AGCVIII kinase in DDYM ( dw2 ) resulted in decreased SbLOB expression, disrupted IM localization, and reduced internode cell proliferation. Transcriptome analysis identified approximately 1000 genes involved in cell proliferation, hormone signaling, and other functions selectively upregulated in the IM compared with a non‐meristematic stem tissue. This cohort of genes is expressed in apical dome stem tissues before localization of the IM at the base of elongating internodes. Gene regulatory network analysis identified connections between genes involved in hormone signaling and cell proliferation. The results indicate that gibberellic acid induces accumulation of growth regulatory factors (GRFs) known to interact with ANGUSTIFOLIA (SbAN3), a master regulator of cell proliferation. GRF:AN3 was predicted to induce SbARF3/ETT expression and regulate SbAN3 expression in an auxin‐dependent manner. GRFs and ARFs regulate genes involved in cytokinin and brassinosteroid signaling and cell proliferation. The results provide a molecular framework for understanding how hormone signaling regulates the expression of genes involved in cell proliferation in the stem IM.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The Supplementation of FloraMax-B11 Did Not Affect the Bile Acid Neosynthesis and the Enterohepatic Circulation in Broiler Chickens

Most probiotics possess bile salt hydrolase enzymes and may increase bile acid excretion and negatively affect fat digestion and absorption. Therefore, the study objective was to determine the time course effects of a commercial probiotic (P) FloraMax-B11 (FM) supplementation on bile acid neosynthesis and enterohepatic circulation in broiler chickens. Fertile Ross 708 eggs were incubated under standard commercial conditions. At hatch, chicks (n = 550) were randomly assigned to 5 treatment groups (n = 5 replicates per treatment group) with 22 birds per pen. The 5 treatment groups consisted of: control group (C, normal water from hatch to 35 days of age without supplements); P3, water supplemented with FM for the first 3 days post-hatch followed by normal water until day 35; P10, water supplemented with FM for the first 10 days post-hatch followed by normal water until day 35; P35, water supplemented with FM from hatch to day 35; and AGP, water supplemented with antibiotic growth promoter (AGP) from hatch until day 35. Ileum, liver, and plasma were collected at hatch, days 3, 10, 21, and 35 post-hatch. The relative mRNA expression of genes involved in bile acid synthesis (CYP7A1, CYP8B1, FXR, FGFR4, and FGF19) and transport (ASBT, I-BABP, OSTα, OSTβ, and BSEP) as well as ileal deoxycholic acid and plasma cholic acid were determined. There was no FM and AGP interaction for any of the response criteria. No FM or AGP effects were observed (p > 0.05) for any genes, except FGF19, which expression was increased (p < 0.0001) in AGP compared to P35. No FM or AGP effects were observed (p > 0.05) for levels of deoxycholic and cholic acids. However, all the genes, deoxycholic acid, and plasma cholic acid were affected by age (p < 0.0001). In general, the data indicate that FM did not negatively impact bile acid metabolism and enterohepatic circulation, which appeared to be age dependent. However, more research should be conducted to confirm these results and investigate the effects of FM on bile acid metabolism, fat digestion, and intestinal microbiota in broiler chickens.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation and metal homeostasis

Copper (Cu) is essential for respiration, neurotransmitter synthesis, oxidative stress response, and transcription regulation, with imbalances leading to neurological, cognitive, and muscular disorders. Here we show the role of a novel Cu-binding protein (Cu-BP) in mammalian transcriptional regulation, specifically on skeletal muscle differentiation using murine primary myoblasts. Utilizing synchrotron X-ray fluorescence-mass spectrometry, we identified murine cysteine-rich intestinal protein 2 (mCrip2) as a key Cu-BP abundant in both nuclear and cytosolic fractions. mCrip2 binds two to four Cu + ions with high affinity and presents limited redox potential. CRISPR/Cas9-mediated deletion of mCrip2 impaired myogenesis, likely due to Cu accumulation in cells. CUT&RUN and transcriptome analyses revealed its association with gene promoters, including MyoD1 and metallothioneins, suggesting a novel Cu-responsive regulatory role for mCrip2. Our work describes the significance of mCrip2 in skeletal muscle differentiation and metal homeostasis, expanding understanding of the Cu-network in myoblasts. Copper (Cu) is essential for various cellular processes, including respiration and stress response, but imbalances can cause serious health issues. This study reveals a new Cu-binding protein (Cu-BP) involved in muscle development in primary myoblasts. Using unbiased metalloproteomic techniques and high throughput sequencing, we identified mCrip2 as a key Cu-BP found in cell nuclei and cytoplasm. mCrip2 binds up to four Cu + ions and has a limited redox potential. Deleting mCrip2 using CRISPR/Cas9 disrupted muscle formation due to Cu accumulation. Further analyses showed that mCrip2 regulates the expression of genes like MyoD1, essential for muscle differentiation, and metallothioneins in response to copper supplementation. This research highlights the importance of mCrip2 in muscle development and metal homeostasis, providing new insights into the Cu-network in cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Metformin attenuates expression of angiogenic and inflammatory genes in human endometriotic stromal cells

Highlights: • Metformin reduced cell migration, and proliferation of endometriotic stromal cells. • Metformin attenuated the expression of angiogenic and inflammatory genes in human endometriotic stromal cells. • Metformin could be used as an effective adjuvant in endometriosis treatment. Endometriosis is a benign gynecological disease that is manifested by the presence and growth of endometrial cells and glands outside the uterine. Active angiogenesis, migration, and invasion of endometrial tissue outside the uterine are critical for the development of endometriosis and lead to the survival and growth of endometriotic lesions. Metformin, as an anti-diabetic agent, represents anti-angiogenic property. Here, we performed a study using human normal endometrial stromal cells (N-ESCs) from healthy endometrial tissue and human eutopic endometrial stromal cells (EU-ESCs) and ectopic endometrial stromal cells (ECT-ESCs) from endometriosis patients. ESCs were cultured and treated with different concentrations of Metformin (0–20 mmol/l) for 72 h to evaluate Metformin effect on cell viability, proliferation, migration was measured by methyl thiazolyl tetrazolium (MTT) assay and scratch test respectively as well as expression of angiogenesis and migration markers. The Metformin reduced cell migration, and proliferation of endometriotic stromal cells in a time and concentration dependently manner. Furthermore, Metformin attenuated the expression of angiogenic and inflammatory genes in human endometriotic stromal cells. The direct anti-proliferative effect on ECT-ESCs combined with the effects of Metformin on inflammatory and angiogenesis-related genes expression supports its therapeutic potential for endometriosis. Metformin could be used as an effective adjuvant in endometriosis treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states

Vancomycin has historically been used as a last-resort treatment for serious bacterial infections. However, vancomycin resistance has become widespread in certain pathogens, presenting a serious threat to public health. Resistance to vancomycin is conferred by a suite of resistance genes, the expression of which is controlled by the VanR–VanS two-component system. VanR is the response regulator in this system; in the presence of vancomycin, VanR accepts a phosphoryl group from VanS, thereby activating VanR as a transcription factor and inducing expression of the resistance genes. This paper presents the X-ray crystal structures of full-length VanR from Streptomyces coelicolor in both the inactive and activated states at resolutions of 2.3 and 2.0 Å, respectively. Comparison of the two structures illustrates that phosphorylation of VanR is accompanied by a disorder-to-order transition of helix 4, which lies within the receiver domain of the protein. This transition generates an interface that promotes dimerization of the receiver domain; dimerization in solution was verified using analytical ultracentrifugation. The inactive conformation of the protein does not appear intrinsically unable to bind DNA; rather, it is proposed that in the activated form DNA binding is enhanced by an avidity effect contributed by the receiver-domain dimerization.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Gamete expression of TALE class HD genes activates the diploid sporophyte program in Marchantia polymorpha

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in gametes primes the haploid-to-diploid transition. In the unicellular chlorophyte alga Chlamydomonas, KNOX and BELL TALE-homeodomain genes mediate this transition. We demonstrate that in the liverwort Marchantia polymorpha, paternal (sperm) expression of three of five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of both MpKNOX1 and MpBELL34 mediate the haploid-to-diploid transition. Loss-of-function alleles of MpKNOX1 result in zygotic arrest, whereas a loss of either maternal or paternal MpBELL234 results in variable zygotic and early embryonic arrest. Expression of MpKNOX1 and MpBELL34 during diploid sporophyte development is consistent with a later role for these genes in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.

59 BASIC BIOLOGICAL SCIENCES↗