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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 919 records · Page 51
All sourcesNASA NTRSDOE OSTI
NASA NTRS2005

Transcriptional profile of a myotube starvation model of atrophy

Skeletal muscle wasting is a pervasive phenomenon that can result from a wide range of pathological conditions as well as from habitual muscular inactivity. The present work describes a cell-culture condition that induces significant atrophy in skeletal muscle C2C12 myotubes. The failure to replenish differentiation media in mature myotubes leads to rapid atrophy (53% in diameter), which is referred to here as starvation. Affymetrix microarrays were used to develop a transcriptional profile of control (fed) vs. atrophied (nonfed) myotubes. Myotube starvation was characterized by an upregulation of genes involved in translational inhibition, amino acid biosynthesis and transport, and cell cycle arrest/apoptosis, among others. Downregulated genes included several structural and regulatory elements of the extracellular matrix as well as several elements of Wnt/frizzled and TGF-beta signaling pathways. Interestingly, the characteristic transcriptional upregulation of the ubiquitin-proteasome system, calpains, and cathepsins known to occur in multiple in vivo models of atrophy were not seen during myotube starvation. With the exception of the downregulation of extracellular matrix genes, serine protease inhibitor genes, and the upregulation of the translation initiation factor PHAS-I, this model of atrophy in cell culture has a transcriptional profile quite distinct from any study published to date with atrophy in whole muscle. These data show that, although the gross morphology of atrophied muscle fibers may be similar in whole muscle vs. myotube culture, the processes by which this phenotype is achieved differ markedly.

NASA Discipline Musculoskeletal↗
DOE OSTI2019

Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism

Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged r andom b arcode t ra n sposon seq uencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both L - and D -lysine metabolism. We first describe three pathway enzymes that catabolize L -2-aminoadipate ( L -2AA) to 2-ketoglutarate (2KG), connecting D -lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of L -lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research. IMPORTANCE P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to D -2-hydroxyglutarate ( D -2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Global Analysis of Chlorella variabilis NC64A mRNA Profiles during the Early Phase of Paramecium bursaria Chlorella Virus-1 Infection

The PBCV-1/Chlorella variabilis NC64A system is a model for studies on interactions between viruses and algae. Here we present the first global analyses of algal host transcripts during the early stages of infection, prior to virus replication. During the course of the experiment stretching over 1 hour, about a third of the host genes displayed significant changes in normalized mRNA abundance that either increased or decreased compared to uninfected levels. The population of genes with significant transcriptional changes gradually increased until stabilizing at 40 minutes post infection. Functional categories including cytoplasmic ribosomal proteins, jasmonic acid biosynthesis and anaphase promoting complex/cyclosomes had a significant excess in upregulated genes, whereas spliceosomal snRNP complexes and the shikimate pathway had significantly more down-regulated genes, suggesting that these pathways were activated or shut-down in response to the virus infection. Lastly, we examined the expression of C. varibilis RNA polymerase subunits, as PBCV-1 transcription depends on host RNA polymerases. Two subunits were up-regulated, RPB10 and RPC34, suggesting that they may function to support virus transcription. These results highlight genes and pathways, as well as overall trends, for further refinement of our understanding of the changes that take place during the early stages of viral infection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Binding profiles for 961 Drosophila and C. elegans transcription factors reveal tissue-specific regulatory relationships

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here, we present the culmination of the efforts of the modENCODE (model organism Encyclopedia of DNA Elements) and modERN (model organism Encyclopedia of Regulatory Networks) consortia to systematically assay TF binding events in vivo in two major model organisms,Drosophila melanogaster(fly) andCaenorhabditis elegans(worm). These data sets comprise 605 TFs identifying 3.6 M sites in the fly and 356 TFs identifying 0.9 M sites in the worm, and represent the majority of the regulatory space in each genome. We demonstrate that TFs associate with chromatin in clusters termed “metapeaks,” that larger metapeaks have characteristics of high-occupancy target (HOT) regions, and that the importance of consensus sequence motifs bound by TFs depends on metapeak size and complexity. Combining ChIP-seq data with single-cell RNA-seq data in a machine-learning model identifies TFs with a prominent role in promoting target gene expression in specific cell types, even differentiating between parent–daughter cells during embryogenesis. These data are a rich resource for the community that should fuel and guide future investigations into TF function. To facilitate data accessibility and utility, all strains expressing green fluorescent protein (GFP)-tagged TFs are available at the stock centers for each organism. The chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center, GEO, and through a direct interface that provides rapid access to processed data sets and summary analyses, as well as widgets to probe the cell-type-specific TF–target relationships.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Conservation and Expansion of Transcriptional Factor Repertoire in the Fusarium oxysporum Species Complex

The Fusarium oxysporum species complex (FOSC) includes both plant and human pathogens that cause devastating plant vascular wilt diseases and threaten public health. Each F. oxysporum genome comprises core chromosomes (CCs) for housekeeping functions and accessory chromosomes (ACs) that contribute to host-specific adaptation. This study inspects global transcription factor profiles (TFomes) and their potential roles in coordinating CC and AC functions to accomplish host-specific interactions. Remarkably, we found a clear positive correlation between the sizes of TFomes and the proteomes of an organism. With the acquisition of ACs, the FOSC TFomes were larger than the other fungal genomes included in this study. Among a total of 48 classified TF families, 14 families involved in transcription/translation regulations and cell cycle controls were highly conserved. Among the 30 FOSC expanded families, Zn2-C6 and Znf_C2H2 were most significantly expanded to 671 and 167 genes per family including well-characterized homologs of Ftf1 (Zn2-C6) and PacC (Znf_C2H2) that are involved in host-specific interactions. Manual curation of characterized TFs increased the TFome repertoires by 3% including a disordered protein Ren1. RNA-Seq revealed a steady pattern of expression for conserved TF families and specific activation for AC TFs. Functional characterization of these TFs could enhance our understanding of transcriptional regulation involved in FOSC cross-kingdom interactions, disentangle species-specific adaptation, and identify targets to combat diverse diseases caused by this group of fungal pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2011

Automated Miniaturized Instrument for Space Biology Applications and the Monitoring of the Astronauts Health Onboard the ISS

Human space travelers experience a unique environment that affects homeostasis and physiologic adaptation. The spacecraft environment subjects the traveler to noise, chemical and microbiological contaminants, increased radiation, and variable gravity forces. As humans prepare for long-duration missions to the International Space Station (ISS) and beyond, effective measures must be developed, verified and implemented to ensure mission success. Limited biomedical quantitative capabilities are currently available onboard the ISS. Therefore, the development of versatile instruments to perform space biological analysis and to monitor astronauts' health is needed. We are developing a fully automated, miniaturized system for measuring gene expression on small spacecraft in order to better understand the influence of the space environment on biological systems. This low-cost, low-power, multi-purpose instrument represents a major scientific and technological advancement by providing data on cellular metabolism and regulation. The current system will support growth of microorganisms, extract and purify the RNA, hybridize it to the array, read the expression levels of a large number of genes by microarray analysis, and transmit the measurements to Earth. The system will help discover how bacteria develop resistance to antibiotics and how pathogenic bacteria sometimes increase their virulence in space, facilitating the development of adequate countermeasures to decrease risks associated with human spaceflight. The current stand-alone technology could be used as an integrated platform onboard the ISS to perform similar genetic analyses on any biological systems from the tree of life. Additionally, with some modification the system could be implemented to perform real-time in-situ microbial monitoring of the ISS environment (air, surface and water samples) and the astronaut's microbiome using 16SrRNA microarray technology. Furthermore, the current system can be enhanced substantially by combining it with other technologies for automated, miniaturized, high-throughput biological measurements, such as fast sequencing, protein identification (proteomics) and metabolite profiling (metabolomics). Thus, the system can be integrated with other biomedical instruments in order to support and enhance telemedicine capability onboard ISS. NASA's mission includes sustained investment in critical research leading to effective countermeasures to minimize the risks associated with human spaceflight, and the use of appropriate technology to sustain space exploration at reasonable cost. Our integrated microarray technology is expected to fulfill these two critical requirements and to enable the scientific community to better understand and monitor the effects of the space environment on microorganisms and on the astronaut, in the process leveraging current capabilities and overcoming present limitations.

Human space travelers↗
DOE OSTI2020

Characterization of a Mo-Nitrogenase Variant Containing a Citrate-Substituted Cofactor

Nitrogenase converts N 2 to NH 3 , and CO to hydrocarbons, at its cofactor site. In this work, we report a biochemical and spectroscopic characterization of a Mo-nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDK Cit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDK Cit reveals a shift of CO reduction from H 2 evolution toward hydrocarbon formation and an opposite shift of N 2 reduction from NH 3 formation toward H 2 evolution. Consistent with a shift in the Mo K-edge energy of NifDK Cit relative to that of its wild-type counterpart, EPR analysis demonstrates a broadening of the line-shape and a decrease in the intensity of the cofactor-originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Metabolic flux, metabolite, and transcript analysis uncover reprogramming of metabolism toward higher seed oil

Overexpression of WRINKLED1 (WRI1), a master regulator of glycolysis and fatty acid biosynthesis, together with DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1), which catalyzes the final step of triacylglycerol assembly, is a promising strategy for enhancing seed oil content. However, how these regulators coordinate system-wide metabolic reprogramming at the levels of gene expression, metabolite pools, and fluxes remains poorly understood. To address this, we performed 13 C-metabolic flux analysis, metabolomics, and transcriptomics on in vitro cultured pennycress (Thlaspi arvense L.) embryos overexpressing the native WRI1 and DGAT1 homologs. Here, in cultured embryos, WRI1/DGAT1 overexpression increased triacylglycerol accumulation by 28% while reducing protein content by 34%, relative to the wild type. Embryos showed ∼20-fold and 50-fold upregulation of WRI1 and DGAT1 along with induction of WRI1 target genes in glycolysis and fatty acid biosynthesis. Genes associated with photosynthesis and Calvin cycle functions were also upregulated, whereas genes encoding ribosomal proteins and seed storage proteins were strongly repressed, consistent with the observed lipid–protein tradeoff. Flux analysis revealed that enhanced triacylglycerol biosynthesis is supported by increased flux through the Rubisco shunt and cytosolic pyruvate kinase, while the oxidative pentose phosphate pathway and malic enzyme contributed little to NADPH or pyruvate supply. Metabolomic profiling revealed extensive perturbations in glycolytic intermediates, tricarboxylic acid cycle metabolites, and amino acids. In plant grown seeds, WRI1/DGAT1 lines also showed a modest but significant increase in total lipid content. Collectively, these findings reveal how WRI1 and DGAT1 reprogram central metabolism to enhance oil accumulation, with relevance to mature seeds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Profiling neuron-autonomous lncRNA changes upon ischemia/reperfusion injury

Highlights: • Tnxa-ps1 is up regulated after OGD/R. • Tnxa-ps1 knockdown promoted neuronal survival after OGD/R. • Tnxa-ps1 up-regulation is associated with the four mRNA changes after OGD/R. Extensive changes of neuronal transcriptome occur post ischemic stroke and during the following reperfusion. Although numerous studies focused on transcriptome changes of mRNAs associated with ischemic stroke, little is known about whether and how long non-coding RNAs (lncRNAs), which play critical roles in cellular homeostasis, are involved in this process. In this study, we performed high throughput screening to analyze expression changes of lncRNAs in primarily cultured hippocampal neurons under an oxygen-glucose deprivation/reperfusion (OGD/R) condition at 0 h, 6 h, 12 h, and 18 h, respectively. Knock down of one validated lncRNAs (Tnxa-ps1) promoted neuronal survival by inhibiting apoptosis. Coding non-coding co-expression network analysis revealed that the expression of Tnxa-ps1 was highly correlated with changes of a particular group of genes, many of which are associated with neural protection. Finally, we showed that down-regulation of Tnxa-ps1 reversed the expression changes of four mRNAs post OGD/R, revealing a regulatory effect between Tnxa-ps1 and selected genes. Together, our data revealed possible participation of lncRNAs in the pathophysiology of OGD/R and thereby provided new insights into the studies of potential therapeutic targets for ischemic stroke.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose

Thermoanaerobacterium saccharolyticum, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by T. saccharolyticum for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (Tsac_1445 through Tsac_1464 for xylan/xylose and Tsac_1344 through Tsac_1349 for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in T. saccharolyticum, and deletion of the Tsac_0361 gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C5 sugars

Currie, Devin↗
DOE OSTI2015

Inhaled ozone (O{sub 3})-induces changes in serum metabolomic and liver transcriptomic profiles in rats

Air pollution has been linked to increased incidence of diabetes. Recently, we showed that ozone (O{sub 3}) induces glucose intolerance, and increases serum leptin and epinephrine in Brown Norway rats. In this study, we hypothesized that O{sub 3} exposure will cause systemic changes in metabolic homeostasis and that serum metabolomic and liver transcriptomic profiling will provide mechanistic insights. In the first experiment, male Wistar Kyoto (WKY) rats were exposed to filtered air (FA) or O{sub 3} at 0.25, 0.50, or 1.0 ppm, 6 h/day for two days to establish concentration-related effects on glucose tolerance and lung injury. In a second experiment, rats were exposed to FA or 1.0 ppm O{sub 3}, 6 h/day for either one or two consecutive days, and systemic metabolic responses were determined immediately after or 18 h post-exposure. O{sub 3} increased serum glucose and leptin on day 1. Glucose intolerance persisted through two days of exposure but reversed 18 h-post second exposure. O{sub 3} increased circulating metabolites of glycolysis, long-chain free fatty acids, branched-chain amino acids and cholesterol, while 1,5-anhydroglucitol, bile acids and metabolites of TCA cycle were decreased, indicating impaired glycemic control, proteolysis and lipolysis. Liver gene expression increased for markers of glycolysis, TCA cycle and gluconeogenesis, and decreased for markers of steroid and fat biosynthesis. Genes involved in apoptosis and mitochondrial function were also impacted by O{sub 3}. In conclusion, short-term O{sub 3} exposure induces global metabolic derangement involving glucose, lipid, and amino acid metabolism, typical of a stress–response. It remains to be examined if these alterations contribute to insulin resistance upon chronic exposure. - Highlights: • Ozone, an ubiquitous air pollutant induces acute systemic metabolic derangement. • Serum metabolomic approach provides novel insights in ozone-induced changes. • Ozone exposure induces leptinemia, hyperglycemia, and glucose intolerance. • Ozone increases serum free fatty acids, branched chain amino acids, cholesterols. • Ozone metabolic derangement is likely mediated by neuronal stress response pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Transcriptional profiling of rat white adipose tissue response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin

Polychlorinated dibenzodioxins are environmental contaminants commonly produced as a by-product of industrial processes. The most potent of these, 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), is highly lipophilic, leading to bioaccumulation. White adipose tissue (WAT) is a major site for energy storage, and is one of the organs in which TCDD accumulates. In laboratory animals, exposure to TCDD causes numerous metabolic abnormalities, including a wasting syndrome. We therefore investigated the molecular effects of TCDD exposure on WAT by profiling the transcriptomic response of WAT to 100 μg/kg of TCDD at 1 or 4 days in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. A comparative analysis was conducted simultaneously in identically treated TCDD-resistant Han/Wistar (Kuopio; H/W) rats one day after exposure to the same dose. We sought to identify transcriptomic changes coinciding with the onset of toxicity, while gaining additional insight into later responses. More transcriptional responses to TCDD were observed at 4 days than at 1 day post-exposure, suggesting WAT shows mostly secondary responses. Two classic AHR-regulated genes, Cyp1a1 and Nqo1, were significantly induced by TCDD in both strains, while several genes involved in the immune response, including Ms4a7 and F13a1 were altered in L-E rats alone. We compared genes affected by TCDD in rat WAT and human adipose cells, and observed little overlap. Interestingly, very few genes involved in lipid metabolism exhibited altered expression levels despite the pronounced lipid mobilization from peripheral fat pads by TCDD in L-E rats. Of these genes, the lipolysis-associated Lpin1 was induced slightly over 2-fold in L-E rat WAT on day 4. - Highlights: • Exposure to TCDD causes wasting syndrome in L-E rats but not in H/W rats. • We examined the transcriptome of TCDD-treated L-E and H/W rat white adipose tissue. • L-E WAT demonstrated altered abundance of several genes involved in immune response. • Few genes had altered abundance in both L-E rat WAT and human adipocytes (hMADS). • Pmepa1 was induced in both L-E WAT and differentiated hMADS cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Combined Imaging and RNA-Seq on a Microfluidic Platform for Viral Infection Studies

The goal of this work was to pioneer a novel, low-overhead protocol for simultaneously assaying cell-surface markers and intracellular gene expression in a single mammalian cell. The purpose of developing such a method is to be able to understand the mechanisms by which pathogens engage with individual mammalian cells, depending on their cell surface proteins, and how both host and pathogen gene expression changes are reflective of these mechanisms. The knowledge gained from such analyses of single cells will ultimately lead to more robust pathogen detection and countermeasures. Our method was aimed at streamlining both the upstream cell sample preparation using microfluidic methods, as well as the actual library making protocol. Specifically, we wanted to implement a random hexamer-based reverse transcription of all RNA within a single cell (as opposed to oligo dT-based which would only capture polyadenylated transcripts), and then use a CRISPR-based method called scDash to deplete ribosomal DNAs (since ribosomal RNAs make up the majority of the RNA in a mammalian cell). After significant troubleshooting, we demonstrate that we are able to prepare cDNA from RNA using the random hexamer primer, and perform the rDNA depletion. We also show that we can visualize individually stained cells, setting up the pipeline for connecting surface markers to RNA-sequencing profiles. Finally, we test a number of devices for various parts of the pipeline, including bead generation, optical barcoding and cell dispensing, and demonstrate that while some of these have potential, more work is needed to optimize this part of the pipeline.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Nuclear import of transcriptional corepressor BCOR occurs through interaction with karyopherin α expressed in human periodontal ligament

Highlights: • Human periodontal ligament cells express karyopherin α (KPNA) of three subclasses. • BCL6 corepressor (BCOR) is translocated to the nucleus by KPNA2, 4, and 6. • BCOR interacts with KPNA through nuclear localization signal 1. • BCOR mutation causing OFCD syndrome downregulates the SMAD4-SHH-NFIC pathways. Mutations in the gene encoding BCL-6 corepressor (BCOR) are responsible for oculofaciocardiodental (OFCD) syndrome, which is a rare X-linked dominant disorder characterized by radiculomegaly of permanent teeth as the most typical symptom. To function as a transcriptional corepressor, BCOR needs to enter the nucleus; however, the molecular pathway for its nuclear translocation during dental root formation remains unclear. The purpose of this study was to determine the mechanism underlying BCOR transport into the nucleus. Our results showed that human periodontal ligament (PDL) cells expressed karyopherin α (KPNA)2, KPNA4, and KPNA6 belonging to a family of nuclear import proteins, which interacted with BCOR in the immunoprecipitation assay. Site-directed mutagenesis targeting the two nuclear localization signals (NLSs) within BCOR reduced its nuclear translocation; however, co-expression of KPNA2, KPNA4, or KPNA6 with BCOR carrying a previously described mutation which eliminated one of the two NLSs significantly increased nuclear accumulation of the mutant BCOR, indicating participation of KPNA in BCOR nuclear translocation. Comparative expression profiling of PDL cells isolated from normal and OFCD patients revealed significant downregulation of SMAD4, GLI1, and nuclear factor 1-C (NFIC) mRNA expression, suggesting that BCOR mutations cause hyperactive root formation in OFCD syndrome by inhibiting SMAD4-Hedgehog-NFIC signaling implicated in dental root development. Our study contributes to understanding of the mechanisms providing nuclear import of BCOR during root formation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Cell wall response of field grown Populus to Septoria infection

Due to its ability to spread quickly and result in tree mortality, Sphaerulina musiva (Septoria) is one of the most severe diseases impacting Populus . Previous studies have identified that Septoria infection induces differential expression of phenylpropanoid biosynthesis genes. However, more extensive characterization of changes to lignin in response to Septoria infection is lacking. To study the changes of lignin due to Septoria infection, four field grown, naturally variant Populus trichocarpa exhibiting visible signs of Septoria infection were sampled at health, infected, and reaction zone regions for cell wall characterization. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and acid hydrolysis were applied to identify changes to the cell wall, and especially lignin. FTIR and subsequent principal component analysis revealed that infected and reaction zone regions were similar and could be distinguished from the non-infected (healthy) region. NMR results indicated the general trend that infected region had a higher syringyl:guaiacyl ratio and lower p -hydroxybenzoate content than the healthy regions from the same genotype. Finally, Klason lignin content in the infected and/or reaction zone regions was shown to be higher than healthy region, which is consistent with previous observations of periderm development and metabolite profiling. These results provide insights on the response of Populus wood characteristics to Septoria infection, especially between healthy and infected region within the same genotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Three differential expression profiles of miRNAs as potential biomarkers for lung adenocarcinoma

Highlights: • To identify the differential miRNAs expression between lung cancer and normal tissues. • Data analysis was based on a computational approach to detect survival-associated signatures. • Three-miRNA signature could be used as a prognostic marker in LACA. Owing to the poor prognosis, novel biomarkers for lung adenocarcinoma (LACA) are needed nowadays. The aim of the study was to identify the differential miRNAs expression between lung cancer and normal tissues and evaluate the prognostic values of the miRNAs. Multidimensional data of 528 samples were retrieved from The Cancer Genome Atlas archive. Data analysis was based on a computational approach to detect survival-associated molecular signatures. A total of 191 differentially expressed miRNAs were identified between LACA tissues and normal tissues, including 88 up-regulated and 103 down-regulated miRNAs. The Kaplan-Meier survival method revealed the prognostic function of the three miRNAs (miR-1293, miR-873 and miR-1914). Cox regression analysis showed that the three-miRNA signature was an independent prognostic factor in LACA. The functional enrichment analysis suggested that the target genes of three miRNAs may be involved in various pathways related to the cancer. This study demonstrated that the three-miRNA signature (miR-1293, miR-873 and miR-1914) could be used as a prognostic marker in LACA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Single-Cell RNA-Seq Reveals Transcriptomic Heterogeneity and Post-Traumatic Osteoarthritis-Associated Early Molecular Changes in Mouse Articular Chondrocytes

Articular cartilage is a connective tissue lining the surfaces of synovial joints. When the cartilage severely wears down, it leads to osteoarthritis (OA), a debilitating disease that affects millions of people globally. The articular cartilage is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes with varying morphology and potentially different functions. Elucidating the molecular and functional profiles of various chondrocyte subtypes and understanding the interplay between these chondrocyte subtypes and other cell types in the joint will greatly expand our understanding of joint biology and OA pathology. Although recent advances in high-throughput OMICS technologies have enabled molecular-level characterization of tissues and organs at an unprecedented resolution, thorough molecular profiling of articular chondrocytes has not yet been undertaken, which may be in part due to the technical difficulties in isolating chondrocytes from dense cartilage ECM. In this study, we profiled articular cartilage from healthy and injured mouse knee joints at a single-cell resolution and identified nine chondrocyte subtypes with distinct molecular profiles and injury-induced early molecular changes in these chondrocytes. We also compared mouse chondrocyte subpopulations to human chondrocytes and evaluated the extent of molecular similarity between mice and humans. This work expands our view of chondrocyte heterogeneity and rapid molecular changes in chondrocyte populations in response to joint trauma and highlights potential mechanisms that trigger cartilage degeneration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T . atroviride exudates. In contrast, P . simiae and P . putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δ tmk 3) gene of T . atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T . atroviride , bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δ tmk3 mutant. Unexpectedly, exudates from wild-type T . atroviride and the Δ tmk 3 mutant rescued purine auxotrophic mutants of H . seropedicae , K . michiganensis and P . simiae . Metabolomic analysis on exudates from wild-type T . atroviride and the Δ tmk 3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma -metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

59 BASIC BIOLOGICAL SCIENCES↗