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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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All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Modification and analysis of context-specific genome-scale metabolic models: methane-utilizing microbial chassis as a case study

ABSTRACT Context-specific genome-scale model (CS-GSM) reconstruction is becoming an efficient strategy for integrating and cross-comparing experimental multi-scale data to explore the relationship between cellular genotypes, facilitating fundamental or applied research discoveries. However, the application of CS modeling for non-conventional microbes is still challenging. Here, we present a graphical user interface that integrates COBRApy, EscherPy, and RIPTiDe, Python-based tools within the BioUML platform, and streamlines the reconstruction and interrogation of the CS genome-scale metabolic frameworks via Jupyter Notebook. The approach was tested using -omics data collected for Methylotuvimicrobium alcaliphilum 20Z R , a prominent microbial chassis for methane capturing and valorization. We optimized the previously reconstructed whole genome-scale metabolic network by adjusting the flux distribution using gene expression data. The outputs of the automatically reconstructed CS metabolic network were comparable to manually optimized i IA409 models for Ca-growth conditions. However, the CS model questions the reversibility of the phosphoketolase pathway and suggests higher flux via primary oxidation pathways. The model also highlighted unresolved carbon partitioning between assimilatory and catabolic pathways at the formaldehyde-formate node. Only a very few genes and only one enzyme with a predicted function in C1 metabolism, a homolog of the formaldehyde oxidation enzyme ( fae1-2 ), showed a significant change in expression in La-growth conditions. The CS-GSM predictions agreed with the experimental measurements under the assumption that the Fae1-2 is a part of the tetrahydrofolate-linked pathway. The cellular roles of the tungsten (W)-dependent formate dehydrogenase ( fdhAB ) and fae homologs ( fae1-2 and fae3 ) were investigated via mutagenesis. The phenotype of the f dhAB mutant followed the model prediction. Furthermore, a more significant reduction of the biomass yield was observed during growth in La-supplemented media, confirming a higher flux through formate. M. alcaliphilum 20Z R mutants lacking fae1-2 did not display any significant defects in methane or methanol-dependent growth. However, contrary to fae1, the fae1-2 homolog failed to restore the formaldehyde-activating enzyme function in complementation tests. Overall, the presented data suggest that the developed computational workflow supports the reconstruction and validation of CS-GSM networks of non-model microbes. IMPORTANCE The interrogation of various types of data is a routine strategy to explore the relationship between genotype and phenotype. An efficient approach for integrating and cross-comparing experimental multi-scale data in the context of whole-genome-based metabolic network reconstruction becomes a powerful tool that facilitates fundamental and applied research discoveries. The present study describes the reconstruction of a context-specific (CS) model for the methane-utilizing bacterium, Methylotuvimicrobium alcaliphilum 20Z R . M. alcaliphilum 20Z R is becoming an attractive microbial platform for the production of biofuels, chemicals, pharmaceuticals, and bio-sorbents for capturing atmospheric methane. We demonstrate that this pipeline can help reconstruct metabolic models that are similar to manually curated networks. Furthermore, the model is able to highlight previously overlooked pathways, thus advancing fundamental knowledge of non-model microbial systems or promoting their development toward biotechnological or environmental implementations.

Kulyashov, M. A.↗
DOE OSTI2025

Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen-fixing photoautotrophs

ABSTRACT The nitrogenase gene cluster of unicellular diazotrophic cyanobacteria, such asCyanothece, is frequently selected by nature for nitrogen-fixing partnerships with eukaryotic phototrophs. The essential cluster components that confer an advantage in such partnerships remain underexplored. To use this cluster for the development of synthetic, phototrophic nitrogen-fixing systems, a thorough and systematic analysis of its constituent genes is necessary. An initial effort to assess the possibility of engineering this cluster into non-diazotrophic phototrophs led to the generation of aSynechocystis6803 strain with significant nitrogenase activity. In the current study, a refactoring approach was taken to determine the dispensability of the non-structural genes in the cluster and define a minimal gene set for constructing a functional nitrogenase for phototrophs. Using a bottom-up strategy, thenifgenes fromCyanothece51142 were re-organized to form new operons. The genes were then seamlessly removed to determine their essentiality in the nitrogen fixation process. We demonstrate that besides the structural genesnifHDK,nifBSUENPVZTXW, as well ashesAB, are important for optimal nitrogenase function in a phototroph. We also show that optimal expression of these genes is crucial for efficient nitrogenase activity. Our findings provide a solid foundation for generating synthetic systems that will facilitate solar-powered conversion of atmospheric nitrogen into nitrogen-rich compounds, a stride toward a greener world. IMPORTANCE Integrating nitrogen fixation genes into various photosynthetic organisms is an exciting strategy for converting atmospheric nitrogen into nitrogen-rich products in a green and energy-efficient way. In order to facilitate this process, it is essential that we understand the fundamentals of the functioning of a prokaryotic nitrogen-fixing machinery in a non-diazotrophic, photoautotrophic cell. This study examines a nitrogenase gene cluster that has been naturally selected on multiple occasions for a nitrogen-fixing partnership by eukaryotic photoautotrophs and provides a basic blueprint for designing a photosynthetic organism with nitrogen-fixing ability.

Microbiology↗
DOE OSTI2021

Identification of a diguanylate cyclase expressed in the presence of plants and its application for discovering candidate gene products involved in plant colonization by Pantoea sp. YR343

Microbial colonization of plant roots is a highly complex process that requires the coordination and regulation of many gene networks, yet the identities and functions of many of these gene products have yet to be discovered. Pantoea sp. YR343, a gamma-proteobacterium isolated from the rhizosphere of Populus deltoides , forms robust biofilms along the root surfaces of Populus and possesses plant growth-promoting characteristics. In this work, we identified three diguanylate cyclases in the plant-associated microbe Pantoea sp. YR343 that are expressed in the presence of plant roots. One of these diguanylate cyclases, DGC2884, localizes to discrete sites in the cells and its overexpression results in reduced motility and increased EPS production and biofilm formation. We performed a genetic screen by expressing this diguanylate cyclase from an inducible promoter in order to identify candidate gene products that may be involved in root colonization by Pantoea sp. YR343. Further, we demonstrate the importance of other domains in DGC2884 to its activity, which in combination with the genes identified by transposon mutagenesis, may yield insights into the mechanisms of plant association as well as the activity and regulation of homologous enzymes in medically and agriculturally relevant microbes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

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

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

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Viral afterlife: SARS-CoV-2 as a reservoir of immunomimetic peptides that reassemble into proinflammatory supramolecular complexes

It is unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to the strong but ineffective inflammatory response that characterizes severe Coronavirus disease 2019 (COVID-19), with amplified immune activation in diverse cell types, including cells without angiotensin-converting enzyme 2 receptors necessary for infection. Proteolytic degradation of SARS-CoV-2 virions is a milestone in host viral clearance, but the impact of remnant viral peptide fragments from high viral loads is not known. Here, we examine the inflammatory capacity of fragmented viral components from the perspective of supramolecular self-organization in the infected host environment. Interestingly, a machine learning analysis to SARS-CoV-2 proteome reveals sequence motifs that mimic host antimicrobial peptides (xenoAMPs), especially highly cationic human cathelicidin LL-37 capable of augmenting inflammation. Such xenoAMPs are strongly enriched in SARS-CoV-2 relative to low-pathogenicity coronaviruses. Moreover, xenoAMPs from SARS-CoV-2 but not low-pathogenicity homologs assemble double-stranded RNA (dsRNA) into nanocrystalline complexes with lattice constants commensurate with the steric size of Toll-like receptor (TLR)-3 and therefore capable of multivalent binding. Such complexes amplify cytokine secretion in diverse uninfected cell types in culture (epithelial cells, endothelial cells, keratinocytes, monocytes, and macrophages), similar to cathelicidin’s role in rheumatoid arthritis and lupus. The induced transcriptome matches well with the global gene expression pattern in COVID-19, despite using <0.3% of the viral proteome. Delivery of these complexes to uninfected mice boosts plasma interleukin-6 and CXCL1 levels as observed in COVID-19 patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Multi-omics analysis reveals the dynamic interplay between Vero host chromatin structure and function during vaccinia virus infection

The genome folds into complex configurations and structures thought to profoundly impact its function. The intricacies of this dynamic structure-function relationship are not well understood particularly in the context of viral infection. To unravel this interplay, here we provide a comprehensive investigation of simultaneous host chromatin structural (via Hi-C and ATAC-seq) and functional changes (via RNA-seq) in response to vaccinia virus infection. Over time, infection significantly impacts global and local chromatin structure by increasing long-range intra-chromosomal interactions and B compartmentalization and by decreasing chromatin accessibility and inter-chromosomal interactions. Local accessibility changes are independent of broad-scale chromatin compartment exchange (~12% of the genome), underscoring potential independent mechanisms for global and local chromatin reorganization. While infection structurally condenses the host genome, there is nearly equal bidirectional differential gene expression. Despite global weakening of intra-TAD interactions, functional changes including downregulated immunity genes are associated with alterations in local accessibility and loop domain restructuring. Therefore, chromatin accessibility and local structure profiling provide impactful predictions for host responses and may improve development of efficacious anti-viral counter measures including the optimization of vaccine design.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Integrative analysis of CAM photosynthesis reveals its impact on primary metabolism in Yucca

Crassulacean Acid Metabolism (CAM) is an adaptation that temporally separates carbon uptake at night from photosynthesis during the day. CAM has evolved repeatedly across vascular plants, as its emergence may depend on simple regulatory changes to deeply conserved metabolic pathways. Modern CAM research relies heavily on interpretation of transcriptomic data, though regulation occurs at multiple levels following transcription. Additionally, while most research to date has focused on a handful of genes and metabolites in the core CAM pathway, the co-option of conserved regulatory and functional genes is bound to have wide-ranging effects on other aspects of primary metabolism. In this study, we integrate transcriptomic, proteomic, and metabolomic data to compare primary metabolism between the CAM species Yucca aloifolia and closely related C 3 species, Y. filamentosa. We observe minimal correlation between protein abundance and mRNA expression, suggesting significant post-transcriptional regulation in CAM species. We also find evidence of shifts in gene expression and metabolite accumulation outside of the central CAM pathway, suggesting that the shift to CAM has cascading effects across primary metabolism, especially nitrogen metabolism. Our findings provide insights into the metabolic shifts associated with CAM evolution, and highlight the complexity of its regulation at multiple biological levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Role of Tensile Stress in DNA Nanoresonators for Epigenetic Studies

The evaluation of epigenetic features such as DNA methylation is becoming increasingly important in many biochemical processes like gene expression and transcription as well as in several diseases like schizophrenia or diabetes. Here, in this work, we report that self-assembled nanomechanical resonators entirely composed of DNA molecules can be used to explore gross changes in DNA methylation levels (0–25–50%), while careful control of tensile stress is needed to reduce the variability of resonance frequency for rigorous quantification. The effect of the tensile stress retained by the suspended DNA nanoresonators on the application of the technique is extensively explored using a combination of laser Doppler vibrometry and atomic force spectroscopy. DNA nanoresonators are real-time, label-free sensors and could avoid chemical functionalization and sample amplification. Therefore, they may represent in the future a key complementary routine tool for global DNA methylation analysis needed to evaluate the consequences of environmental stresses on the human genome.

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

Visualization and modeling of inhibition of IL-1β and TNF-α mRNA transcription at the single-cell level

Abstract IL-1β and TNF-α are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1β and TNF-α at the single-cell level. The amount of IL-1β and TNF-α mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1β and TNF-α mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1β is activated by both NF-κB and C/EBP, while TNF-α is predominantly activated by NF-κB. Our combined single-cell experimental and modeling efforts show the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Deletion of the cytochrome bc complex from Heliobacterium modesticaldum results in viable but non-phototrophic cells

The heliobacteria, a family of anoxygenic phototrophs, possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria can also grow chemotrophically via pyruvate metabolism in the dark. In the heliobacteria, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c 553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for the mobile cytochrome c 553 other than the photochemical reaction center. We have, therefore, hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. Here, we used a two-step method for CRISPR-based genome editing in Heliobacterium modesticaldum to delete the genes encoding the four major subunits of the cytochrome bc complex. Genotypic analysis verified the deletion of the petCBDA gene cluster encoding the catalytic components of the complex. Spectroscopic studies revealed that re-reduction of cytochrome c 553 after flash-induced photo-oxidation was over 100 times slower in the petCBDA mutant compared to the wild-type. Steady-state levels of oxidized P 800 (the primary donor of the photochemical reaction center) were much higher in the petCBDA mutant at every light level, consistent with a limitation in electron flow to the reaction center. The petCBDA mutant was unable to grow phototrophically on acetate plus CO 2 but could grow chemotrophically on pyruvate as a carbon source similar to the wild-type strain in the dark. The mutants could be complemented by reintroduction of the petCBDA gene cluster on a plasmid expressed from the clostridial eno promoter.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

De novo transcriptome sequencing of Capsicum frutescens . L and comprehensive analysis of salt stress alleviating mechanism by Bacillus atrophaeus WU ‐9

Abstract Salt stress, as one of the most severe environmental stresses, can cause a series of changes in plants. However, the explanation of plant salt stress alleviating mechanism of plant growth–promoting rhizobacteria (PGPR) was hindered by the limited availability of transcriptomic information for salt stress‐treated plants grown in a microorganism‐controlled environment. Our previous reports have selected Bacillus atrophaeus WU‐9 as PGPR significantly alleviating pepper ( Capsicum frutescens . L) salt stress. In this work, the RNA‐seq analysis of salt stress‐treated and untreated plants, grown with and without WU‐9 in a microorganism‐controlled environment, was used to reveal the plant salt stress alleviating mechanisms of WU‐9. Twelve sequencing libraries, prepared by treating with WU‐9 and salt (150 mM NaCl for 36 h), were constructed by RNA‐Seq technique. Non‐inoculated seedlings mainly respond to salt stress through regulation of signal transduction, such as ethylene‐activated signaling pathway, signaling and cell communication, etc. And ethylene signal participated in salt stress response in pepper through regulating defense responses, fruit ripening and senescence. WU‐9 inoculation under salt stress mainly improves salt tolerance and plant growth by regulating salt stress‐responding ethylene and auxin signal transduction, utilization of proline, photosynthesis, antioxidant enzyme activities and cell enlargement. Furthermore, 86 differentially expressed genes and 20 transcription factors were identified as associated with salt stress response and tolerance. Thus, this innovative transcriptomic study identified the salt stress response and alleviation in C. frutescens . L with PGPR inoculation. This result provided novel insights into the salinity alleviation in pepper regulated by PGPR.

Wang, Wenfei↗
DOE OSTI2022

Analysis of ionizing radiation induced DNA damage response in human adult stem cells and differentiated neurons

Findings of neurodegenerative features associated with human radiosensitive syndromes such as Ataxia telangiectasia suggest that DNA repair efficiency is crucial for maintaining the functional integrity of central nervous system. To gain a better understanding of ionizing radiation (IR) induced DNA damage response in undifferentiated and differentiated neural cell types and to evaluate the role of ATM in DNA double strand break (DSB) repair, an in vitro human neural cell differentiation model system was utilized in this study. As compared to adult stem cells, differentiated neurons displayed an attenuated DSB repair response (as judged by the persistence of 53BP1 foci) after IR exposure and the attenuation was even more pronounced in stem cells and neurons after suppression of ATM (Ataxia Telangiectasia Mutated) gene product suggesting the importance of ATM for an optimal DSB repair efficiency in human neural cell types. In corroboration with an attenuated DNA damage response, a sharp decline in the expression levels of several DSB repair genes was observed in neurons. Furthermore, our results suggest that cellular differentiation modulates the expression of several genes thereby compromising the DSB repair fidelity in post mitotic neurons. Further studies are required to verify whether or not ATM mediated exacerbation of DNA repair deficiency in differentiated neurons leads to neurodegeneration.

53 binding protein 1↗
DOE OSTI2024

Multilevel analysis between Physcomitrium patens and Mortierellaceae endophytes explores potential long‐standing interaction among land plants and fungi

SUMMARY The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant–fungal interactions today, it is hypothesized these kingdoms have a long‐standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free‐living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia , interact with P. patens in coculture. We also assess how Mollicute ‐related or Burkholderia ‐related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high‐throughput phenomics, microscopy, RNA‐sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high‐throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant–fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens .

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Iron rescues glucose-mediated photosynthesis repression during lipid accumulation in the green alga Chromochloris zofingiensis

Energy status and nutrients regulate photosynthetic protein expression. The unicellular green alga Chromochloris zofingiensis switches off photosynthesis in the presence of exogenous glucose (+Glc) in a process that depends on hexokinase (HXK1). Here, we show that this response requires that cells lack sufficient iron (–Fe). Cells grown in –Fe+Glc accumulate triacylglycerol (TAG) while losing photosynthesis and thylakoid membranes. However, cells with an iron supplement (+Fe+Glc) maintain photosynthesis and thylakoids while still accumulating TAG. Proteomic analysis shows that known photosynthetic proteins are most depleted in heterotrophy, alongside hundreds of uncharacterized, conserved proteins. Photosynthesis repression is associated with enzyme and transporter regulation that redirects iron resources to (a) respiratory instead of photosynthetic complexes and (b) a ferredoxin-dependent desaturase pathway supporting TAG accumulation rather than thylakoid lipid synthesis. Combining insights from diverse organisms from green algae to vascular plants, we show how iron and trophic constraints on metabolism aid gene discovery for photosynthesis and biofuel production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

MaizeMine: A Data Mining Warehouse for the Maize Genetics and Genomics Database

MaizeMine is the data mining resource of the Maize Genetics and Genome Database (MaizeGDB; http://maizemine.maizegdb.org). It enables researchers to create and export customized annotation datasets that can be merged with their own research data for use in downstream analyses. MaizeMine uses the InterMine data warehousing system to integrate genomic sequences and gene annotations from the Zea mays B73 RefGen_v3 and B73 RefGen_v4 genome assemblies, Gene Ontology annotations, single nucleotide polymorphisms, protein annotations, homologs, pathways, and precomputed gene expression levels based on RNA-seq data from the Z. mays B73 Gene Expression Atlas. MaizeMine also provides database cross references between genes of alternative gene sets from Gramene and NCBI RefSeq. MaizeMine includes several search tools, including a keyword search, built-in template queries with intuitive search menus, and a QueryBuilder tool for creating custom queries. The Genomic Regions search tool executes queries based on lists of genome coordinates, and supports both the B73 RefGen_v3 and B73 RefGen_v4 assemblies. The List tool allows you to upload identifiers to create custom lists, perform set operations such as unions and intersections, and execute template queries with lists. When used with gene identifiers, the List tool automatically provides gene set enrichment for Gene Ontology (GO) and pathways, with a choice of statistical parameters and background gene sets. With the ability to save query outputs as lists that can be input to new queries, MaizeMine provides limitless possibilities for data integration and meta-analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

EMC3 regulates trafficking and pulmonary toxicity of the SFTPC I73T mutation associated with interstitial lung disease

The most common mutation in surfactant protein C gene (SFTPC), SFTPC I73T , causes interstitial lung disease with few therapeutic options. We previously demonstrated that EMC3, an important component of the multiprotein endoplasmic reticulum membrane complex (EMC), is required for surfactant homeostasis in alveolar type 2 epithelial (AT2) cells at birth. In the present study, we investigated the role of EMC3 in the control of SFTPC I73T metabolism and its associated alveolar dysfunction. Using a knock-in mouse model phenocopying the I73T mutation, we demonstrated that conditional deletion of Emc3 in AT2 cells rescued alveolar remodeling/simplification defects in neonatal and adult mice. Proteomic analysis revealed that Emc3 depletion reversed the disruption of vesicle trafficking pathways and rescued the mitochondrial dysfunction associated with I73T mutation. Affinity mass spectrometry analysis identified potential EMC3 interacting proteins in lung AT2 cells, including Valosin Containing Protein (VCP) and its interactors. Treatment of Sftpc I73T knock-in mice and SFTPC I73T expressing iAT2 cells derived from SFTPC I73T patient-specific iPSCs with the specific VCP inhibitor CB5083 restored alveolar structure and SFTPC I73T trafficking respectively. Taken together, the present work identifies the EMC complex and VCP in the metabolism of the disease-associated SFTPC I73T mutant, providing novel therapeutical targets for SFTPC I73T -associated interstitial lung disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Exploring Plant Cis –Regulatory Elements at Single–Cell Resolution: Overcoming Biological and Computational Challenges to Advance Plant Research

Cis-regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall have limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Furthermore, advances in single cell epigenomics have revolutionized the field identifying cell-type-specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single-cell research, challenges and common pitfalls in analysis of plant single-cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single-cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Statistical relationships across epigenomes using large-scale hierarchical clustering

Recent advances in genomics and sequencing platforms have revolutionized our ability to create immense data sets, particularly for studying epigenetic regulation of gene expression. However, the avalanche of epigenomic data is difficult to parse for biological interpretation given nonlinear complex patterns and relationships. This attractive challenge in epigenomic data lends itself to machine learning for discerning infectivity and susceptibility. In this study, we explore over 3000 epigenomes of uninfected individuals and provide a framework to characterize the relationships among epigenetic modifiers, their modifiers, genetic loci, and specific immune cell types across all chromosomes using hierarchical clustering. Hierarchical clustering of epigenomic data revealed consistent epigenetic patterns across chromosomes, demonstrating that variation due to epigenetic modifiers is greater than variation between cell types. Gene Ontology and KEGG pathway analyses indicated significant enrichment of genes involved in chromatin remodeling, mRNA splicing, immune responses, and the regulation of microRNAs and snoRNAs. Epigenetic modifiers frequently formed biologically relevant clusters, including the cohesin complex, RNA Polymerase II transcription factors, and PRC2 complex members. These clustering behaviors remained consistent across all chromosomes, supported by entropy analysis and high Adjusted Rand Index scores, indicating robust cross-chromosomal similarity. Co-occurrence analysis further revealed specific sets of modifiers that consistently appeared together within clusters, reflecting shared biological functions and interactions. Validation using another dataset confirmed the reproducibility of these clustering patterns and modifier co-occurrence relationships, underscoring the reliability and generalizability of the methodology.

97 MATHEMATICS AND COMPUTING↗