SEARCH · Search NASA

Results for “RNA”

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.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9

All sources NASA NTRS DOE OSTI

DOE OSTI2026

Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics

Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Polyyne production is regulated by the transcriptional regulators PgnC and GacA in Pseudomonas protegens Pf-5

ABSTRACT Polyynes produced by bacteria have promising applications in agriculture and medicine due to their potent antimicrobial activities. Polyyne biosynthetic genes have been identified inPseudomonasandBurkholderia. However, the molecular mechanisms underlying the regulation of polyyne biosynthesis remain largely unknown. In this study, we used a soil bacteriumPseudomonas protegensPf-5, which was recently reported to produce polyyne called protegenin, as a model to investigate the regulation of bacterial polyyne production. Our results show that Pf-5 controls polyyne production at both the pathway-specific level and a higher global level. Mutation ofpgnC, a transcriptional regulatory gene located in the polyyne biosynthetic gene cluster, abolished polyyne production. Gene expression analysis revealed that PgnC directly activates the promoter of polyyne biosynthetic genes. The production of polyyne also requires a global regulator GacA. Mutation ofgacAdecreased the translation of PgnC, which is consistent with the result thatpgnCleader mRNA bound directly to RsmE, an RNA-binding protein negatively regulated by GacA. These results suggest that GacA induces the expression of the PgnC regulator, which in turn activates polyyne biosynthesis. Additionally, the polyyne-producing strain of Pf-5, but not the polyyne-nonproducing strain, could inhibit a broad spectrum of bacteria including both Gram-negative and Gram-positive bacteria. IMPORTANCE Antimicrobial metabolites produced by bacteria are widely used in agriculture and medicine to control plant, animal, and human pathogens. Although bacteria-derived polyynes have been identified as potent antimicrobials for decades, the molecular mechanisms by which bacteria regulate polyyne biosynthesis remain understudied. In this study, we found that polyyne biosynthesis is directly activated by a pathway-specific regulator PgnC, which is induced by a global regulator GacA through the RNA-binding protein RsmE inPseudomonas protegens. To our knowledge, this work is the first comprehensive study of the regulatory mechanisms of bacterial polyyne biosynthesis at both pathway-specific level and global level. The discovered molecular mechanisms can help us optimize polyyne production for agricultural or medical applications.

Biotechnology & Applied Microbiology↗

DOE OSTI2025

Physiological roles of an Acinetobacter -specific σ factor

ABSTRACT The Gram-negative pathogen Acinetobacter baumannii is considered an “urgent threat” to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF “SigAb”; however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE-like ECFs. We combine promoter mutagenesis, motif scanning, and chromatin immunoprecipitation-sequencing (ChIP-seq) to define the direct SigAb regulon, which consists of genes encoding SigAb itself, the stringent response mediator, RelA, and the uncharacterized small RNA, “SabS.” However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper-induced SigAb-dependent transcription. Furthermore, we found that two uncharacterized genes in the sigAb operon, “ aabA ” and “ aabB ,” have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb , aabA , and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii . IMPORTANCE Acinetobacter baumannii is a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding how A. baumannii senses and responds to stress may uncover novel routes to treat infections. Here, we examine how the Acinetobacter -specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically relevant contexts.

Bacon, Emily E. (ORCID:0000000180907689)↗

DOE OSTI2024

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira

ABSTRACT Leptospirosis, caused by pathogenic bacteria from the genusLeptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation ofLeptospirahas been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal toLeptospiracells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3′ end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenicLeptospirafrom multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulateLeptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis. IMPORTANCE Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenicLeptospiraspp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system toLeptospiraenhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique inLeptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in theLeptospira borgpeterseniibackground, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms ofLeptospiraand the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Microbiology↗

DOE OSTI2025

Separate, separated, and together: the transcriptional program of the Clostridium acetobutylicum-Clostridium ljungdahlii syntrophy leading to interspecies cell fusion

ABSTRACT Syntrophic cocultures (hitherto assumed to be commensalistic) of Clostridium acetobutylicum and Clostridium ljungdahlii , whereby CO 2 and H 2 produced by the former feed the latter, result in interspecies cell fusion involving large-scale exchange of protein, RNA, and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane-separated versus -unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in C. ljungdahlii’s autotrophic Wood-Ljungdahl pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by C. acetobutylicum and are metabolized by C. ljungdahlii to its growth advantage. These and other findings, and notably upregulation of C. acetobutylicum ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO 2 and H 2 , C. acetobutylicum feeds C. ljungdahlii with growth-boosting amino acids, while benefiting from the H 2 utilization by C. ljungdahlii . IMPORTANCE The construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies “touching” on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

Willis, Noah B. (ORCID:0009000689365955)↗

DOE OSTI2025

A Melanoma Brain Metastasis CTC Signature and CTC:B-cell Clusters Associate with Secondary Liver Metastasis: A Melanoma Brain–Liver Metastasis Axis

Melanoma brain metastasis is linked to dismal prognosis and low overall survival and is detected in up to 80% of patients at autopsy. Circulating tumor cells (CTC) are the smallest functional units of cancer and precursors of fatal metastasis. We previously used an unbiased multilevel approach to discover a unique ribosomal protein large/small subunit (RPL/RPS) CTC gene signature associated with melanoma brain metastasis. In this study, we hypothesized that CTC-driven melanoma brain metastasis secondary metastasis (“metastasis of metastasis” per clinical scenarios) has targeted organ specificity for the liver. We injected parallel cohorts of immunodeficient and newly developed humanized NBSGW (huNBSGW) mice with cells from CTC-derived melanoma brain metastasis to identify secondary metastatic patterns. We found the presence of a melanoma brain–liver metastasis axis in huNBSGW mice. Furthermore, RNA sequencing analysis of tissues showed a significant upregulation of the RPL/RPS CTC gene signature linked to metastatic spread to the liver. Additional RNA sequencing of CTCs from huNBSGW blood revealed extensive CTC clustering with human B cells in these mice. CTC:B-cell clusters were also upregulated in the blood of patients with primary melanoma and maintained either in CTC-driven melanoma brain metastasis or melanoma brain metastasis CTC–derived cells promoting liver metastasis. CTC-generated tumor tissues were interrogated at single-cell gene and protein expression levels (10x Genomics Xenium and HALO spatial biology platforms, respectively). Collectively, our findings suggest that heterotypic CTC:B-cell interactions can be critical at multiple stages of metastasis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2026

Poxvirus infection triggers remodeling of host m⁶A epitranscriptome and benefits from the m⁶A regulatory responses

Understanding how host gene regulation responds to viral infection is essential for developing effective antiviral strategies. Emerging evidence suggests that host transcripts undergo dynamic chemical modifications to counteract viral invasion. Conversely, viruses that rely on nuclear transcription exploit host RNA methyltransferases to enhance mRNA export and translation. Orthopoxviruses, however, complete their entire replication cycle within compartmentalized cytoplasmic “factories” utilizing enzymes encoded by their large double-stranded viral DNA genomes. The dynamic interplay between host and poxviral epitranscriptome remains poorly characterized. Using a temporally resolved model of Vaccinia virus (VV) infection, we investigated host-virus interactions through transcriptome and N6-methyladenosine (m⁶A) epitranscriptome whole genome sequencing. We found that host m⁶A modifications respond rapidly to VV infection, preceding the delayed transcriptional changes that emerge at later stages. Early m⁶A signatures included key innate immunity factors as well as host genes involved in transcriptional regulation, post-transcriptional modification, and protein ubiquitination. Functional assays validated two host factors with early m⁶A modification changes that are essential for VV infection: a m⁶A reader, YTHDF1, and a component of the SCF E3 ubiquitin ligase complex, FBXO31. The m⁶A gain on YTHDF1 enhanced its protein expression and promoted efficient VV replication. In addition, we identified previously unrecognized roles of FBXO31 and the SCF E3 ligase complex in supporting VV infection. Temporal profiling of the m⁶A epitranscriptome reveals how VV exploits host post-transcriptional regulatory pathways, specifically m⁶A RNA modification and protein ubiquitination. These findings highlight critical host factors co-opted during poxvirus infection and identify potential targets for therapeutic intervention.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Circularization of 23S rRNA but not 16S rRNA within archaeal ribosomes

Background Processing of archaeal 16S and 23S rRNAs is believed to involve excision of individual rRNAs from polycistronic precursors, circularization of excised rRNAs, and re-linearization before the incorporation into ribosomes. However, all the knowledge is derived from several isolated species, leaving open the possibility that different processes may occur in other archaeal groups. Results Here, we investigate rRNAs from diverse and mostly uncultivated archaea. Sequencing of total cellular RNA from eight phylum-level lineages indicates that archaeal circular 23S rRNA transcript abundances vastly exceed those of linear counterparts, and linear versions are often undetectable. As the majority of rRNAs derive from mature ribosomes, the data suggest that ribosomes contain circular 23S rRNAs. Thus, we directly sequence RNA extracted from isolated ribosomes of a model archaeon, Methanosarcina acetivorans, and confirm that the 23S rRNAs in the ribosomes are circular. Structural modeling places the 5′ and 3′ ends of the linear precursors of archaeal 23S rRNAs in close proximity to form a GNRA tetraloop (in which N is A, C, G, or U and R is A or G), consistent with their existence as circular molecules. We also confirm the existence of circular 16S rRNA intermediates in transcriptomes of most archaea, yet a circular form is not evident in some distinct archaeal groups, suggesting that certain archaea do not circularize 16S rRNA during processing. Conclusions Our findings uncover unexpected variations in the processing required to generate mature rRNAs and the conformation of functional molecules in archaeal ribosomes.

Archaea↗

DOE OSTI2026

Data for Sugar Accumulation Enhancement in Sorghum Stem is Associated with Reduced Reproductive Sink Strength and Increased Phloem Unloading Activity

Sweet sorghum has emerged as a promising source of bioenergy mainly due to its high biomass and high soluble sugar yield in stems. Studies have shown that loss-of-function Dry locus alleles have been selected during sweet sorghum domestication, and decapitation can further boost sugar accumulation in sweet sorghum, indicating that the potential for improving sugar yields is yet to be fully realized. To maximize sugar accumulation, it is essential to gain a better understanding of the mechanism underlying the massive accumulation of soluble sugars in sweet sorghum stems in addition to the Dry locus. We performed a transcriptomic analysis upon decapitation of near-isogenic lines for mutant (d, juicy stems, and green leaf midrib) and functional (D, dry stems and white leaf midrib) alleles at the Dry locus. Our analysis revealed that decapitation suppressed photosynthesis in leaves, but accelerated starch metabolic processes in stems. SbbHLH093 negatively correlates with sugar levels supported by genotypes (DD vs. dd), treatments (control vs. decapitation), and developmental stages post anthesis (3d vs.10d). D locus gene SbNAC074A and other programmed cell death-related genes were down regulated by decapitation, while sugar transporter-encoding gene SbSWEET1A was induced. Both SbSWEET1A and Invertase 5 were detected in phloem companion cells by RNA in situ assay. Loss of the SbbHLH093 homolog, AtbHLH093, in Arabidopsis led to a sugar accumulation increase. This study provides new insights into sugar accumulation enhancement in bioenergy crops, which can be potentially achieved by reducing reproductive sink strength and enhancing phloem unloading.

Transcriptomics↗

DOE OSTI2024

Data for Rapid and Efficient in planta Genome Editing in Sorghum Using Foxtail Mosaic Virus-mediated sgRNA Delivery

The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor . We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase (PDS), Magnesium-chelatase subunit I (MgCh), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, orthologs of maize Lemon white1 (Lw1) or GFP. The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum.

Feedstock Production↗

DOE OSTI2025

Long-read sequencing transcriptome quantification with lr-kallisto

RNA abundance quantification has become routine and affordable thanks to high-throughput “short-read” technologies that provide accurate molecule counts at the gene level. Similarly accurate and affordable quantification of definitive full-length, transcript isoforms has remained a stubborn challenge, despite its obvious biological significance across a wide range of problems. “Long-read” sequencing platforms now produce data-types that can, in principle, drive routine definitive isoform quantification. However some particulars of contemporary long-read datatypes, together with isoform complexity and genetic variation, present bioinformatic challenges. We show here, using ONT data, that fast and accurate quantification of long-read data is possible and that it is improved by exome capture. To perform quantifications we developed lr-kallisto, which adapts the kallisto bulk and single-cell RNA-seq quantification methods for long-read technologies.

Loving, Rebekah K. (ORCID:0000000187250376)↗

DOE OSTI2026

Multi-omics data resource: Data package 25 (Pck025)

This data package comprises omics datasets from human pancreatic islets treated with IL-1β + IFNγ or with estrogen (E2) for 18 h. Two RNA-seq datasets are available: the first is a discovery dataset involving human islets treated with or without IL-1β + IFNγ for 18 hours; the second is a validation dataset, where human islets are treated with or without IL-1β + IFNγ or E2 for 18 hours. DIA proteomic analysis was performed on the same validation dataset samples. Data contributors: Kiersten L. Webster, Sarah Tersey & Raghavendra G. Mirmir: Kovler Diabetes Center and Department of Medicine, The University of Chicago, Chicago, IL, 60637, USA. Soumyadeep Sarkar, Raghavendra Mirmira, Ernesto S. Nakayasu: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, 99354, USA. Data repository: RNA-seq: GSE310965 Proteomics: MSV000101892 Publication: PMID 41279069

Sarkar, Soumyadeep [Pacific Northwest National Lab↗

DOE OSTI2024

Human Host Cellular Response to HCoV-229E Infection Proteomics (ACS-JM-DP2)

The purpose of this experiment was to evaluate the human host cellular response to wild-type Human coronavirus strain 229E (HCoV-229E) infection. Sample data was obtained for mock and infected immortalized human lung epithelial cells (A549) (MOI 5) nuclear extracts, immortalized human lung fibroblasts cells (MRC5) (MOI5) nuclear extracts, and primary human airway epithelial (HAE) (MOI 3) cells from lung tissue and processed for proteome analysis. Processed datasets are openly accessible from the download button and contain secondary processed proteomic results files and supporting metadata materials. Experimental proteomics samples were prepared using Limited Proteolysis (LiP) methods for Label-free quantification (LFQ) and global proteomic evaluation. Sample data was acquired using a Q-Exactive HF-X mass spectrometer and was processed and compiled using MaxQuant software (v.1.6.17.0). Processed proteomic data downloads include a sample naming key, processed MaxQuant results/parameters, and protein annotated relative abundance files. See corresponding primary data accessions below and Viral Experiment LiP Analysis source code supporting data transparency and reuse. Experimental transcriptomics samples were collected in parallel and processed for RNA sequencing (RNA-Seq) as summarized under ACS-DP1 (https://data.pnnl.gov/group/nodes/dataset/34069).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

HtPIP: High-throughput phage isolation platform increases diversity and reduces isolation time using multiple bacteria

Bacteriophages are ubiquitous in nature, but relatively few have been isolated and characterized compared to the number of bacterial strains. Phage biotechnology applications benefit from a diverse library of isolated phages to kill or transfer genetic material to a bacterium of interest. However, scaling up phage discovery for diverse bacterial hosts can be time-consuming and costly. Here, we developed an approach to capture novel phages for multiple bacterial strains in parallel from an environmental sample using commercially available 0.2-μM filter plates. Using this High-throughput Phage Isolation Platform (HtPIP), 12 novel phages were isolated spanning 9 diverse bacterial host genera. Eleven of the isolated phages define new phage species, with nine also defining new genera. The HtPIP was used to discover both DNA and RNA phages, including a Tectiviridae infecting Pseudomonas putida mt-2 and a Leviviricetes infecting a Microbacterium isolate, which represents the first cultured RNA phage infecting a host outside of Proteobacteria. Using a metagenomic approach, we demonstrate that the HtPIP captures a higher proportion of novel phages compared to traditional low-throughput methods.

High-throughput↗

DOE OSTI2024

Eucalyptus Wood Smoke Extract Elicits a Dose-Dependent Effect in Brain Endothelial Cells

The frequency, duration, and size of wildfires have been increasing, and the inhalation of wildfire smoke particles poses a significant risk to human health. Epidemiological studies have shown that wildfire smoke exposure is positively associated with cognitive and neurological dysfunctions. However, there is a significant gap in knowledge on how wildfire smoke exposure can affect the blood–brain barrier and cause molecular and cellular changes in the brain. Our study aims to determine the acute effect of smoldering eucalyptus wood smoke extract (WSE) on brain endothelial cells for potential neurotoxicity in vitro. Primary human brain microvascular endothelial cells (HBMEC) and immortalized human brain endothelial cell line (hCMEC/D3) were treated with different doses of WSE for 24 h. WSE treatment resulted in a dose-dependent increase in IL-8 in both HBMEC and hCMEC/D3. RNA-seq analyses showed a dose-dependent upregulation of genes involved in aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (NRF2) pathways and a decrease in tight junction markers in both HBMEC and hCMEC/D3. When comparing untreated controls, RNA-seq analyses showed that HBMEC have a higher expression of tight junction markers compared to hCMEC/D3. In summary, our study found that 24 h WSE treatment increases IL-8 production dose-dependently and decreases tight junction markers in both HBMEC and hCMEC/D3 that may be mediated through the AhR and NRF2 pathways, and HBMEC could be a better in vitro model for studying the effect of wood smoke extract or particles on brain endothelial cells.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Transcriptomic and Proteomic Insights into Host Immune Responses in Pediatric Severe Malarial Anemia: Dysregulation in HSP60-70-TLR2/4 Signaling and Altered Glutamine Metabolism

Severe malarial anemia (SMA, Hb < 6.0 g/dL) is a leading cause of childhood morbidity and mortality in holoendemic Plasmodium falciparum transmission zones. This study explored the entire expressed human transcriptome in whole blood from 66 Kenyan children with non-SMA (Hb ≥ 6.0 g/dL, n = 41) and SMA (n = 25), focusing on host immune response networks. RNA-seq analysis revealed 6862 differentially expressed genes, with equally distributed up-and down-regulated genes, indicating a complex host immune response. Deconvolution analyses uncovered leukocytic immune profiles indicative of a diminished antigenic response, reduced immune priming, and polarization toward cellular repair in SMA. Weighted gene co-expression network analysis revealed that immune-regulated processes are central molecular distinctions between non-SMA and SMA. A top dysregulated immune response signaling network in SMA was the HSP60-HSP70-TLR2/4 signaling pathway, indicating altered pathogen recognition, innate immune activation, stress responses, and antigen recognition. Validation with high-throughput gene expression from a separate cohort of Kenyan children (n = 50) with varying severities of malarial anemia (n = 38 non-SMA and n = 12 SMA) confirmed the RNA-seq findings. Proteomic analyses in 35 children with matched transcript and protein abundance (n = 19 non-SMA and n = 16 SMA) confirmed dysregulation in the HSP60-HSP70-TLR2/4 signaling pathway. Additionally, glutamine transporter and glutamine synthetase genes were differentially expressed, indicating altered glutamine metabolism in SMA. This comprehensive analysis underscores complex immune dysregulation and novel pathogenic features in SMA.

Microbiology↗

DOE OSTI2025

The Importance of Solution Studies for the Structural Characterization of the Enterovirus 5’ Cloverleaf

Enteroviruses initiate genomic replication via a highly conserved mechanism that is controlled by an RNA platform, also known as the 5’ cloverleaf (5’CL). Here, we present a biophysical analysis of the 5’CL conformation of three enterovirus serotypes under various ionic conditions, utilizing CD spectroscopy, size-exclusion chromatography, and small-angle X-ray scattering. In general, a tendency toward a smaller monomeric hydrodynamic radius in the presence of salts was observed, but the exact structural signature of each 5’CL varied depending upon the serotype. Rhinovirus B14 (RVB14) exhibited at least two monomeric conformations and a low propensity for dimerization, while poliovirus 1 (PV1) showed a high propensity for dimerization, which was enhanced by the presence of salts. Enterovirus D70 was observed to be somewhat intermediate, with primarily a monomeric structure, but possessing some potential for dimerization. The equilibrium between the two monomeric and the dimeric conformations is also discussed. These results indicate that the 5’CL conformation may be more complex than the current literature suggests, thus underscoring the need for a combined crystal and solution approach for the accurate representation of the 5’CL conformation, and the conformation of other RNA structural elements, under native conditions.

Virology↗

DOE OSTI2024

Expression of dehydroshikimate dehydratase in poplar induces transcriptional and metabolic changes in the phenylpropanoid pathway

Abstract Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In planta expression of a bacterial 3-dehydroshikimate dehydratase in poplar trees reduced lignin content and altered the monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we acquired fundamental knowledge on lignin-modified poplar expressing 3-dehydroshikimate dehydratase using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibited the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. The changes affected predominantly the shikimate and phenylpropanoid pathways as well as secondary cell wall metabolism, and resulted in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

59 BASIC BIOLOGICAL SCIENCES↗